Bta-miR-124a Affects Lipid Metabolism by Regulating PECR Genedownloads.hindawi.com/journals/bmri/2019/2596914.pdf · bta-miR-124a mimics miR-shNC ∗∗ (a) Cellular Triglyceride

Research ArticleBta-miR-124a Affects Lipid Metabolism byRegulating PECR Gene

Binglei Shen 12 Zhuonina Yang 1 Shuo Han1 Ziwen Zou1 Juan Liu1 Lian Nie1

Wentao Dong1 E Li1 Shengjun Liu1 Zhihui Zhao3 and RuiWu 12

1College of Animal Science and Veterinary Medicine Heilongjiang Bayi Agricultural University 163319 Heilongjiang Daqing China2Heilongjiang Provincial Key Laboratory of Prevention and Control of Bovine Diseases 163319 Heilongjiang China3College of Agricultural Guangdong Ocean University 524088 Zhanjiang China

Correspondence should be addressed to Binglei Shen binglei514163com Zhuonina Yang yznn1225163comand Rui Wu fuhewu126com

Received 21 January 2019 Accepted 27 June 2019 Published 28 July 2019

Academic Editor Marco Fichera

Copyright copy 2019 Binglei Shen 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

According to our previous studies bta-miR-124a was differentially expressed in breast tissue between high-fat and low-fat dairycows However the function of bta-miR-124a in lipid metabolism of dairy cows and the identification of its target genes have notbeen reported Therefore this study will identify the target gene of bta-miR-124a and explore its role in the regulation of milk lipidmetabolism First preliminary bioinformatics prediction of bta-miR-124a candidate target genes was performed and quantitativereal-time polymerase chain reaction (qRT-PCR) was used to analyze relative expression changes of bta-miR-124a and its candidatetarget genes and the expression level of the downstream gene of the target gene in the lipid metabolism signaling pathway in dairymammary epithelial cell lines (Mac-T) using the dual luciferase reporter system for the identification of the targeting relationshipbetween bta-miR-124a and the candidate target gene Then the effect of transfection of bta-miR-124a mimics and inhibitors ontriglyceride (TG) and free fatty acid (FFA) levels was analyzed The results indicate that bta-miR-124a directly interacts with the31015840-untranslated region of peroxisomal trans-2-enoyl-CoA reductase (PECR) to downregulate its expression inMac-T cells Furtherbta-mir-124a regulates the expression of PECR and the downstream gene extension of very long chain fatty acid protein 2 (ELOVL2)through an unsaturated fatty acid biosynthesis signaling pathway In conclusion bta-miR-124a is involved in lipid metabolism bydirectly downregulating the PECR gene and affecting the expression of the downstream gene ELOVL2 and regulates the content ofsome key secretory elements such as TG and FFAThe function of bta-miR-124a has a certain effect on the synthesis and secretionof milk fat in the mammary epithelial cells of dairy cows

1 Introduction

As peoples dietary needs increase consumers are increas-ingly paying attention to the fat content of dairy productswhich has prompted livestock farmers to produce morediverse dairy products to meet consumer demand Milk fatis an important nutrient component of milk and a majorindicator used to measure milk quality in dairy production

MiRNAs are noncoding small RNAs of about 20nucleotides in length that mediate posttranscriptional reg-ulation by forming RNA-induced silencing complexes withrelated proteins thereby playing an important role in celldifferentiation and proliferation [1ndash3]

Numerous studies have confirmed thatmanymicroRNAs(miRNAs) have an effect on milk fat metabolism in dairycows Lipid metabolism-related miRNAs have been reportedto be involved in lipid synthesis transport and oxidation toregulate lipidmetabolism [4 5]ThemiRNAs and their targetgenes are cross-regulated and such regulatory features leadto the complexity of lipid metabolism regulation involvedin miRNAs [6] At present some studies have shown thatmiRNAs can participate in the development of breast prolif-eration and differentiation of breast cells milk fat synthesislactation and other processes This strongly suggests theimportance of miRNAs in milk fat metabolism

HindawiBioMed Research InternationalVolume 2019 Article ID 2596914 9 pageshttpsdoiorg10115520192596914

2 BioMed Research International

Our previous studies have identified several lipidmetabolism-related miRNAs Bta-miR-224 could affectapoptosis and triglyceride (TG) synthesis by regulating acyl-CoA dehydrogenase medium chain (ACADM) and aldehydedehydrogenase 2 (ALDH2) which are two important genesinvolved in lipid metabolism in Mac-T cells [7] In anotherstudy bta-miR-33a negatively regulated the expression ofelongation of very long chain fatty acids protein 5 (ELOVL5)elongation of very long chain fatty acids protein 6 (ELOVL6)and sterol-C4-methyl oxidase (SC4MOL) in mammarygland with low milk fat content (MG-LL) bta-miR-152 wasdownregulated in MG-LL and the significantly upregulatedgenes were prostaglandin-endoperoxide synthase 2 (PTGS2)protein kinase AMP-activated catalytic subunit alpha1 (PRKAA1) and uncoupling protein 3 (UCP3) [8] Inaddition another research reported that the overexpressionof miR-130a significantly decreased cellular triacylglycerol(TG) expression level and suppressed lipid droplet formationwhereas the inhibition of miR-130a resulted in greater lipiddroplet formation and TG accumulation in bovinemammaryepithelial cells [9]

MiR-124 is a small RNA molecule located on chromo-some 8 After comparing the precursor sequence and maturesequence of miR-124 we found that the miR-124 sequence ishighly conserved among species MiR-124 has been studiedfor many years most studies were related to brain andneurons development Researchers found that the tumorsuppressor activity of miR-124 could be partly due to itsinhibitory effects on glioma stem-like traits and invasivenessthrough Snail homolog 2 (SNAI2) [10] Silber et al reportedthatmiR-124 inhibit proliferation of glioblastomamultiformecells and induce differentiation of brain tumor stem cells [11]Eugene V et al reported that miR-124 promotes neuronaldifferentiation by triggering brain-specific alternative pre-mRNA splicing [12]

