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Research ArticleAltered DNA Methylation of Long Noncoding RNA uc167Inhibits Cell Differentiation in Heart Development
Anwen Yin1 Mengwen Feng1 Zijie Cheng1 Qijun Zhang2 Hua Li1
Jia Xu1 Hao Zhang1 Yun Li 3 and Lingmei Qian 1
1Department of Cardiology The First Affiliated Hospital of Nanjing Medical University Nanjing China2Department of Cardiology Yinzhou Hospital Medical School of Ningbo University Ningbo China3Department of Pharmacy Obstetrics and Gynecology Hospital Nanjing Medical University Nanjing China
Correspondence should be addressed to Yun Li liyunwater163com and Lingmei Qian lmqiannjmueducn
Received 19 December 2017 Revised 3 April 2018 Accepted 10 April 2018 Published 30 May 2018
Academic Editor Shoichiro Ono
Copyright copy 2018 Anwen Yin et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
In previous studies we have demonstrated the function of uc167 in the heart development DNA methylation plays a crucial rolein regulating the expression of developmental genes during embryonic development In this study the methylomic landscapewas investigated in order to identify the DNA methylation alterations Methylated DNA immunoprecipitation (MeDIP) wasperformed to examine the differences in methylation status of overexpressed uc167 in P19 cells GO and KEGG pathway analysesof differentially methylated genes were also conducted We found that the distribution of differentially methylated regions (DMRs)peaks in different components of genome was mainly located in intergenic regions and intron The biological process associatedwith uc167 was focal adhesion and Rap1 signaling pathway MEF2C was significantly decreased in uc167 overexpressed groupsuggesting that uc167 may influence the P19 differentiation through MEF2C reduction Taken together our findings revealed thatthe effect of uc167 on P19 differentiation may be attributed to the altered methylation of specific genes
1 Introduction
Congenital heart disease (CHD) one of the most com-mon congenital malformations worldwide is caused by thedysplasia of heart and vessels in fetal period The mostprevalent CHD subtype is the ventricular septal defects(VSD) making up about 40 of CHD [1] These are relatedto a combination of environmental and genetic factors suchas NKX25 GATA4 TBX5 and MEF2C which are simplycaused by environmental factors only accounting for only2ndash5 [2] Mutations of different genes can lead to differentsubtypes of CHD which puts heavy economic and socialburdens on the family and society [3]
Epigenetics is regulated by various mechanismsincluding histone modifications DNA methylation ATP-dependent chromatin-remodeling complexes and smallRNAs like siRNA miRNAs and other noncoding RNAs[4] DNA methylation is an epigenetic modificationassociated with transcriptional changes [5] As an underlyingmechanism for birth defects including CHD derangements
in epigenetic regulated by DNA methylation can lead todisturbed embryogenesis [6]
Ultraconserved elements (UCEs) are a specific type oflong noncoding RNAs and are absolutely conserved betweenorthologous regions of the human rat and mouse genomes[7] LncRNAs have been shown to influence gene expressionby being recruited to gene promoters andmodifying the geneimprinting which is associated with DNA methylation Ithas been revealed that changes in DNA methylation maypartly disrupt the control of specific promoter usage incancers [8] However the DNAmethylation dynamic patternof long noncoding RNA has remained unknown in heartdevelopment
In previous studies we have demonstrated the impactof lncRNA uc167 on the development of embryonic heartand cardiomyocytesrsquo growth proliferation apoptosis anddifferentiation [9] Analyzing the methylation changes incardiomyocytes may provide an insight into the mechanismof uc167 regulation in CHD In this study methylatedDNA immunoprecipitation sequencing (MeDIP-seq) was
HindawiBioMed Research InternationalVolume 2018 Article ID 4658024 9 pageshttpsdoiorg10115520184658024
2 BioMed Research International
performed to provide extensive coverage of the methylomiclandscape in order to identify the different distributionlevels of genome-wide methylation regions In addition afteroverexpressing uc167 in P19 cells the related biologicalfunctions and pathways are discussed Our research providedevidence for the mechanism underlying uc167 in the heartdevelopment
2 Materials and Methods
21 Cell Culture and Plasmid Transfection P19 cells wereobtained from the American Type Culture Collection(ATCC Manassas VA USA) The cells were cultivated inAlpha Minimum Essential Medium (120572-MEM Gibco Carls-bad CA USA) containing 10 fetal bovine serum (FBSGibco) 100Uml penicillin and 50 ugml streptomycin at37∘C in 5 CO2 Cells were seeded into 6-well cultureplates and grown to 50ndash70 confluence before transfec-tion which was performed according to the Lipofectamine2000 DNA transfection reagent protocol (Life TechnologiesInc Waltham MA USA) The plasmid for overexpression(pCDNA31(+)) was constructed by GenePharma (ShanghaiChina) The same empty vector plasmid was used as controlgroup
22 Real-Time PCR Primers were designed in National Cen-ter for Biotechnology Information (NCBI) and synthesized byGeneray (Shanghai China) (Table S1) The RNA extraction(Tiangen) reverse transcription (Takara 047A) and qRT-PCR (SYBR Green PCR Kit (RR420A)) were performedaccording to the manufacturerrsquos guidelines The expressionsof uc167 were quantified to confirm successful overexpres-sion The data were analyzed using the 2minusΔΔ119862119879 method
23 Nuclear and Cytoplasmic Fraction Nuclear and cyto-plasmic fractions were divided using a PARIS Kit (ThermoFisher Scientific) At least 107 cultured cells in total werecollected and then 500 ul ice-cold cell fractionation buffer(CFB) was added The pellet was gently resuspended bypipetting and continuously incubated on ice for 10minThe nuclear pellet fraction was separated from cytoplasmicfraction after samples were centrifuged at 500119892 for 5minThe pellet was washed twice in ice-cold CFB to prevent thestructure of cytoplasmic fraction from contaminating thenuclear fraction
24 DNA Extraction and Library Preparation DNA fromtransfected P19 cells was extracted using TissueLyser andAllPrep DNARNAProtein Mini Kit (Qiagen) accordingto the manufacturerrsquos protocols Samples were quantifiedthrough 1 agarose gel electrophoresis with Omega TissueDNA Kit (200) (D3396-02)
25 Methylated DNA Immunoprecipitation Sequencing Pro-cedure Covaris S2 system was used to sonicate genomicDNA (3 120583g) for 55 s at intensity of 4 for 200 cyclesper burst Illumina library preparation was conducted byusing the Sample Preparation Kit Fragmented DNA was
end-repaired and ATP-tailed and the adaptor was ligatedThen the adaptor-ligated DNA was recovered by AMPureXP Beads (Beckman Coulter) denatured and then subjectedto methylated DNA immunoprecipitation (MeDIP) 5 g of amonoclonal antibody against 5-methylcytosine (Eurogentec)coupled to magnetic Dynabeads with M-280 sheep antibodyagainst mouse IgG (Thermo Fisher Scientific) was used tocarry out MeDIP Sequencing libraries were denatured at95∘C for 10min and incubation with the beads in the IPBuffer (140mMNaCl 025 Triton times100 and 10Mm sodiumphosphate buffer (pH 70)) was performed at 4∘C for 4 hThe IP buffer was used to wash the beads for three timesand DNA was eluted in the elution buffer (10mM EDTA1 SDS and 50mM Tris-HCl (pH 75)) for 15min at 65∘CThen the beads were conducted with proteinase K at 55∘C for2 h and methylated DNA was recovered with the QIA quickPCR Purification Kit (Qiagen) Quantitative PCR (qPCR)was used to assess the efficiency of MeDIP After MeDIPenrichment remaining DNA was amplified with sequencingprimers through PCR and sequenced with the HiSeq 2500platform (Illumina)