In recent years miR-124 has also been shown to beinvolved in lipid metabolism and affect lipid synthesis Daset al reported that miR-124a attenuates RNA and proteinexpression of the major TG hydrolase adipose TG lipase(ATGLPNPLA2) and its coactivator comparative gene iden-tification 58 (CGI-58ABHD5) [13] Holmes et al found miR-124506 andmiR-108 binding sites in the 31015840-noncoding regionofmRNA including peroxisomeproliferator-activated recep-tor alpha (PPARA) and peroxisome proliferative activatedreceptor gamma (PPARG) which are key regulators of genesencoding enzymes of lipid metabolism [14] Those provideda basis for the study of miR-124a in lipid metabolism and alsoindicated that miR-124a plays an important role in fatty acidmetabolism However the study ofmiR-124a involved in fattyacid metabolism in dairy cows is limited We had reportedthat bta-miR-124a was differentially expressed between high-fat and low-fat dairy cow primary mammary epithelialcells with high significance [8] Therefore this experimentattempted to identify the target gene of bta-miR-124a andexplore its effect on lipid metabolism in dairy cows in orderto provide a basic reference for molecular-oriented breedingof dairy cows

Peroxisomal trans-2-enoyl-CoA reductase (PECR) is alsocalled TERP and TECR which is located on chromosome

q35 (chr2215996329-216082955) with 86627 bases (httpswwwgenecardsorg) It can participate in carbon chainelongation in fatty acid metabolism and catalyze the lastreaction of four long chain fatty acid elongation cycles Eachcycle of PECR adds two carbons to the long chain andvery long chain fatty acid (VLCFA) chains reducing theintermediate of trans-23-enoyl coenzyme A fatty acid to acylcoenzyme A which can further extend the carbon chain byentering a new elongation cycle [15 16] Meanwhile PECRis a peroxisome protein involved in fatty acid synthesis andplays an important role in milk fat synthesis Studies haveshown that the gene is closely related to the late develop-ment of TG [17] Therefore in this study we reported theexpression of bta-miR-124a in Mac-T cells which can affectlipid metabolism by regulating PECR gene which provideda reference for further study on the regulation mechanism ofmilk fat in dairy cows

2 Materials and Methods

21 Bioinformatics Prediction of Candidate Target Genes ofbta-miR-124a The mature sequences of bta-miR-124a wereobtained on miRBase (httpwwwmirbaseorgindexshtml)and homology analysis was performed The possibletarget genes for bta-miR-124a are predicted by TargetScan(httpwwwtargetscanorg) The bioinformatics websiteStarbase (httpstarbasesysueducn) was used to predictthe extent of nonspecific pairing of bta-miR-124a with itscandidate target genes

22 Cells Culture and Transfection Mac-T cells were from thelaboratory inHeilongjiangBayiAgriculturalUniversityMac-T cells were cultured in Dulbeccorsquos modified Eaglersquos mediumand F-12 (DMEMF12) (Gibco Grand Island NY) with 10fetal bovine serum (FBS) (Gibco Grand Island NY USA) 100UmL penicillin and 100 120583gmL streptomycin with or with-out 250 ngmL amphotericin B (Invitrogen Carlsbad CAUSA) digestive cells with 025 trypsin (Solarbio BeijingChina) and incubated at 37∘C in a humidified atmosphere of5 CO

2

The indicated cells reaching 60 to 80 confluence weretransfected with 10nM of bta-miR-124a mimics or inhibitorsand their corresponding control group constructs (Mir-shNC Anti-NC) (Sangon Biotech Shanghai China) in 6-well using Lipofectamine 3000 (Invitrogen Life TechnologiesCarlsbad CAUSA)Themediumwas changed to regular cellculture medium after 4-6 h Cells were collected for analysisat 24-48 h after transfection

23 Total RNA Isolation and Quantitative Polymerase ChainReaction (qPCR) Assays Forty-eight hours after transfectionwith the miR-124a mimicsinhibitors total RNA of the Mac-T cells was extracted using trizol reagent (Invitrogen) Thequantity of RNA was verified by NanoDrop1000 (ThermoScientific) ensuring the optical density (OD) 260nm280nmvaluewas between 18 and 20 ComplementaryDNA (cDNA)was synthesized using the Primer Script reverse transcriptionPCR (RT-PCR) kit (Takara) The expressions of miR-124aand target genes were detected on a Bio-Rad CFX Connect

BioMed Research International 3

Table 1 Primer sequences of genes for quantitative real-time PCR

Symbol Primer Sequence (51015840-31015840) Amplicon size

Bta-miR-124aR-T GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTGGC

F CGCGTAAGGCACGCGGTGAATR ATCCAGTGCAGGGTCCGAGG

U6R-T CGCTTCACGAATTTGCGTGTCATF GCTTCGGCAGCACATATACTAAAATR CGCTTCACGAATTTGCGTGTCAT

PECR F CCAGCAGTGGAGTAAGGATCAATAGTG 181bpR GCAGGAGACAGCAGGAAGCATAC

GADPH F CGGCACAGTCAAGGCAGAGAAC 116bpR CCACATACTCAGCACCAGCATCAC

ELOVL2 F GGCTGCCTCATCTTCCAGTCTTC 169bpR TTACTCCGTTCGCTGTGCTGAAG

Note The genes in the above table are all from dairy cow species

Predicted consequential pairing of targetregion (top) and miRNA (bottom)

Position 35-42 of PECR

3rsquoUTR bta-miR-124a

5rsquo-GUCUUUUGUCCCCAAGUGCCUUA-3rsquo 8mer

Site type

3rsquo-GAACCGUAAGUGGCGCACGGAAU-5rsquo


3rsquoUTR bta-miR-124b

5rsquo-GUCUUUUGUCCCCAAGUGCCUUA-3rsquo


8mer

Figure 1 Predicted binding sites of bta-miR-124a on 31015840-untranslated region (31015840-UTR) of peroxisomal trans-2-enoyl-CoA reductase (PECR)

instrument (Biorad America) using the SYBRGreen I (TOY-OBO Japan) Primer 60 (Premier) was used to design fluo-rescently labeled primers and reverse transcription primersfor quantitative analysis of miR-124a PECR and ELOVL2(enhanced extension of very long chain fatty acid protein2) The expression of U6 and GADPH (glyceraldehyde-3-phosphate dehydrogenase) was used as a loading control Allprimers were synthesized by Shanghai Sangon BiotechnologyCompany (see Table 1) The relative gene expression wascalculated by the 2minusΔΔCt method

24 Vectors Construction Vectors construction of miR-124amimics miR-124a inhibitors and corresponding controlgroup (Mir-NC Anti-NC) were purchased from ShanghaiSangon Biotech Company The PECR-mutWTsi vectorswere purchased from GenePharma Company

25 Luciferase Activity Assay Mac-T cells were cultured in6-well plates the day prior to transfection All transfectionswere carried out with Lipofectamine 3000 (Invitrogen) ThePECR mutWTsi vectors were cotransfected with bta-miR-124a into Mac-T cells After 48 h of transfection luciferaseactivities were measured using the Dual-Luciferase ReporterAssay System (Promega) and normalized to renilla luciferaseactivity The experiments were performed in triplicate

26 Detection of TG and FFA Detection Content After 48 hof transfection cultured cells were collected The expression

levels of TG and FFA in Mac-T cells were assayed usingthe cell TG assay kit and FFA assay kit (Jiancheng NanjingChina) All of the above experiments were performed accord-ing to the manufacturerrsquos recommended protocol

27 Statistical Analysis The results were analyzed using Ran-dom Design of Single Factor Analysis of Variance betweengroups based on SPSS 220 software All results were pre-sented as the means plusmn standard error of mean (SEM)of separate experiments (n gt=3) Statistical significance ispresented as lowastplt 005 lowastlowastplt 001

3 Results

31 Sequence Homology Analysis of Bta-miR-124 In the miR-Base database the bta-miR-124a base sequence was obtainedand the mature sequence of bta-miR-124 was found to behighly conserved among species Using the predictive soft-ware TargetScan 72 results showed that the miR-124a targetsite is present at 36-43 nt of the PECR 31015840 UTR (see Figure 1)Based on above findings the regulatory relationship analysisof bta-miR-124a and its candidate target gene PECR wasperformed in Mac-T cells

32 Bta-miR-124a Downregulated Its Candidate TargetGene PECR After transfection with bta-miR-124a mimicsinhibitors and control group (Mir-shNC and Anti-NC) intoMac-T cells respectively the corresponding expression levels

4 BioMed Research InternationalRe

lativ

e exp

ress

ion

ofbt

a-m

iR-1

24a

012345

500600700800900

1000

bta-miR-124amimics

miR-shNC

lowast

(a)

Rela

tive e

xpre

ssio

n of

bta-

miR

-124

a

00

05

10

15

bta-miR-124ainhibitor

Anti-shNC

lowastlowast

(b)

00

05

10

15

bta-miR-124amimics

miR-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(c)

00

05

10

15

20


Anti-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(d)

Figure 2 Expression levels of bta-miR-124a and PECR after transient transfection with bta-miR-124a mimicsinhibitors in Mac-T cells (ab) The expressions of bta-miR-124a in Mac-T cells that transfected bta-miR-124a mimics bta-miR-124a inhibitors control group (Mir-shNCand Anti-NC) (lowastp lt005 lowastlowastp lt001) (c d) The mRNA expressions of PECR gene in Mac-T cells that transfected bta-miR-124a mimicsbta-miR-124a inhibitors and control group (Mir-shNC and Anti-NC) (lowastp lt005)

were detected by quantitative real-time PCR (qPCR) whoseprimer sequence was designed by primer 60 The resultsshowed that the expression of bta-miR-124a mimics wassignificantly higher than that of the control group and thebta-miR-124a inhibitors were lower than the control group(see Figures 2(a) and 2(b)) Comparedwith the control groupthe expression of PECR gene decreased after the transfectionof bta-miR-124a mimics (see Figure 2(c)) and increasedslightly after the transfection of bta-miR-124a inhibitors (seeFigure 2(d)) Furthermore the 31015840-UTR of wild-type PECRwas linked to pmirGlo (Promega) and examined in Mac-Tcells that had been transfected with bta-miR-124amimics or acontrol group A significant decrease in reporter activity wasobserved when using the bta-miR-124a mimics transfectedwith wild type 31015840 UTR-reporter of PECR compared to thecontrolThe transfection with bta-miR-124amimics had littleeffect on the luciferase activity of themutant 31015840 UTR-reporterof PECR compared with the control (see Figure 3)The above

results indicated bta-miR-124a could directly target PECRgene

33 Bta-miR-124aRegulates DownstreamGenes of PECR Theinteraction between miRNAs and target genes may affecta series of downstream genes Therefore we obtained thelipid metabolism signal transduction pathway involved inPECR from the KEGG database The PECR downstreamgene ELOVL2was observed in the lipid metabolism pathwayAfter transfection of bta-miR-124amimics and inhibitors andtheir control group it was noticed that the expression ofELOVL2 increased after transfection by bta-miR-124amimics(see Figure 4) and the expression of ELOVL2 decreased aftertransfection by bta-miR-124a inhibitors (see Figure 4) Theabove results suggest that bta-miR-124a plays an importantrole in the biosynthesis of unsaturated fatty acids signalingpathway

BioMed Research International 5Re

lativ

e luc

ifera

se ac

tivity

00

05

10

15

lowast

mim

ics+

PECR

-3-U

TR-M

ut

mim

ics+

PECR

-3-U

TR-W

T

mim

ics+

PECR

-3-U

TR-S

i

Figure 3 Luciferase activity assay report target relationshipLuciferase activities were detected in Mac-T cells that miR-124acotransfected with PECR-mutWTNC vector (lowastp lt005)

Rela

tive e

xpre

ssio

n of

ELO

VL2

00

05

10

15

bta-miR-124amimics

miR-shNC bta-miR-124ainhibitor

Anti-shNC

lowast

lowastlowast

Figure 4 Expression of ELOVL2 after the overexpression andinhibition of bta-miR-124a in Mac-T cells mRNA level of ELOVL2after treatment with bta-miR-124a mimics and control group (miR-NC) in Mac-T cells mRNA level of ELOVL2 during treatment withbta-miR-124a inhibitors and control group (Anti-NC) inMac-T cells(lowastp lt005 lowastlowastp lt001)

34 Overexpression of Bta-miR-124a Upregulates TG and FFALevels The synthesis and secretion of lipids in mammaryepithelial cells are regulated by lipid metabolism-relatedgenes Therefore the contents of TG and FFA in Mac-T cellswere examined after the overexpression and inhibition of bta-miR-124a Compared with the control group the content ofTG and FFA in Mac-T cells transfected with mimics wasincreased and the difference was significantly (lowastlowastp lt001)The content of TGandFFA in the transfected inhibitors groupwas decreased compared with the control group as well but

there was no significant difference in TG content (see Figures5 and 6)