26 Data Preprocessed and Quality Control Raw readsincluding overall the low-quality reads more than 20 basesand proportion less than 50 less than 20 bases in 31015840 endjoint sequence reads less than 20 in length and reads witha nitrogenous preprocessed base were removed (FASTX-Toolkit) (version 0013) Then we used Bowtie (version0128) to conduct genomemapping the reads were aligned tothe reference genomes and then the efficiency of enrichmentwas calculated The relationships between the coverage anddifferent depth in the area of CpG island promoter andthe whole genome were analyzed The saturation and theefficiency of the methylated enrichment should satisfy thequality control
27 Methylation Analysis MeDIP-Seq peaks were countedusing MACS (version 142) Control (vector) versus uc167peaks were recognized as hypomethylation peaks and uc167versus control (vector) peaks were recognized as hyperme-thylation peaks The distribution of peaks in the CpG islanddifferent gene functional components differences betweengroups based on RPKM (reads per kilobase per millionmapped reads) and the correlations of enrichment betweensamples were analyzed
28 Bisulfate Sequencing PCR (BSP) Genomic DNAwas firsttreated by bisulfate according to the DNA Bisulfate Con-version Kit protocol (TIANGEN) BSP was performed usingmethylation-specific primers and PCR Kit (Tiangen) PCRamplifications were performed in 30 uL reaction mixturescontaining 2 uL of bisulfite-treated genomic DNA and thereaction conditions were 5min at 95∘C 10 cycles for 30 s at94∘C 30 s at 60∘C and 30 s at 72∘C 25 cycles for 30 s at94∘C 30 s at 50∘C and 30 s at 72∘C and finally at 60∘C for30minThe PCR products were separated on 2 agarose gelsand cloned into a pCRII vector using TA Cloning Kit DualPromoter (Invitrogen) Plasmids DNA from 10 colonies weresequenced Primers were listed in Table S1
BioMed Research International 3
Rela
tive d
ensit
y lowastlowastlowast
NCuc167
0
1
2
3
4
5
80
100
120
140
(a)
NC uc167
(b)
45S 12S
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
00
05
10
15
Relat
ive d
ensit
y
cytoplasmnucleus
uc167
(c)
d0 d4 d8 d10
NKX25
lowastlowastlowastlowast lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
MEF2C
lowast
lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
d0 d4 d8 d10
lowastlowastlowast
lowastlowast
cTnT
0
10
20
30
40
Rela
tive d
ensit
y
d0 d4 d8 d10
NCuc167
NCuc167
NCuc167
(d)
Figure 1 Locations and functions of uc167 (a) uc167 expression levels in P19 cells were significantly higher than those of the NC groupslowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 Studentrsquos 119905-test (b) Optical microscopy and fluorescence microscopy (times100) were used to observe lentivirus
transfection efficiency via GFP expression of the uc167 overexpression vector and control vector in P19 cells after 48 h transfection (c) qRT-PCR analyses of uc167 in cytoplasmic and nuclear fractions from P19 cells uc167 mainly located in the nucleus (d) Cardiac markers weredecreased in the uc167 overexpressed group compared with control group lowast119875 lt 005 versus NC groups Studentrsquos 119905-test
29 Gene Ontology (GO) Analysis Data were analyzed basedon GO terms The GO stats package was applied to testoverrepresented GO terms [10] The hypergeometric test wasapplied to filter the significant enrichment of differentiallymethylated region (DMR) [11] The calculated 119875 value iscorrected by Bonferroni test and the GO term that satisfiesthe condition that corrected 119875 value (FDR) le 005 (FDR = 1minus NkT) [12] is defined as the GO term that is significantlyenriched in the expression of the differential genes (Nkthe number of Fisherrsquos test 119875 values less than v2 test 119875values) GO analysis was used to analyze the main functionof the differential expression genes according to the GOdatabase that offers the key functional classifications toNCBI
210 Pathway Analysis Pathway analysis is a functionalanalysis mapping genes to KEGG pathways We select thesignificant pathway based on the 119875 value taht denotes thesignificance of the pathway correlated to the conditions [12]The lower the 119875 value is the more important the pathwayis This approach also identifies functional relationshipsamong genes such as upregulation downregulation or directbinding
211 Statistical Analysis Using SPSS 130 software all thevalues are presented as the means plusmn standard error ofthe mean (SEM) Statistical analyses were conducted withStudentrsquos 119905-test The statistical significance was 119875 lt 005
3 Results
31 The Establishment of Stable P19 Cells Lines Overexpresseduc167 To explore the methylation effects of uc167 weconstructed uc167 overexpression plasmidsThe transfectionefficiency was detected through quantitative analysis thatdemonstrated that there was approximately 80-fold excess inP19 cells overexpressing uc167 compared to control group(Figures 1(a) and 1(b))
32 Location of uc167 Nuclear and cytoplasmic fractiona-tion of P19 cells manifested that uc167 was mainly local-ized in the nucleus compartment (Figure 1(c)) As a con-trol the 45S rRNA precursor was primarily localized tothe nuclear fraction whereas the mitochondrial 12S rRNAgene was mainly found in the cytoplasmic compartmentOverexpressed uc167 influenced P19 cells differentiation tocardiomyocytes and caused a significant decrease of cardiac
4 BioMed Research International
4108
229
4133
206
1051
065208
genetic features (total number 250771)
promoterfive UTRCDSthree UTR
intronTTRintergenic
(a)
genetic features (total number 293107)
4117
231
199
1003
06207
4183promoterfive UTRCDSthree UTR
intronTTRintergenic
(b)
Chromosome Graph
delta beta Hypermethylation Hypomethylation
chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
chrY
68
minus68
68
minus68
64
minus64
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
68
minus68
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
63
minus63
37
minus37
33minus33
(c)
Figure 2 Differential methylated regions and chromosomes (a) DMRs peaks distribution in NC group (b) DMRs peaks distribution inuc167 overexpressed group (c) Hypomethylated and hypermethylated peaks distribution in chromosomes
markers including cTnT (d8d10) MEF2C (d8 d10) andNKX25 (d4 d8 d10) (Figure 1(d))
33 Characterization of Differential Methylated Regions Toidentify the differentially methylated genes in uc167 over-expression cells we initially classified the CpGs accordingto their methylation status The DMRs were located in
promoter 31015840UTR 51015840UTR CDS intron TTR and intergenicregions respectivelyTheDMRs peaks distributed in differentcomponents of genome showed that reads in intergenicregions (4133 in NC group and 4183 in overexpresseduc167 group) and intron (4108 in NC group and 4117 inoverexpressed uc167 group) had a relatively higher methyla-tion level than others (Figures 2(a) and 2(b))The distribution
BioMed Research International 5
Dif Gene Sig GO
5 10 15 200
minusLg
calcium ion transmembrane transportcalcium ion transport
heart development nervous system development
protein phosphorylation intracellular signal transduction
transportphosphorylation
cell adhesion homophilic cell adhesion
P
(a)
Dif Gene Sig GO
regulation of membrane potentialbrain development
phosphorylation ion transport
axon guidance transport
nervous system developmentintracellular signal transduction
cell adhesion homophilic cell adhesion
5 10 150
minusLg P
(b)
Dif Gene Sig Pathway
PI3K-Akt signaling pathwayCircadian entrainment
Tight junctionRap1 signaling pathway
Adherens junctionGlutamatergic synapse
Oxytocin signaling pathwayPathways in cancer
ARVCFocal adhesion
2 4 6 80
minusLg P
(c)
Dif Gene Sig Pathway
2 4 6 80 10
Glutamatergic synapseInsulin secretion
Cholinergic synapse Retrograde endocannabinoid signaling
Axon guidance Adherens junction
Phosphatidylinositol signaling systemPathways in cancer
Focal adhesion Rap1 signaling pathway
minusLg P
(d)
Figure 3 GO and KEGG analysis for the differentially methylated genes in uc167 overexpressed groups (a) GO analysis of hypermethylatedgenes (b) GO analysis of hypomethylated genes (c) KEGG pathways analysis of hypermethylated genes (d) KEGG pathways analysis ofhypomethylated genes
of differentially methylated genes in chromosome was alsoshown in Figure 2(c)