4 Discussion

Lipid metabolism is one of the three major nutrientsmetabolisms which is mainly involved in the energy supplyand storage of the body the composition of biofilm and itsfunction As a regulator of lipid metabolism and lactation indairy cows miRNAs play an important role in the growthand development of dairy cows and milk production It hasbeen confirmed that miR-33 miR-548p and miR-30c aremainly involved in lipid synthesis and transport processwhile miR-122 and miR-370 are mainly involved in lipid syn-thesis and oxidation utilization [18ndash23] In Chinese Holsteincows research has been reported that bta-miR-181a regulatesthe biosynthesis of bovine milk fat by targeting acyl-CoAsynthetase long chain family member 1 (ACSL1) [24] Bta-miR-130a regulates the biosynthesis of bovine milk fat bytargeting peroxisome proliferator-activated receptor gamma[9] Das et al have shown that ectopic expression of miR-124ain adipocytes can lead to decreased fat decomposition andincreased cell TG accumulation [25]

According to previous research miR-124a was domi-nantly shown to be a potential prognostic factor for breastcancer myelodysplastic syndrome and prostate cancer [2627] Meanwhile miR-124a regulates the expression of mul-tiple genes miR-124a is particularly highly expressed indifferentiated and mature neurons but is lowly in neuralstem cells neural precursor cells and glial cells It is also themost expressed miRNAs in the mammalian nervous systemaccounting for 5048 of the total miRNA in themammaliancerebral cortex [11] In recent years the research of miR-124awas not limited to the brain but also involved in the lipidmetabolism process Research showed that the 31015840-noncodingregions of humanATGL gene contains miR-124 binding sitesincluding transcription factor binding sites such as PPARAand PPARG which are key regulatory factors for encodinglipid metabolism enzyme genes [14] Pan et al reported thatmiR-124-3p inhibit adipogenesis by targeting branched chainketo acid dehydrogenase E1 alpha polypeptide (BCKDHA)mRNA which not only revealed the role of miR-124-3p inregulating intramuscular fat formation but also provided areference for understanding the role of BCKDHA in ovineadipogenesis [28] However dairy lipid metabolism hasobvious difference from total lipidmetabolism the regulationfunction ofmiR-124 in dairy lipid for dairy cow is still limited

Our previous study revealed that bta-miR-124a was dif-ferentially expressed between high-fat and low-fat dairy cowprimary mammary epithelial cells with high significance[8] Therefore miR-124a may be a potential regulator ofmilk fat synthesis in dairy cows However the detailedfunctional mechanism is still unknown In this experimentthe candidate target gene PECR was quantitatively analyzedby fluorescence we found that the overexpression or silencingof bta-miR-124a in dairy cow resulted in downregulationor upregulation of the PECR gene which suggest that bta-miR-124a can target the 31015840UTR of PECR gene to regulate itsexpression At the same time PECR gene is involved in fatty

6 BioMed Research InternationalC

ellu

lar T

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cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

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ride

(m

olL

)

025

020

015

010

005

000

bta-miR-124ainhibitors

Anti-NC

(b)

Figure 5 The cellular triglyceride in Mac-T cells that transfected bta-miR-124a mimics bta-miR-124a inhibitors and control group (Mir-shNC and Anti-NC) (lowastlowastp lt001)

0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)

Figure 6The cellular free fat acids content in Mac-T cells that transfected bta-miR-124a mimics bta-miR-124a inhibitors and control group(Mir-shNC and Anti-NC) (lowastp lt005)

acid metabolism pathway suggesting that bta-miR-124a mayindirectly affect the synthesis of milk fat by PECR gene

PECR is a peroxisomal NADPH-specific trans-2-enoyl-CoA reductase that catalyzes the reduction of trans-2-enoyl-CoAs of varying chain lengths from 61 to 161 having maxi-mum activity with 101 CoA It plays a critical role in fatty acidelongation via the conversion of 2E-Octadecenoyl-coA tostearoyl-CoA [29] It has been shown that PECR is expressedin CD4CD8 T cells B cells dendritic cells endothelial cellssmooth muscle kidneys liver and adipocytes [30] Dinavahiet al reported that PECR plays an important role in the latesynthesis of TG and in the treatment of fatty diseases [15] Atpresent studies have shown that miRNA can participate infatty acidmetabolism biosynthesis of unsaturated fatty acidsand peroxisomes by regulated of PECR In 2015 HuangHY etal studied the downregulation of PECR gene in chicken lipid

metabolism by gga-miR-125b-3p gga-miR-130b-3p and gga-miR-26a-5p [31]

For the synthetic process of cows milk fat is significantlydifferent fromother animalsThe precursor of dairy cowmilkfat synthesis is not mainly derived from the glucose producedby the decomposition of starch but through the rumen fer-mentation the final product of cellulose hemicellulose andlignin the final product of acetic acid butyric acid throughthe rumen wall Β-hydroxybutyric acid formed by oxidationof raw processes Furthermore mammary epithelial cellsuse 120573-hydroxybutyric acid as a precursor to synthesizefatty acids eventually producing TG [32] Acetic acid ismainly converted to acetyl-CoA by means of acetyl-CoAsynthetase and then acetyl-CoA is converted to malonyl-CoA by carboxylation This step is the rate-limiting stepof milk fat synthesis [33] Fatty acid uses malonyl-CoA