34 GO Analysis The ontologies and gene associationscan be accessible from the GO website (httpwwwgene-ontologyorg) Interestingly we observed that the genes thatwere hypermethylated in uc167 overexpression P19 cellswere potentially relevant to (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion(3) phosphorylation (4) transport (5) intracellular signaltransduction (6) protein phosphorylation (7) nervous sys-tem development (8) heart development (9) calcium iontransport and (10) calcium ion transmembrane transport(Figure 3(a)) Meanwhile the enriched GO terms associatedwith the hypomethylated genes in uc167 overexpressionP19 cells were involved in (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion (3)intracellular signal transduction (4) nervous systemdevelop-ment (5) transport (6) axon guidance (7) ion transport (8)phosphorylation (9) brain development and (10) regulationof membrane potential (Figure 3(b)) Overall these resultssuggest that these biological processes are epigeneticallyregulated in uc167 overexpression P19 cells
35 Pathway Analysis To further characterize the func-tional significance of the differentially methylated geneswe performed a systematic analysis and searched pathwaysthat were significantly enriched in the uc167 overexpressedgroups The pathway diagrams are used to catch how genes
regulate and interact with each other KEGGpathway analysisshowed that hypermethylated genes were implicated in thefollowing pathways (1) focal adhesion (2) arrhythmogenicright ventricular cardiomyopathy (ARVC) (3) pathways incancer (4) oxytocin signaling pathway (5) glutamatergicsynapse (6) adherens junction (7) Rap1 signaling pathway(8) tight junction (9) circadian entrainment and (10) PI3K-Akt signaling pathway (Figure 3(c)) KEGG pathway analysisalso showed that hypomethylated genes weremainly involvedthe following pathways (1) Rap1 signaling pathway (2) focaladhesion (3) pathways in cancer (4) phosphatidylinositolsignaling system (5) adherens junction (6) axon guidance(7) retrograde endocannabinoid signaling (8) cholinergicsynapse (9) insulin secretion and (10) glutamatergic synapse(Figure 3(d)) Based on this computed signaling networkwe found that multiple signaling procedures includingRap1 PI3K-Akt and phosphatidylinositol signaling systemwere important to the formation of this pathway network(Figure 4)
36 Analysis of DNA Methylation in P19 Cells To confirmthat the microarray data correctly reflected the differences inmethylation we examined the levels of promotermethylationfor several genes We quantitatively measured site-specificCpG methylation upstream of Opcm1 Mmp2 Hspa13MEF2C Strap and Sufu Microarray analysis predicted thepromoter regions of Opcm1 Mmp2 and Hspa13 to behypermethylated in uc167 overexpression groups while thepromoter regions of MEF2C Strap and Sufu were predicted
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
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2 BioMed Research International
performed to provide extensive coverage of the methylomiclandscape in order to identify the different distributionlevels of genome-wide methylation regions In addition afteroverexpressing uc167 in P19 cells the related biologicalfunctions and pathways are discussed Our research providedevidence for the mechanism underlying uc167 in the heartdevelopment
2 Materials and Methods
21 Cell Culture and Plasmid Transfection P19 cells wereobtained from the American Type Culture Collection(ATCC Manassas VA USA) The cells were cultivated inAlpha Minimum Essential Medium (120572-MEM Gibco Carls-bad CA USA) containing 10 fetal bovine serum (FBSGibco) 100Uml penicillin and 50 ugml streptomycin at37∘C in 5 CO2 Cells were seeded into 6-well cultureplates and grown to 50ndash70 confluence before transfec-tion which was performed according to the Lipofectamine2000 DNA transfection reagent protocol (Life TechnologiesInc Waltham MA USA) The plasmid for overexpression(pCDNA31(+)) was constructed by GenePharma (ShanghaiChina) The same empty vector plasmid was used as controlgroup
22 Real-Time PCR Primers were designed in National Cen-ter for Biotechnology Information (NCBI) and synthesized byGeneray (Shanghai China) (Table S1) The RNA extraction(Tiangen) reverse transcription (Takara 047A) and qRT-PCR (SYBR Green PCR Kit (RR420A)) were performedaccording to the manufacturerrsquos guidelines The expressionsof uc167 were quantified to confirm successful overexpres-sion The data were analyzed using the 2minusΔΔ119862119879 method
23 Nuclear and Cytoplasmic Fraction Nuclear and cyto-plasmic fractions were divided using a PARIS Kit (ThermoFisher Scientific) At least 107 cultured cells in total werecollected and then 500 ul ice-cold cell fractionation buffer(CFB) was added The pellet was gently resuspended bypipetting and continuously incubated on ice for 10minThe nuclear pellet fraction was separated from cytoplasmicfraction after samples were centrifuged at 500119892 for 5minThe pellet was washed twice in ice-cold CFB to prevent thestructure of cytoplasmic fraction from contaminating thenuclear fraction
24 DNA Extraction and Library Preparation DNA fromtransfected P19 cells was extracted using TissueLyser andAllPrep DNARNAProtein Mini Kit (Qiagen) accordingto the manufacturerrsquos protocols Samples were quantifiedthrough 1 agarose gel electrophoresis with Omega TissueDNA Kit (200) (D3396-02)
25 Methylated DNA Immunoprecipitation Sequencing Pro-cedure Covaris S2 system was used to sonicate genomicDNA (3 120583g) for 55 s at intensity of 4 for 200 cyclesper burst Illumina library preparation was conducted byusing the Sample Preparation Kit Fragmented DNA was
end-repaired and ATP-tailed and the adaptor was ligatedThen the adaptor-ligated DNA was recovered by AMPureXP Beads (Beckman Coulter) denatured and then subjectedto methylated DNA immunoprecipitation (MeDIP) 5 g of amonoclonal antibody against 5-methylcytosine (Eurogentec)coupled to magnetic Dynabeads with M-280 sheep antibodyagainst mouse IgG (Thermo Fisher Scientific) was used tocarry out MeDIP Sequencing libraries were denatured at95∘C for 10min and incubation with the beads in the IPBuffer (140mMNaCl 025 Triton times100 and 10Mm sodiumphosphate buffer (pH 70)) was performed at 4∘C for 4 hThe IP buffer was used to wash the beads for three timesand DNA was eluted in the elution buffer (10mM EDTA1 SDS and 50mM Tris-HCl (pH 75)) for 15min at 65∘CThen the beads were conducted with proteinase K at 55∘C for2 h and methylated DNA was recovered with the QIA quickPCR Purification Kit (Qiagen) Quantitative PCR (qPCR)was used to assess the efficiency of MeDIP After MeDIPenrichment remaining DNA was amplified with sequencingprimers through PCR and sequenced with the HiSeq 2500platform (Illumina)
26 Data Preprocessed and Quality Control Raw readsincluding overall the low-quality reads more than 20 basesand proportion less than 50 less than 20 bases in 31015840 endjoint sequence reads less than 20 in length and reads witha nitrogenous preprocessed base were removed (FASTX-Toolkit) (version 0013) Then we used Bowtie (version0128) to conduct genomemapping the reads were aligned tothe reference genomes and then the efficiency of enrichmentwas calculated The relationships between the coverage anddifferent depth in the area of CpG island promoter andthe whole genome were analyzed The saturation and theefficiency of the methylated enrichment should satisfy thequality control
27 Methylation Analysis MeDIP-Seq peaks were countedusing MACS (version 142) Control (vector) versus uc167peaks were recognized as hypomethylation peaks and uc167versus control (vector) peaks were recognized as hyperme-thylation peaks The distribution of peaks in the CpG islanddifferent gene functional components differences betweengroups based on RPKM (reads per kilobase per millionmapped reads) and the correlations of enrichment betweensamples were analyzed
28 Bisulfate Sequencing PCR (BSP) Genomic DNAwas firsttreated by bisulfate according to the DNA Bisulfate Con-version Kit protocol (TIANGEN) BSP was performed usingmethylation-specific primers and PCR Kit (Tiangen) PCRamplifications were performed in 30 uL reaction mixturescontaining 2 uL of bisulfite-treated genomic DNA and thereaction conditions were 5min at 95∘C 10 cycles for 30 s at94∘C 30 s at 60∘C and 30 s at 72∘C 25 cycles for 30 s at94∘C 30 s at 50∘C and 30 s at 72∘C and finally at 60∘C for30minThe PCR products were separated on 2 agarose gelsand cloned into a pCRII vector using TA Cloning Kit DualPromoter (Invitrogen) Plasmids DNA from 10 colonies weresequenced Primers were listed in Table S1
BioMed Research International 3
Rela
tive d
ensit
y lowastlowastlowast
NCuc167
0
1
2
3
4
5
80
100
120
140
(a)
NC uc167
(b)
45S 12S
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
00
05
10
15
Relat
ive d
ensit
y
cytoplasmnucleus
uc167
(c)
d0 d4 d8 d10
NKX25
lowastlowastlowastlowast lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
MEF2C
lowast
lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
d0 d4 d8 d10
lowastlowastlowast
lowastlowast
cTnT
0
10
20
30
40
Rela
tive d
ensit
y
d0 d4 d8 d10
NCuc167
NCuc167
NCuc167
(d)
Figure 1 Locations and functions of uc167 (a) uc167 expression levels in P19 cells were significantly higher than those of the NC groupslowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 Studentrsquos 119905-test (b) Optical microscopy and fluorescence microscopy (times100) were used to observe lentivirus
transfection efficiency via GFP expression of the uc167 overexpression vector and control vector in P19 cells after 48 h transfection (c) qRT-PCR analyses of uc167 in cytoplasmic and nuclear fractions from P19 cells uc167 mainly located in the nucleus (d) Cardiac markers weredecreased in the uc167 overexpressed group compared with control group lowast119875 lt 005 versus NC groups Studentrsquos 119905-test
29 Gene Ontology (GO) Analysis Data were analyzed basedon GO terms The GO stats package was applied to testoverrepresented GO terms [10] The hypergeometric test wasapplied to filter the significant enrichment of differentiallymethylated region (DMR) [11] The calculated 119875 value iscorrected by Bonferroni test and the GO term that satisfiesthe condition that corrected 119875 value (FDR) le 005 (FDR = 1minus NkT) [12] is defined as the GO term that is significantlyenriched in the expression of the differential genes (Nkthe number of Fisherrsquos test 119875 values less than v2 test 119875values) GO analysis was used to analyze the main functionof the differential expression genes according to the GOdatabase that offers the key functional classifications toNCBI
210 Pathway Analysis Pathway analysis is a functionalanalysis mapping genes to KEGG pathways We select thesignificant pathway based on the 119875 value taht denotes thesignificance of the pathway correlated to the conditions [12]The lower the 119875 value is the more important the pathwayis This approach also identifies functional relationshipsamong genes such as upregulation downregulation or directbinding
211 Statistical Analysis Using SPSS 130 software all thevalues are presented as the means plusmn standard error ofthe mean (SEM) Statistical analyses were conducted withStudentrsquos 119905-test The statistical significance was 119875 lt 005
3 Results
31 The Establishment of Stable P19 Cells Lines Overexpresseduc167 To explore the methylation effects of uc167 weconstructed uc167 overexpression plasmidsThe transfectionefficiency was detected through quantitative analysis thatdemonstrated that there was approximately 80-fold excess inP19 cells overexpressing uc167 compared to control group(Figures 1(a) and 1(b))
32 Location of uc167 Nuclear and cytoplasmic fractiona-tion of P19 cells manifested that uc167 was mainly local-ized in the nucleus compartment (Figure 1(c)) As a con-trol the 45S rRNA precursor was primarily localized tothe nuclear fraction whereas the mitochondrial 12S rRNAgene was mainly found in the cytoplasmic compartmentOverexpressed uc167 influenced P19 cells differentiation tocardiomyocytes and caused a significant decrease of cardiac
4 BioMed Research International
4108
229
4133
206
1051
065208
genetic features (total number 250771)
promoterfive UTRCDSthree UTR
intronTTRintergenic
(a)
genetic features (total number 293107)
4117
231
199
1003
06207
4183promoterfive UTRCDSthree UTR
intronTTRintergenic
(b)
Chromosome Graph
delta beta Hypermethylation Hypomethylation
chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
chrY
68
minus68
68
minus68
64
minus64
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
68
minus68
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
63
minus63
37
minus37
33minus33
(c)
Figure 2 Differential methylated regions and chromosomes (a) DMRs peaks distribution in NC group (b) DMRs peaks distribution inuc167 overexpressed group (c) Hypomethylated and hypermethylated peaks distribution in chromosomes
markers including cTnT (d8d10) MEF2C (d8 d10) andNKX25 (d4 d8 d10) (Figure 1(d))
33 Characterization of Differential Methylated Regions Toidentify the differentially methylated genes in uc167 over-expression cells we initially classified the CpGs accordingto their methylation status The DMRs were located in
promoter 31015840UTR 51015840UTR CDS intron TTR and intergenicregions respectivelyTheDMRs peaks distributed in differentcomponents of genome showed that reads in intergenicregions (4133 in NC group and 4183 in overexpresseduc167 group) and intron (4108 in NC group and 4117 inoverexpressed uc167 group) had a relatively higher methyla-tion level than others (Figures 2(a) and 2(b))The distribution
BioMed Research International 5
Dif Gene Sig GO
5 10 15 200
minusLg
calcium ion transmembrane transportcalcium ion transport
heart development nervous system development
protein phosphorylation intracellular signal transduction
transportphosphorylation
cell adhesion homophilic cell adhesion
P
(a)
Dif Gene Sig GO
regulation of membrane potentialbrain development
phosphorylation ion transport
axon guidance transport
nervous system developmentintracellular signal transduction
cell adhesion homophilic cell adhesion
5 10 150
minusLg P
(b)
Dif Gene Sig Pathway
PI3K-Akt signaling pathwayCircadian entrainment
Tight junctionRap1 signaling pathway
Adherens junctionGlutamatergic synapse
Oxytocin signaling pathwayPathways in cancer
ARVCFocal adhesion
2 4 6 80
minusLg P
(c)
Dif Gene Sig Pathway
2 4 6 80 10
Glutamatergic synapseInsulin secretion
Cholinergic synapse Retrograde endocannabinoid signaling
Axon guidance Adherens junction
Phosphatidylinositol signaling systemPathways in cancer
Focal adhesion Rap1 signaling pathway
minusLg P
(d)
Figure 3 GO and KEGG analysis for the differentially methylated genes in uc167 overexpressed groups (a) GO analysis of hypermethylatedgenes (b) GO analysis of hypomethylated genes (c) KEGG pathways analysis of hypermethylated genes (d) KEGG pathways analysis ofhypomethylated genes
of differentially methylated genes in chromosome was alsoshown in Figure 2(c)
34 GO Analysis The ontologies and gene associationscan be accessible from the GO website (httpwwwgene-ontologyorg) Interestingly we observed that the genes thatwere hypermethylated in uc167 overexpression P19 cellswere potentially relevant to (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion(3) phosphorylation (4) transport (5) intracellular signaltransduction (6) protein phosphorylation (7) nervous sys-tem development (8) heart development (9) calcium iontransport and (10) calcium ion transmembrane transport(Figure 3(a)) Meanwhile the enriched GO terms associatedwith the hypomethylated genes in uc167 overexpressionP19 cells were involved in (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion (3)intracellular signal transduction (4) nervous systemdevelop-ment (5) transport (6) axon guidance (7) ion transport (8)phosphorylation (9) brain development and (10) regulationof membrane potential (Figure 3(b)) Overall these resultssuggest that these biological processes are epigeneticallyregulated in uc167 overexpression P19 cells