as a substrate to undergo four cycles of dehydrogenationhydration dehydrogenation and thioester lysis The carbonchain is gradually extended to synthesize the two main fattyacids in the body stearic acid and palmitic acid And stearicacid other fatty acids on the basis of C16 and C18 areextended and desaturated Afterwards we hypothesized thatbta-miR-124a could affect the expression of the ELOVL2gene downstream of PECR in the lipid metabolism signalingpathway ELOVL2 gene is a protein-coding gene and themaingene of long chain fatty acid family It can participate in themetabolism of linolenic acid and the prolongation of fattyacids and is one of the important genes in lipid metabolismAs a downstream gene of PECR it can extend the carbonchain with PECR gene thus forming long chain fatty acids Tofurther identify the function of bta-miR-124a on the effect oflipidmetabolism the expression change of ELOVL2 gene wasdetected The results showed that the expression of ELOVL2was significantly increased after transfection of bta-miR-124a mimics and significantly decreased after transfection ofbta-miR-124a inhibitors The PECR gene is involved in theelongation of the carbon chain in fatty acidmetabolism Eachcycle of PECR adds 2 carbons to the long chain and verylong chain fatty acid chains and reduces the trans-23-enoyl-CoA fatty acid intermediate to acyl-Coenzyme A [15 16] Byentering a new extended cycle and regulating the expressionof ELOVL2 ELOVL2 has been shown to be involved inC20 and C22 polyunsaturated fatty acid extensions In factour study confirmed that ELOVL2 expression changes inresponse to PECR and PECR expression appears to bedependent on bta-miR-124a levels Overexpression of bta-miR-124a inhibits the expression of PECR at the mRNAthereby increasing the expression level of ELOVL2 The lipidmetabolism process is a complex process in which PECR canparticipate in multiple carbon chain extensions in the lipidmetabolism pathway In the present study when bta-miR-124a was overexpressed the PECR of the trans-23-enoyl CoAfatty acid intermediate decreased and ELOVL2 accumulatedWhen bta-miR-124a is inhibited the expression of PECR isincreased and the amount of ELOVL2 is decreased This maybe due to the fact that the lipid metabolism process is oftenthe result of multiple factors indicating that ELOVL2 is notonly a downstream gene of PECR but also regulated by otherfactors so when PECR is inhibited ELOVL2 expression canbe activated by other pathways

In addition we examined TG levels in mammary epithe-lial cells of dairy cows after the overexpression and inhibitionof bta-miR-124a The results showed that compared with thecontrol group the content of TG in transfected cells increasedsignificantly (p lt001) Compared with the control group thecontent of TG in the transfection inhibitor group decreasedbut there was no significant difference (p lt005) Bta-miR-124a mimics are mature double strand of chemically synthe-sized miRNAs which can enhance the function of endoge-nous miRNAs The bta-miR-124a inhibitors are chemicallysynthesized methoxy-modified mature miRNA complemen-tary single-strands which specifically target inhibitors ofspecific target miRNAs in cells and can effectively inhibit theactivity of endogenousmiRNAs in vivoWe speculate that theformation of TGmay be affected only when the expression of

bta-miR-124a is high Further analysis after inhibiting bta-miR-124a there were significant differences in the level oftarget genes mRNA which proved that bta-miR-124a had aregulatory effect on target gene However the content of TGis not obvious which may be due to the regulation of TG bymany genes In addition to the PECR gene our undetectedgenes are involved in the regulation of TG Overexpressionof bta-miR-124a can inhibit the production of multiple targetgenes Because of a dose compensation effect in the bodygenes of TG synthesis pathway in cells actively mobilize toreach the original target gene level and reduce the impactof bta-miR-124a At the same time after the detection offree fatty acids it was found that inhibition of bta-miR-124acould reduce the content of intracellular FFA suggesting thatbta-miR-124a may regulate lipid transport in the transportprocess but had no significant effect on the synthesis of TG

FFA is mainly decomposed from neutral fats Generallysingle-stomach animals cannot absorb and digest FFA wellbut cows as ruminants have evolved efficient fatty acidmetabolism mechanism During the feeding process of dairycows unsaturated fatty acids in roughage are hydrolyzed toFFA through rumen At the same time TG or a small portionof full-length chain fatty acids in feed are converted intoFFA before absorption which can be absorbed in the smallintestine As a kind of fatty acid FFA can also affect thelipid synthesis of dairy cows through the mechanism of fattyacid metabolism As a small biological molecule miRNAscan indirectly regulate the secretion of FFA and can also beaffected by FFA In this study we detected the level of FFA inmammary epithelial cells of dairy cows after transfectionTheresults showed that the content of FFA inmammary epithelialcells increased after the overexpression of bta-miR-124a andthe difference was significant (p lt 005) Compared with thecontrol group the FFA in the bta-miR-124a inhibitor groupdecreased with no significant difference (p lt 005) Accord-ing to the results of intracellular TG analysis since FFA arethe intermediate products of TG metabolism in vivo thereis a close relationship between FFA and TG Bta-miR-124aplays an important role in regulating the content of TG andFFAWhen bta-miR-124a was overexpressed themetabolismof TG increased and the content of FFA increasedWhen bta-miR-124a was inhibited the content of TG did not changesignificantly but the content of FFA decreased This studyshows that bta-miR-124a can participate in the transport oflipids affecting the transformation of intracellular TG andfatty acids thereby affecting the production of milk fat

In conclusion bta-miR-124a is involved in lipidmetabolism by directly downregulating the PECR geneand affecting the expression of the downstream geneELOVL2 and regulates the content of some key secretoryelements such as TG and FFA

5 Conclusion

In conclusion bta-miR-124a is involved in lipid metabolismby directly downregulating the PECR gene and affecting theexpression of the downstream gene ELOVL2 and regulatesthe content of some key secretory elements such as TG andFFAThe function of bta-miR-124a has a certain effect on the


synthesis and secretion of milk fat in the mammary epithelialcells of dairy cows

Data Availability

The data used to support the findings of this study areincluded within the article

Disclosure

The author states that no competitive economic benefits maybe considered to undermine the impartiality of the reportedresearch

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Binglei Shen and Zhuonina Yang contributed equally to thiswork and share the first authorship

Acknowledgments

This research was supported by China Postdoctoral ScienceFoundation Project (2018M631970) Heilongjiang ProvincePostdoctoral Startup Fund Project (LBH-Z16166) Hei-longjiang Provincial Department of Education Science andTechnology Research Project (12521365) Heilongjiang BayiAgricultural University Youth Innovation Talent Project(CXRC-2016-06) Heilongjiang Bayi Agricultural Univer-sity Postdoctoral Startup Fund Project (XDB-2017-06) Hei-longjiang Provincial Key Laboratory of Prevention andControl of Bovine Diseases (PCBD201708) National MajorSpecial Project on New Varieties Cultivation for TransgenisOrganisms (2016ZX08009003-006) National Natural Sci-ence Foundation of China (31472249 31772562)

References

[1] Z Yang T Cappello and L Wang ldquoEmerging role of microR-NAs in lipid metabolismrdquo Acta Pharmaceutica Sinica B (APSB)vol 5 no 2 pp 145ndash150 2015

[2] W Chen X M Fan Mao L et al ldquoMicroRNA-224 as a poten-tial target for miR-based therapy of cancerrdquo Tumour Biologythe Journal of the International Society for OncodevelopmentalBiology amp Medicine vol 36 no 9 pp 6645ndash6652 2015

[3] Z Wang J Han Y Cui X Zhou and K Fan ldquomiRNA-21inhibition enhances RANTES and IP-10 release in MCF-7 viaPIAS3 and STAT3 signalling and causes increased lymphocytemigrationrdquo Biochemical and Biophysical Research Communica-tions vol 439 no 3 pp 384ndash389 2013

[4] A Davalos L Goedeke P Smibert et al ldquomiR-33ab contributeto the regulation of fatty acidmetabolism and insulin signalingrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 108 no 22 pp 9232ndash9237 2011

[5] S Dunner N Sevane D Garcia et al ldquo Genes involved inmuscle lipid composition in 15 European Bos taurus breeds rdquoAnimal Genetics vol 44 no 5 pp 493ndash501 2013

[6] K C Vickers B M Shoucri M G Levin et al ldquoMicroRNA-27b is a regulatory hub in lipid metabolism and is altered indyslipidemiardquo Hepatology vol 57 no 2 pp 533ndash542 2013

[7] B Shen Q Pan Y Yang et al ldquomiR-224 affects mammaryepithelial cell apoptosis and triglyceride production by down-regulating ACADM and ALDH2 genesrdquo DNA and Cell Biologyvol 36 no 1 pp 26ndash33 2017

[8] B Shen L Zhang C Lian et al ldquoDeep sequencing andscreening of differentially expressedMicroRNAs related tomilkfat metabolism in bovine primary mammary epithelial cellsrdquoInternational Journal of Molecular Sciences vol 17 no 2 p 2002016

[9] W C Yang W L Guo L S Zan Y N Wang and K Q TangldquoBta-miR-130a regulates the biosynthesis of bovine milk fat bytargeting peroxisome proliferator-activated receptor gammardquoJournal of Animal Science vol 95 no 7 pp 2898ndash2906 2017

[10] H Xia W K C Cheung S S Ng et al ldquoLoss of brain-enrichedmiR-124 microRNA enhances stem-like traits and invasivenessof glioma cellsrdquoe Journal of Biological Chemistry vol 287 no13 pp 9962ndash9971 2012

[11] J Silber D A Lim C Petritsch et al ldquomiR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cellsand induce differentiation of brain tumor stem cellsrdquo BMCMedicine vol 6 article 14 2008

[12] E V Makeyev J Zhang M A Carrasco and T ManiatisldquoThe microRNA miR-124 promotes neuronal differentiationby triggering brain-specific alternative pre-mRNA splicingrdquoMolecular Cell vol 27 no 3 pp 435ndash448 2007

[13] S Das E Stadelmeyer S Schauer et al ldquoMicro RNA-124aregulates lipolysis via adipose triglyceride lipase and compar-ative gene identification 58rdquo International Journal of MolecularSciences vol 16 no 4 pp 8555ndash8568 2015

[14] R S Holmes J L VandeBerg and L A Cox ldquoVertebrateendothelial lipase comparative studies of an ancient gene andprotein in vertebrate evolutionrdquoGenetica vol 139 no 3 pp 291ndash304 2011

[15] K Abe Y Ohno T Sassa et al ldquoMutation for nonsyndromicmental retardation in the trans-2-enoyl-CoA reductase TERgene involved in fatty acid elongation impairs the enzymeactivity and stability leading to change in sphingolipid profilerdquoe Journal of Biological Chemistry vol 288 no 51 pp 36741ndash36749 2013

[16] T Wakashima K Abe and A Kihara ldquoDual functions ofthe trans-2-enoyl-CoA reductase TER in the sphingosine 1-phosphate metabolic pathway and in fatty acid elongationrdquoeJournal of Biological Chemistry vol 289 no 36 pp 24736ndash24748 2014

[17] R Dinavahi A George A Tretin et al ldquoAntibodies reactiveto non-HLA antigens in transplant glomerulopathyrdquo Journal ofthe American Society of Nephrology vol 22 no 6 pp 1168ndash11782011

[18] R O Benatti A M Melo F O Borges et al ldquo Maternal high-fat diet consumption modulates hepatic lipid metabolism andmicroRNA-122 ( miR-122 ) and microRNA-370 ( miR-370 )expression in offspring rdquo British Journal of Nutrition vol 111no 12 pp 2112ndash2122 2014

[19] S Elhanati R Ben-Hamo Y Kanfi et al ldquoReciprocal regulationbetween SIRT6 and miR-122 controls liver metabolism and


predicts hepatocarcinoma prognosisrdquo Cell Reports vol 14 no2 pp 234ndash242 2016

[20] L Goedeke F M Vales-Lara M Fenstermaker et al ldquoA regu-latory role for MicroRNA 33 in controlling lipid metabolismgene expressionrdquoMolecular and Cellular Biology vol 33 no 11pp 2339ndash2352 2013

[21] D Iliopoulos K Drosatos Y Hiyama I J Goldberg and V IZannis ldquoMicroRNA-370 controls the expression ofMicroRNA-122 and Cpt1120572 and affects lipid metabolismrdquo Journal of LipidResearch vol 51 no 6 pp 1513ndash1523 2010

[22] J Soh J Iqbal J Queiroz C Fernandez-Hernando andM M Hussain ldquoMicroRNA-30c reduces hyperlipidemia andatherosclerosis in mice by decreasing lipid synthesis andlipoprotein secretionrdquo Nature Medicine vol 19 no 7 pp 892ndash900 2013

[23] L Zhou and M M Hussain ldquoHuman MicroRNA-548pdecreases hepatic apolipoprotein B secretion and lipid synthe-sisrdquo Arteriosclerosis rombosis and Vascular Biology vol 37no 5 pp 786ndash793 2017

[24] S Lian J Guo X Nan L Ma J Loor and D Bu ldquoMicroRNABta-miR-181a regulates the biosynthesis of bovine milk fat bytargeting ACSL1rdquo Journal of Dairy Science vol 99 no 5 pp3916ndash3924 2016


[26] X Lv Y Jiao YQing et al ldquomiR-124 suppressesmultiple steps ofbreast cancer metastasis by targeting a cohort of pro-metastaticgenes in vitrordquoChinese Journal of Cancer vol 30 no 12 pp 821ndash830 2011

[27] R Ben Gacem O Ben Abdelkrim S Ziadi M Ben Dhiaband M Trimeche ldquoMethylation of miR-124a-1 miR-124a-2and miR-124a-3 genes correlates with aggressive and advancedbreast cancer diseaserdquo Tumor Biology vol 35 no 5 pp 4047ndash4056 2014