35 Pathway Analysis To further characterize the func-tional significance of the differentially methylated geneswe performed a systematic analysis and searched pathwaysthat were significantly enriched in the uc167 overexpressedgroups The pathway diagrams are used to catch how genes
regulate and interact with each other KEGGpathway analysisshowed that hypermethylated genes were implicated in thefollowing pathways (1) focal adhesion (2) arrhythmogenicright ventricular cardiomyopathy (ARVC) (3) pathways incancer (4) oxytocin signaling pathway (5) glutamatergicsynapse (6) adherens junction (7) Rap1 signaling pathway(8) tight junction (9) circadian entrainment and (10) PI3K-Akt signaling pathway (Figure 3(c)) KEGG pathway analysisalso showed that hypomethylated genes weremainly involvedthe following pathways (1) Rap1 signaling pathway (2) focaladhesion (3) pathways in cancer (4) phosphatidylinositolsignaling system (5) adherens junction (6) axon guidance(7) retrograde endocannabinoid signaling (8) cholinergicsynapse (9) insulin secretion and (10) glutamatergic synapse(Figure 3(d)) Based on this computed signaling networkwe found that multiple signaling procedures includingRap1 PI3K-Akt and phosphatidylinositol signaling systemwere important to the formation of this pathway network(Figure 4)
36 Analysis of DNA Methylation in P19 Cells To confirmthat the microarray data correctly reflected the differences inmethylation we examined the levels of promotermethylationfor several genes We quantitatively measured site-specificCpG methylation upstream of Opcm1 Mmp2 Hspa13MEF2C Strap and Sufu Microarray analysis predicted thepromoter regions of Opcm1 Mmp2 and Hspa13 to behypermethylated in uc167 overexpression groups while thepromoter regions of MEF2C Strap and Sufu were predicted
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
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Disease Markers
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PPAR Research
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Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 3
Rela
tive d
ensit
y lowastlowastlowast
NCuc167
0
1
2
3
4
5
80
100
120
140
(a)
NC uc167
(b)
45S 12S
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
00
05
10
15
Relat
ive d
ensit
y
cytoplasmnucleus
uc167
(c)
d0 d4 d8 d10
NKX25
lowastlowastlowastlowast lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
MEF2C
lowast
lowastlowast
0
5
10
15
20
25
Rela
tive d
ensit
y
d0 d4 d8 d10
lowastlowastlowast
lowastlowast
cTnT
0
10
20
30
40
Rela
tive d
ensit
y
d0 d4 d8 d10
NCuc167
NCuc167
NCuc167
(d)
Figure 1 Locations and functions of uc167 (a) uc167 expression levels in P19 cells were significantly higher than those of the NC groupslowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 Studentrsquos 119905-test (b) Optical microscopy and fluorescence microscopy (times100) were used to observe lentivirus
transfection efficiency via GFP expression of the uc167 overexpression vector and control vector in P19 cells after 48 h transfection (c) qRT-PCR analyses of uc167 in cytoplasmic and nuclear fractions from P19 cells uc167 mainly located in the nucleus (d) Cardiac markers weredecreased in the uc167 overexpressed group compared with control group lowast119875 lt 005 versus NC groups Studentrsquos 119905-test
29 Gene Ontology (GO) Analysis Data were analyzed basedon GO terms The GO stats package was applied to testoverrepresented GO terms [10] The hypergeometric test wasapplied to filter the significant enrichment of differentiallymethylated region (DMR) [11] The calculated 119875 value iscorrected by Bonferroni test and the GO term that satisfiesthe condition that corrected 119875 value (FDR) le 005 (FDR = 1minus NkT) [12] is defined as the GO term that is significantlyenriched in the expression of the differential genes (Nkthe number of Fisherrsquos test 119875 values less than v2 test 119875values) GO analysis was used to analyze the main functionof the differential expression genes according to the GOdatabase that offers the key functional classifications toNCBI
210 Pathway Analysis Pathway analysis is a functionalanalysis mapping genes to KEGG pathways We select thesignificant pathway based on the 119875 value taht denotes thesignificance of the pathway correlated to the conditions [12]The lower the 119875 value is the more important the pathwayis This approach also identifies functional relationshipsamong genes such as upregulation downregulation or directbinding
211 Statistical Analysis Using SPSS 130 software all thevalues are presented as the means plusmn standard error ofthe mean (SEM) Statistical analyses were conducted withStudentrsquos 119905-test The statistical significance was 119875 lt 005
3 Results
31 The Establishment of Stable P19 Cells Lines Overexpresseduc167 To explore the methylation effects of uc167 weconstructed uc167 overexpression plasmidsThe transfectionefficiency was detected through quantitative analysis thatdemonstrated that there was approximately 80-fold excess inP19 cells overexpressing uc167 compared to control group(Figures 1(a) and 1(b))
32 Location of uc167 Nuclear and cytoplasmic fractiona-tion of P19 cells manifested that uc167 was mainly local-ized in the nucleus compartment (Figure 1(c)) As a con-trol the 45S rRNA precursor was primarily localized tothe nuclear fraction whereas the mitochondrial 12S rRNAgene was mainly found in the cytoplasmic compartmentOverexpressed uc167 influenced P19 cells differentiation tocardiomyocytes and caused a significant decrease of cardiac
4 BioMed Research International
4108
229
4133
206
1051
065208
genetic features (total number 250771)
promoterfive UTRCDSthree UTR
intronTTRintergenic
(a)
genetic features (total number 293107)
4117
231
199
1003
06207
4183promoterfive UTRCDSthree UTR
intronTTRintergenic
(b)
Chromosome Graph
delta beta Hypermethylation Hypomethylation
chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
chrY
68
minus68
68
minus68
64
minus64
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
68
minus68
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
63
minus63
37
minus37
33minus33
(c)
Figure 2 Differential methylated regions and chromosomes (a) DMRs peaks distribution in NC group (b) DMRs peaks distribution inuc167 overexpressed group (c) Hypomethylated and hypermethylated peaks distribution in chromosomes
markers including cTnT (d8d10) MEF2C (d8 d10) andNKX25 (d4 d8 d10) (Figure 1(d))
33 Characterization of Differential Methylated Regions Toidentify the differentially methylated genes in uc167 over-expression cells we initially classified the CpGs accordingto their methylation status The DMRs were located in
promoter 31015840UTR 51015840UTR CDS intron TTR and intergenicregions respectivelyTheDMRs peaks distributed in differentcomponents of genome showed that reads in intergenicregions (4133 in NC group and 4183 in overexpresseduc167 group) and intron (4108 in NC group and 4117 inoverexpressed uc167 group) had a relatively higher methyla-tion level than others (Figures 2(a) and 2(b))The distribution
BioMed Research International 5
Dif Gene Sig GO
5 10 15 200
minusLg
calcium ion transmembrane transportcalcium ion transport
heart development nervous system development
protein phosphorylation intracellular signal transduction
transportphosphorylation
cell adhesion homophilic cell adhesion
P
(a)
Dif Gene Sig GO
regulation of membrane potentialbrain development
phosphorylation ion transport
axon guidance transport
nervous system developmentintracellular signal transduction
cell adhesion homophilic cell adhesion
5 10 150
minusLg P
(b)
Dif Gene Sig Pathway
PI3K-Akt signaling pathwayCircadian entrainment
Tight junctionRap1 signaling pathway
Adherens junctionGlutamatergic synapse
Oxytocin signaling pathwayPathways in cancer
ARVCFocal adhesion
2 4 6 80
minusLg P
(c)
Dif Gene Sig Pathway
2 4 6 80 10
Glutamatergic synapseInsulin secretion
Cholinergic synapse Retrograde endocannabinoid signaling
Axon guidance Adherens junction