[28] Y Pan J Jing L Qiao et al ldquomiR-124-3p affects the formationof intramuscular fat through alterations in branched chainamino acid consumption in sheeprdquo Biochemical and BiophysicalResearch Communications vol 495 no 2 pp 1769ndash1774 2018

[29] A K Das M D Uhler and A K Hajra ldquoMolecular cloningand expression of mammalian peroxisomal trans-2-enoyl-coenzyme A reductase cDNAsrdquoe Journal of Biological Chem-istry vol 275 no 32 pp 24333ndash24340 2000

[30] C Wu C Orozco J Boyer et al ldquoBioGPS an extensibleand customizable portal for querying and organizing geneannotation resourcesrdquo Genome Biology vol 10 no 11 articleR130 2009

[31] H Y Huang R R Liu G P Zhao et al ldquoIntegrated analysisof microRNA and mRNA expression profiles in abdominaladipose tissues in chickensrdquo Scientific Reports vol 5 no 1Article ID 16132 2015

[32] J Ha and K H Kim ldquoInhibition of fatty acid synthesis byexpression of an acetyl-CoA carboxylase-specific ribozymegenerdquo Proceedings of the National Acadamy of Sciences of theUnited States of America vol 91 no 21 pp 9951ndash9955 1994

[33] R J Mansbridge and J S Blake ldquoNutritional factors affectingthe fatty acid composition of bovine milkrdquo British Journal ofNutrition vol 78 no 1 pp S37ndashS47 1997

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018


Genetics Research International


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Virolog y Stem Cells International



Enzyme Research



MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom


Our previous studies have identified several lipidmetabolism-related miRNAs Bta-miR-224 could affectapoptosis and triglyceride (TG) synthesis by regulating acyl-CoA dehydrogenase medium chain (ACADM) and aldehydedehydrogenase 2 (ALDH2) which are two important genesinvolved in lipid metabolism in Mac-T cells [7] In anotherstudy bta-miR-33a negatively regulated the expression ofelongation of very long chain fatty acids protein 5 (ELOVL5)elongation of very long chain fatty acids protein 6 (ELOVL6)and sterol-C4-methyl oxidase (SC4MOL) in mammarygland with low milk fat content (MG-LL) bta-miR-152 wasdownregulated in MG-LL and the significantly upregulatedgenes were prostaglandin-endoperoxide synthase 2 (PTGS2)protein kinase AMP-activated catalytic subunit alpha1 (PRKAA1) and uncoupling protein 3 (UCP3) [8] Inaddition another research reported that the overexpressionof miR-130a significantly decreased cellular triacylglycerol(TG) expression level and suppressed lipid droplet formationwhereas the inhibition of miR-130a resulted in greater lipiddroplet formation and TG accumulation in bovinemammaryepithelial cells [9]

MiR-124 is a small RNA molecule located on chromo-some 8 After comparing the precursor sequence and maturesequence of miR-124 we found that the miR-124 sequence ishighly conserved among species MiR-124 has been studiedfor many years most studies were related to brain andneurons development Researchers found that the tumorsuppressor activity of miR-124 could be partly due to itsinhibitory effects on glioma stem-like traits and invasivenessthrough Snail homolog 2 (SNAI2) [10] Silber et al reportedthatmiR-124 inhibit proliferation of glioblastomamultiformecells and induce differentiation of brain tumor stem cells [11]Eugene V et al reported that miR-124 promotes neuronaldifferentiation by triggering brain-specific alternative pre-mRNA splicing [12]

In recent years miR-124 has also been shown to beinvolved in lipid metabolism and affect lipid synthesis Daset al reported that miR-124a attenuates RNA and proteinexpression of the major TG hydrolase adipose TG lipase(ATGLPNPLA2) and its coactivator comparative gene iden-tification 58 (CGI-58ABHD5) [13] Holmes et al found miR-124506 andmiR-108 binding sites in the 31015840-noncoding regionofmRNA including peroxisomeproliferator-activated recep-tor alpha (PPARA) and peroxisome proliferative activatedreceptor gamma (PPARG) which are key regulators of genesencoding enzymes of lipid metabolism [14] Those provideda basis for the study of miR-124a in lipid metabolism and alsoindicated that miR-124a plays an important role in fatty acidmetabolism However the study ofmiR-124a involved in fattyacid metabolism in dairy cows is limited We had reportedthat bta-miR-124a was differentially expressed between high-fat and low-fat dairy cow primary mammary epithelialcells with high significance [8] Therefore this experimentattempted to identify the target gene of bta-miR-124a andexplore its effect on lipid metabolism in dairy cows in orderto provide a basic reference for molecular-oriented breedingof dairy cows

Peroxisomal trans-2-enoyl-CoA reductase (PECR) is alsocalled TERP and TECR which is located on chromosome

q35 (chr2215996329-216082955) with 86627 bases (httpswwwgenecardsorg) It can participate in carbon chainelongation in fatty acid metabolism and catalyze the lastreaction of four long chain fatty acid elongation cycles Eachcycle of PECR adds two carbons to the long chain andvery long chain fatty acid (VLCFA) chains reducing theintermediate of trans-23-enoyl coenzyme A fatty acid to acylcoenzyme A which can further extend the carbon chain byentering a new elongation cycle [15 16] Meanwhile PECRis a peroxisome protein involved in fatty acid synthesis andplays an important role in milk fat synthesis Studies haveshown that the gene is closely related to the late develop-ment of TG [17] Therefore in this study we reported theexpression of bta-miR-124a in Mac-T cells which can affectlipid metabolism by regulating PECR gene which provideda reference for further study on the regulation mechanism ofmilk fat in dairy cows

2 Materials and Methods

21 Bioinformatics Prediction of Candidate Target Genes ofbta-miR-124a The mature sequences of bta-miR-124a wereobtained on miRBase (httpwwwmirbaseorgindexshtml)and homology analysis was performed The possibletarget genes for bta-miR-124a are predicted by TargetScan(httpwwwtargetscanorg) The bioinformatics websiteStarbase (httpstarbasesysueducn) was used to predictthe extent of nonspecific pairing of bta-miR-124a with itscandidate target genes