Phosphatidylinositol signaling systemPathways in cancer
Focal adhesion Rap1 signaling pathway
minusLg P
(d)
Figure 3 GO and KEGG analysis for the differentially methylated genes in uc167 overexpressed groups (a) GO analysis of hypermethylatedgenes (b) GO analysis of hypomethylated genes (c) KEGG pathways analysis of hypermethylated genes (d) KEGG pathways analysis ofhypomethylated genes
of differentially methylated genes in chromosome was alsoshown in Figure 2(c)
34 GO Analysis The ontologies and gene associationscan be accessible from the GO website (httpwwwgene-ontologyorg) Interestingly we observed that the genes thatwere hypermethylated in uc167 overexpression P19 cellswere potentially relevant to (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion(3) phosphorylation (4) transport (5) intracellular signaltransduction (6) protein phosphorylation (7) nervous sys-tem development (8) heart development (9) calcium iontransport and (10) calcium ion transmembrane transport(Figure 3(a)) Meanwhile the enriched GO terms associatedwith the hypomethylated genes in uc167 overexpressionP19 cells were involved in (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion (3)intracellular signal transduction (4) nervous systemdevelop-ment (5) transport (6) axon guidance (7) ion transport (8)phosphorylation (9) brain development and (10) regulationof membrane potential (Figure 3(b)) Overall these resultssuggest that these biological processes are epigeneticallyregulated in uc167 overexpression P19 cells
35 Pathway Analysis To further characterize the func-tional significance of the differentially methylated geneswe performed a systematic analysis and searched pathwaysthat were significantly enriched in the uc167 overexpressedgroups The pathway diagrams are used to catch how genes
regulate and interact with each other KEGGpathway analysisshowed that hypermethylated genes were implicated in thefollowing pathways (1) focal adhesion (2) arrhythmogenicright ventricular cardiomyopathy (ARVC) (3) pathways incancer (4) oxytocin signaling pathway (5) glutamatergicsynapse (6) adherens junction (7) Rap1 signaling pathway(8) tight junction (9) circadian entrainment and (10) PI3K-Akt signaling pathway (Figure 3(c)) KEGG pathway analysisalso showed that hypomethylated genes weremainly involvedthe following pathways (1) Rap1 signaling pathway (2) focaladhesion (3) pathways in cancer (4) phosphatidylinositolsignaling system (5) adherens junction (6) axon guidance(7) retrograde endocannabinoid signaling (8) cholinergicsynapse (9) insulin secretion and (10) glutamatergic synapse(Figure 3(d)) Based on this computed signaling networkwe found that multiple signaling procedures includingRap1 PI3K-Akt and phosphatidylinositol signaling systemwere important to the formation of this pathway network(Figure 4)
36 Analysis of DNA Methylation in P19 Cells To confirmthat the microarray data correctly reflected the differences inmethylation we examined the levels of promotermethylationfor several genes We quantitatively measured site-specificCpG methylation upstream of Opcm1 Mmp2 Hspa13MEF2C Strap and Sufu Microarray analysis predicted thepromoter regions of Opcm1 Mmp2 and Hspa13 to behypermethylated in uc167 overexpression groups while thepromoter regions of MEF2C Strap and Sufu were predicted
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
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Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
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Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
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OphthalmologyJournal of
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Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
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Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 BioMed Research International
4108
229
4133
206
1051
065208
genetic features (total number 250771)
promoterfive UTRCDSthree UTR
intronTTRintergenic
(a)
genetic features (total number 293107)
4117
231
199
1003
06207
4183promoterfive UTRCDSthree UTR
intronTTRintergenic
(b)
Chromosome Graph
delta beta Hypermethylation Hypomethylation
chr1
chr2
chr3
chr4
chr5
chr6
chr7
chr8
chr9
chr10
chr11
chr12
chr13
chr14
chr15
chr16
chr17
chr18
chr19
chrX
chrY
68
minus68
68
minus68
64
minus64
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
68
minus68
64
minus64
69
minus69
69
minus69
69
minus69
65
minus65
65
minus65
67
minus67
63
minus63
37
minus37
33minus33
(c)
Figure 2 Differential methylated regions and chromosomes (a) DMRs peaks distribution in NC group (b) DMRs peaks distribution inuc167 overexpressed group (c) Hypomethylated and hypermethylated peaks distribution in chromosomes
markers including cTnT (d8d10) MEF2C (d8 d10) andNKX25 (d4 d8 d10) (Figure 1(d))
33 Characterization of Differential Methylated Regions Toidentify the differentially methylated genes in uc167 over-expression cells we initially classified the CpGs accordingto their methylation status The DMRs were located in
promoter 31015840UTR 51015840UTR CDS intron TTR and intergenicregions respectivelyTheDMRs peaks distributed in differentcomponents of genome showed that reads in intergenicregions (4133 in NC group and 4183 in overexpresseduc167 group) and intron (4108 in NC group and 4117 inoverexpressed uc167 group) had a relatively higher methyla-tion level than others (Figures 2(a) and 2(b))The distribution
BioMed Research International 5
Dif Gene Sig GO
5 10 15 200
minusLg
calcium ion transmembrane transportcalcium ion transport
heart development nervous system development
protein phosphorylation intracellular signal transduction
transportphosphorylation
cell adhesion homophilic cell adhesion
P
(a)
Dif Gene Sig GO
regulation of membrane potentialbrain development
phosphorylation ion transport
axon guidance transport
nervous system developmentintracellular signal transduction
cell adhesion homophilic cell adhesion
5 10 150
minusLg P
(b)
Dif Gene Sig Pathway
PI3K-Akt signaling pathwayCircadian entrainment
Tight junctionRap1 signaling pathway
Adherens junctionGlutamatergic synapse
Oxytocin signaling pathwayPathways in cancer
ARVCFocal adhesion
2 4 6 80
minusLg P
(c)
Dif Gene Sig Pathway
2 4 6 80 10
Glutamatergic synapseInsulin secretion
Cholinergic synapse Retrograde endocannabinoid signaling
Axon guidance Adherens junction
Phosphatidylinositol signaling systemPathways in cancer
Focal adhesion Rap1 signaling pathway
minusLg P
(d)
Figure 3 GO and KEGG analysis for the differentially methylated genes in uc167 overexpressed groups (a) GO analysis of hypermethylatedgenes (b) GO analysis of hypomethylated genes (c) KEGG pathways analysis of hypermethylated genes (d) KEGG pathways analysis ofhypomethylated genes
of differentially methylated genes in chromosome was alsoshown in Figure 2(c)
34 GO Analysis The ontologies and gene associationscan be accessible from the GO website (httpwwwgene-ontologyorg) Interestingly we observed that the genes thatwere hypermethylated in uc167 overexpression P19 cellswere potentially relevant to (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion(3) phosphorylation (4) transport (5) intracellular signaltransduction (6) protein phosphorylation (7) nervous sys-tem development (8) heart development (9) calcium iontransport and (10) calcium ion transmembrane transport(Figure 3(a)) Meanwhile the enriched GO terms associatedwith the hypomethylated genes in uc167 overexpressionP19 cells were involved in (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion (3)intracellular signal transduction (4) nervous systemdevelop-ment (5) transport (6) axon guidance (7) ion transport (8)phosphorylation (9) brain development and (10) regulationof membrane potential (Figure 3(b)) Overall these resultssuggest that these biological processes are epigeneticallyregulated in uc167 overexpression P19 cells
35 Pathway Analysis To further characterize the func-tional significance of the differentially methylated geneswe performed a systematic analysis and searched pathwaysthat were significantly enriched in the uc167 overexpressedgroups The pathway diagrams are used to catch how genes
regulate and interact with each other KEGGpathway analysisshowed that hypermethylated genes were implicated in thefollowing pathways (1) focal adhesion (2) arrhythmogenicright ventricular cardiomyopathy (ARVC) (3) pathways incancer (4) oxytocin signaling pathway (5) glutamatergicsynapse (6) adherens junction (7) Rap1 signaling pathway(8) tight junction (9) circadian entrainment and (10) PI3K-Akt signaling pathway (Figure 3(c)) KEGG pathway analysisalso showed that hypomethylated genes weremainly involvedthe following pathways (1) Rap1 signaling pathway (2) focaladhesion (3) pathways in cancer (4) phosphatidylinositolsignaling system (5) adherens junction (6) axon guidance(7) retrograde endocannabinoid signaling (8) cholinergicsynapse (9) insulin secretion and (10) glutamatergic synapse(Figure 3(d)) Based on this computed signaling networkwe found that multiple signaling procedures includingRap1 PI3K-Akt and phosphatidylinositol signaling systemwere important to the formation of this pathway network(Figure 4)
36 Analysis of DNA Methylation in P19 Cells To confirmthat the microarray data correctly reflected the differences inmethylation we examined the levels of promotermethylationfor several genes We quantitatively measured site-specificCpG methylation upstream of Opcm1 Mmp2 Hspa13MEF2C Strap and Sufu Microarray analysis predicted thepromoter regions of Opcm1 Mmp2 and Hspa13 to behypermethylated in uc167 overexpression groups while thepromoter regions of MEF2C Strap and Sufu were predicted
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 5
Dif Gene Sig GO
5 10 15 200
minusLg
calcium ion transmembrane transportcalcium ion transport
heart development nervous system development
protein phosphorylation intracellular signal transduction
transportphosphorylation
cell adhesion homophilic cell adhesion
P
(a)
Dif Gene Sig GO
regulation of membrane potentialbrain development
phosphorylation ion transport
axon guidance transport
nervous system developmentintracellular signal transduction
cell adhesion homophilic cell adhesion
5 10 150
minusLg P
(b)
Dif Gene Sig Pathway
PI3K-Akt signaling pathwayCircadian entrainment
Tight junctionRap1 signaling pathway
Adherens junctionGlutamatergic synapse
Oxytocin signaling pathwayPathways in cancer
ARVCFocal adhesion
2 4 6 80
minusLg P
(c)
Dif Gene Sig Pathway
2 4 6 80 10
Glutamatergic synapseInsulin secretion
Cholinergic synapse Retrograde endocannabinoid signaling
Axon guidance Adherens junction
Phosphatidylinositol signaling systemPathways in cancer
Focal adhesion Rap1 signaling pathway
minusLg P
(d)
Figure 3 GO and KEGG analysis for the differentially methylated genes in uc167 overexpressed groups (a) GO analysis of hypermethylatedgenes (b) GO analysis of hypomethylated genes (c) KEGG pathways analysis of hypermethylated genes (d) KEGG pathways analysis ofhypomethylated genes
of differentially methylated genes in chromosome was alsoshown in Figure 2(c)
34 GO Analysis The ontologies and gene associationscan be accessible from the GO website (httpwwwgene-ontologyorg) Interestingly we observed that the genes thatwere hypermethylated in uc167 overexpression P19 cellswere potentially relevant to (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion(3) phosphorylation (4) transport (5) intracellular signaltransduction (6) protein phosphorylation (7) nervous sys-tem development (8) heart development (9) calcium iontransport and (10) calcium ion transmembrane transport(Figure 3(a)) Meanwhile the enriched GO terms associatedwith the hypomethylated genes in uc167 overexpressionP19 cells were involved in (1) homophilic cell adhesion viaplasma membrane adhesion molecules (2) cell adhesion (3)intracellular signal transduction (4) nervous systemdevelop-ment (5) transport (6) axon guidance (7) ion transport (8)phosphorylation (9) brain development and (10) regulationof membrane potential (Figure 3(b)) Overall these resultssuggest that these biological processes are epigeneticallyregulated in uc167 overexpression P19 cells
35 Pathway Analysis To further characterize the func-tional significance of the differentially methylated geneswe performed a systematic analysis and searched pathwaysthat were significantly enriched in the uc167 overexpressedgroups The pathway diagrams are used to catch how genes
regulate and interact with each other KEGGpathway analysisshowed that hypermethylated genes were implicated in thefollowing pathways (1) focal adhesion (2) arrhythmogenicright ventricular cardiomyopathy (ARVC) (3) pathways incancer (4) oxytocin signaling pathway (5) glutamatergicsynapse (6) adherens junction (7) Rap1 signaling pathway(8) tight junction (9) circadian entrainment and (10) PI3K-Akt signaling pathway (Figure 3(c)) KEGG pathway analysisalso showed that hypomethylated genes weremainly involvedthe following pathways (1) Rap1 signaling pathway (2) focaladhesion (3) pathways in cancer (4) phosphatidylinositolsignaling system (5) adherens junction (6) axon guidance(7) retrograde endocannabinoid signaling (8) cholinergicsynapse (9) insulin secretion and (10) glutamatergic synapse(Figure 3(d)) Based on this computed signaling networkwe found that multiple signaling procedures includingRap1 PI3K-Akt and phosphatidylinositol signaling systemwere important to the formation of this pathway network(Figure 4)
36 Analysis of DNA Methylation in P19 Cells To confirmthat the microarray data correctly reflected the differences inmethylation we examined the levels of promotermethylationfor several genes We quantitatively measured site-specificCpG methylation upstream of Opcm1 Mmp2 Hspa13MEF2C Strap and Sufu Microarray analysis predicted thepromoter regions of Opcm1 Mmp2 and Hspa13 to behypermethylated in uc167 overexpression groups while thepromoter regions of MEF2C Strap and Sufu were predicted
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
6 BioMed Research International
Figure 4 Interactions between the differentially methylated genes in uc167 overexpressed group and control group as identified using thebiological network analysis function of Ingenuity Pathway Analysis
to be hypomethylated BSP confirmed that the site-specificCpGs of Opcm1 Mmp2 and Hspa13 were hypermethylated(Figure 5(a)) and the relative mRNA levels were significantlydecreased in the uc167 overexpression group (Figure 5(b))We also verified the site-specific CpGs of MEF2C Strapand Sufu which were hypomethylated (Figure 5(c)) and therelative mRNAs level increased in uc167 overexpressiongroups (Figure 5(d)) although the absolute differences weregenerally small
4 Discussion
The molecular network of embryonic heart developmenthowever is still unclear In previous research we havedemonstrated that overexpression of uc167 promoted apop-tosis and inhibited proliferation in embryonic myocardialcells [9] In this study we analyzed the DNA methylationalterations in uc167 overexpressed P19 cells and revealedmethylation alterations in specific genes participating inthe mechanism underlying the function of uc167 in heartdevelopment
Three main epigenetic processes are represented byDNA methylation posttranslational histone modificationsand RNA-based mechanisms [13] Dysregulated epigeneticmodifications may lead to irregular development of diseaseIndeed DNA methylation has been associated with insulinsensitivity [14] coronary heart disease [15 16] obesity [17]and breast cancer [18] DNAmethylation is usually associatedwith transcriptional suppression while demethylation isassociated with transcriptional activation thus affecting geneexpression by modifying DNA promoter approachabilityto RNA polymerase and gene level The decrease in DNAmethylation in the promoter region of lncRNA H19 also wasassociated with calcific aortic valve disease (CAVD) [19]Such changes were shown to promote an osteogenic programby interfering with the expression of NOTCH1 The alteredpromotermethylation in genes relevant tomyocyte apoptosisfibrosis and contractility was revealed in patients with heartfailure [20] The present findings shed new light on the firsttime that lncRNA uc167 is dysregulated and contributesto the aberrant heart development LncRNA uc167 couldrepresent a novel target in congenital heart disease