22 Cells Culture and Transfection Mac-T cells were from thelaboratory inHeilongjiangBayiAgriculturalUniversityMac-T cells were cultured in Dulbeccorsquos modified Eaglersquos mediumand F-12 (DMEMF12) (Gibco Grand Island NY) with 10fetal bovine serum (FBS) (Gibco Grand Island NY USA) 100UmL penicillin and 100 120583gmL streptomycin with or with-out 250 ngmL amphotericin B (Invitrogen Carlsbad CAUSA) digestive cells with 025 trypsin (Solarbio BeijingChina) and incubated at 37∘C in a humidified atmosphere of5 CO

2

The indicated cells reaching 60 to 80 confluence weretransfected with 10nM of bta-miR-124a mimics or inhibitorsand their corresponding control group constructs (Mir-shNC Anti-NC) (Sangon Biotech Shanghai China) in 6-well using Lipofectamine 3000 (Invitrogen Life TechnologiesCarlsbad CAUSA)Themediumwas changed to regular cellculture medium after 4-6 h Cells were collected for analysisat 24-48 h after transfection

23 Total RNA Isolation and Quantitative Polymerase ChainReaction (qPCR) Assays Forty-eight hours after transfectionwith the miR-124a mimicsinhibitors total RNA of the Mac-T cells was extracted using trizol reagent (Invitrogen) Thequantity of RNA was verified by NanoDrop1000 (ThermoScientific) ensuring the optical density (OD) 260nm280nmvaluewas between 18 and 20 ComplementaryDNA (cDNA)was synthesized using the Primer Script reverse transcriptionPCR (RT-PCR) kit (Takara) The expressions of miR-124aand target genes were detected on a Bio-Rad CFX Connect















Site type






8mer








3 Results




lativ

e exp

ress

ion

ofbt

a-m

iR-1

24a

012345

500600700800900

1000

bta-miR-124amimics

miR-shNC

lowast

(a)

Rela

tive e

xpre

ssio

n of

bta-

miR

-124

a

00

05

10

15


Anti-shNC

lowastlowast

(b)

00

05

10

15

bta-miR-124amimics

miR-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(c)

00

05

10

15

20


Anti-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(d)






lativ

e luc

ifera

se ac

tivity

00

05

10

15

lowast

mim

ics+

PECR

-3-U

TR-M

ut

mim

ics+

PECR

-3-U

TR-W

T

mim

ics+

PECR

-3-U

TR-S

i


Rela

tive e

xpre

ssio

n of

ELO

VL2

00

05

10

15

bta-miR-124amimics


Anti-shNC

lowast

lowastlowast




4 Discussion





ellu

lar T

rigly

cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

lyce

ride

(m

olL

)

025

020

015

010

005

000


Anti-NC

(b)


0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)












5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018
















Site type






8mer








3 Results




lativ

e exp

ress

ion

ofbt

a-m

iR-1

24a

012345

500600700800900

1000

bta-miR-124amimics

miR-shNC

lowast

(a)

Rela

tive e

xpre

ssio

n of

bta-

miR

-124

a

00

05

10

15


Anti-shNC

lowastlowast

(b)

00

05

10

15

bta-miR-124amimics

miR-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(c)

00

05

10

15

20


Anti-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(d)






lativ

e luc

ifera

se ac

tivity

00

05

10

15

lowast

mim

ics+

PECR

-3-U

TR-M

ut

mim

ics+

PECR

-3-U

TR-W

T

mim

ics+

PECR

-3-U

TR-S

i


Rela

tive e

xpre

ssio

n of

ELO

VL2

00

05

10

15

bta-miR-124amimics


Anti-shNC

lowast

lowastlowast




4 Discussion





ellu

lar T

rigly

cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

lyce

ride

(m

olL

)

025

020

015

010

005

000


Anti-NC

(b)


0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)












5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



lativ

e exp

ress

ion

ofbt

a-m

iR-1

24a

012345

500600700800900

1000

bta-miR-124amimics

miR-shNC

lowast

(a)

Rela

tive e

xpre

ssio

n of

bta-

miR

-124

a

00

05

10

15


Anti-shNC

lowastlowast

(b)

00

05

10

15

bta-miR-124amimics

miR-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(c)

00

05

10

15

20


Anti-shNC

lowast

Rela

tive e

xpre

ssio

n of

PECR

(d)






lativ

e luc

ifera

se ac

tivity

00

05

10

15

lowast

mim

ics+

PECR

-3-U

TR-M

ut

mim

ics+

PECR

-3-U

TR-W

T

mim

ics+

PECR

-3-U

TR-S

i


Rela

tive e

xpre

ssio

n of

ELO

VL2

00

05

10

15

bta-miR-124amimics


Anti-shNC

lowast

lowastlowast




4 Discussion





ellu

lar T

rigly

cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

lyce

ride

(m

olL

)

025

020

015

010

005

000


Anti-NC

(b)


0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)












5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



lativ

e luc

ifera

se ac

tivity

00

05

10

15

lowast

mim

ics+

PECR

-3-U

TR-M

ut

mim

ics+

PECR

-3-U

TR-W

T

mim

ics+

PECR

-3-U

TR-S

i


Rela

tive e

xpre

ssio

n of

ELO

VL2

00

05

10

15

bta-miR-124amimics


Anti-shNC

lowast

lowastlowast




4 Discussion





ellu

lar T

rigly

cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

lyce

ride

(m

olL

)

025

020

015

010

005

000


Anti-NC

(b)


0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)












5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018



ellu

lar T

rigly

cerid

e(

mol

L)

008

006

004

002

000

bta-miR-124amimics

miR-shNC

lowastlowast

(a)

Cel

lula

r Trig

lyce

ride

(m

olL

)

025

020

015

010

005

000


Anti-NC

(b)


0025

0020

0015

0010

0005

0000

Cel

lula

r Fre

e fat

acid

(m

olL

)

bta-miR-124amimics

miR-shNC

lowast

(a)

Cel

lula

r Fre

e fat

acid

(m

olL

)


Anti-shNC

008

006

004

002

000

(b)












5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018








5 Conclusion




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Data Availability


Disclosure






Acknowledgments


References






































Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




















Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018




Volume 2018

Zoology











Volume 2018

















Advances in




Enzyme Research





Volume 2018


Documents

Bta-miR-124a Affects Lipid Metabolism by Regulating PECR Genedownloads.hindawi.com/journals/bmri/2019/2596914.pdf · bta-miR-124a mimics miR-shNC ∗∗ (a) Cellular Triglyceride