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 7
NCuc167
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
NC uc167
lowast
Opcml
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Hspa13
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
) 40
30
20
10
0
Mmp2
NCuc167
NC uc167
lowast
(a)
Opcml
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
Mmp2Re
lativ
e den
sity
15
10
05
00
NCuc167
NC uc167
lowast
Hspa13
Rela
tive d
ensit
y
15
10
05
00
NCuc167
NC uc167
lowast
(b)M
ethy
latio
n le
vels
()
30
20
10
0
50
40
Sufu
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
30
20
10
0
50
40
Strap
NCuc167
NC uc167
lowast
Met
hyla
tion
leve
ls (
)
Mef2c30
20
10
0
NCuc167
NC uc167
lowast
(c)
Relat
ive d
ensit
y
Mef2c4
3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Strap3
2
1
0
NCuc167
NC uc167
lowast
Relat
ive d
ensit
y
Sufu3
2
1
0
NCuc167
NC uc167
lowast
(d)
Figure 5 The levels of promoter methylation were measured by BSP in uc167 overexpressed and control cells (a) The methylation level wasincreased in Opcm1 Mmp2 and Hspa13 (b) The mRNA level was decreased in Opcm1 Mmp2 and Hspa13 (c) The methylation level wasdecreased in MEF2C Strap and Sufu (d) The mRNA level was increased in MEF2C Strap and Sufu
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 BioMed Research International
During embryo development the heart is one of themostsignificant organs and a series of complex morphogeneticinteractions contribute to the development of the heart [21] Abalance between cardiomyocyte apoptosis and proliferationdetermines the growth of the embryonic fetal heart [22] Inmammalian cells DNA methylation is catalyzed by DNMTsDNA methylation has a crucial impact on the regulationof gene expression and maintaining genomic integrity [23]LncRNAs are known to regulate the expression of adjacentgenes as cis-acting regulatory elements [24] In the previousstudy expression of uc167 showed an opposite correlationwith MEF2C in the process of P19 cells differentiation [9]Overexpression of MEF2C partially reverses the negativeeffects of uc167 on proliferation apoptosis and differenti-ation In this study the methylation level of MEF2C wasdecreased compared with control groups suggesting thatuc167 may influence the P19 differentiation through alteringMEF2C methylation status
The genome-wide epigenetic studies will allow identifica-tion of unique regulatory pathways giving new insight intothe etiology of congenital heart disease such as ventricularseptal defects
Conflicts of Interest
The authors declare that there are no financial conflicts ofinterest
Authorsrsquo Contributions
Yun Li and Lingmei Qian designed the research HaoZhang Jia Xu and Anwen Yin performed the experimentsHua Li and Qijun Zhang analyzed the data Zijie Chengand Mengwen Feng wrote this manuscript Lingmei Qiansupervised this work Anwen Yin Mengwen Feng and ZijieCheng contributed equally to this work All authors read andapproved the final manuscript
Acknowledgments
This project was supported by grants from the NationalNatural Science Foundation of China (nos 81370278 and81570209)
Supplementary Materials
Table S1 primers used in this study (Supplementary Materials)
References
[1] H Mollmann H M Nef S Kostin et al ldquoNatural tissueengineering inside a ventricular septum defect occluderrdquo Cir-culation vol 113 no 16 pp e718ndashe719 2006
[2] S A Obermann-Borst L M J W Van Driel W A Helbinget al ldquoCongenital heart defects and biomarkers of methylationin children A case-control studyrdquo European Journal of ClinicalInvestigation vol 41 no 2 pp 143ndash150 2011
[3] B S Marino P H Lipkin J W Newburger et al ldquoNeurodevel-opmental outcomes in children with congenital heart disease
Evaluation and management a scientific statement from theamerican heart associationrdquoCirculation vol 126 no 9 pp 1143ndash1172 2012
[4] J C Chuang and P A Jones ldquoEpigenetics and microRNAsrdquoPediatric Research vol 61 no 5 part 2 pp 24Rndash29R 2007
[5] I Beerman C Bock B S Garrison et al ldquoProliferation-dependent alterations of the DNA methylation landscapeunderlie hematopoietic stem cell agingrdquo Cell Stem Cell vol 12no 4 pp 413ndash425 2013
[6] M M Suzuki and A Bird ldquoDNA methylation landscapesprovocative insights from epigenomicsrdquo Nature Reviews Genet-ics vol 9 no 6 pp 465ndash476 2008
[7] G Bejerano M Pheasant I Makunin et al ldquoUltraconservedelements in the human genomerdquo Science vol 304 no 5675 pp1321ndash1325 2004
[8] G C Hon R D Hawkins O L Caballero et al ldquoGlobal DNAhypomethylation coupled to repressive chromatin domain for-mation and gene silencing in breast cancerrdquo Genome Researchvol 22 no 2 pp 246ndash258 2012
[9] G Song Y Shen Z Ruan et al ldquoLncRNA-uc167 influencescell proliferation apoptosis and differentiation of P19 cells byregulating Mef2crdquo Gene vol 590 no 1 pp 97ndash108 2016
[10] S Falcon and R Gentleman Using GOstats to test gene lists forGO term association Oxford University Press 2007 257-258
[11] D I Rodenhiser J Andrews W Kennette et al ldquoEpigeneticmapping and functional analysis in a breast cancer metasta-sis model using whole-genome promoter tiling microarraysrdquoBreast Cancer Research vol 10 no 4 article no R62 2008
[12] D Dupuy et al ldquoGenome-scale analysis of in vivo spatiotem-poral promoter activity in Caenorhabditis elegansrdquo Naturebiotechnology vol 25 663 no 6 p 668 2007
[13] L Metzinger S De Franciscis and R Serra ldquoThe managementof cardiovascular risk through epigenetic biomarkersrdquo BioMedResearch International vol 2017 Article ID 9158572 2017
[14] L Yang M-L Tong X Chi M Zhang C-M Zhang andX-R Guo ldquoGenomic DNA methylation changes in NYGGF4-overexpression 3T3-L1 adipocytesrdquo International Journal ofMolecular Sciences vol 13 no 12 pp 15575ndash15587 2012
[15] R Murray J Bryant P Titcombe et al ldquoDNA methylationat birth within the promoter of ANRIL predicts markers ofcardiovascular risk at 9 yearsrdquo Clinical Epigenetics vol 8 no1 article no 90 2016
[16] A Fernandez-Sanles S Sayols-Baixeras I Subirana I RDegano and R Elosua ldquoAssociation betweenDNAmethylationand coronary heart disease or other atherosclerotic events asystematic reviewrdquo Atherosclerosis 2017
[17] J Zhu ldquoDifferential DNA Methylation Status Between HumanPreadipocytes and Mature Adipocytesrdquo Cell Biochemistry Bio-physics vol 63 no 1 p 15 2012
[18] C Zhang X Wang X Li et al ldquoThe landscape of DNAmethylation-mediated regulation of long non-coding RNAs inbreast cancerrdquo Oncotarget vol 8 no 31 pp 51134ndash51150 2017
[19] F Hadji M-C Boulanger S-P Guay et al ldquoAltered DNAMethylation of Long Noncoding RNA H19 in Calcific AorticValve Disease PromotesMineralization by Silencing NOTCH1rdquoCirculation vol 134 no 23 pp 1848ndash1862 2016
[20] M Movassagh A Vujic and R Foo ldquoGenome-wide DNAmethylation in human heart failurerdquo Epigenomics vol 3 no 1pp 103ndash109 2011
[21] L Bouchard R Rabasa-Lhoret M Faraj et al ldquoDifferentialepigenomic and transcriptomic responses in subcutaneous
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
BioMed Research International 9
adipose tissue between low and high responders to caloricrestrictionrdquo American Journal of Clinical Nutrition vol 91 no2 pp 309ndash320 2010
[22] S Falcon andR Gentleman ldquoUsingGOstats to test gene lists forGO term associationrdquo Bioinformatics vol 23 no 2 pp 257-2582007
[23] N Khyzha A Alizada M D Wilson and J E Fish ldquoEpige-netics of Atherosclerosis EmergingMechanisms andMethodsrdquoTrends in Molecular Medicine vol 23 no 4 pp 332ndash347 2017
[24] I Ulitsky and D P Bartel ldquoXLincRNAs genomics evolutionand mechanismsrdquo Cell vol 154 no 1 pp 26ndash46 2013
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Recommended