Research Article Whole Genome Expression Profiling and Signal Pathway …downloads.hindawi.com/journals/sci/2014/913050.pdf · 2019-07-31 · Research Article Whole Genome Expression

Research ArticleWhole Genome Expression Profiling and Signal PathwayScreening of MSCs in Ankylosing Spondylitis

Yuxi Li1 Peng Wang1 Zhongyu Xie1 Lin Huang1 Rui Yang1 Liangbin Gao1 Yong Tang1

Xin Zhang1 Jichao Ye1 Keng Chen1 Zhaopeng Cai1 Yanfeng Wu2 and Huiyong Shen1

1Department of Orthopedics Sun Yat-sen Memorial Hospital Sun Yat-sen University No 107 Yanjiang Road WestGuangzhou 510120 China2Center for Biotherapy Sun Yat-sen Memorial Hospital Sun Yat-sen University No 107 Yanjiang Road WestGuangzhou 510120 China

Correspondence should be addressed to Yanfeng Wu wuyanfengcn126com and Huiyong Shen shenhuiyongaliyuncom

Received 23 June 2014 Revised 26 October 2014 Accepted 12 November 2014 Published 3 December 2014

Academic Editor Eva Mezey

Copyright copy 2014 Yuxi Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The pathogenesis of dysfunctional immunoregulation of mesenchymal stem cells (MSCs) in ankylosing spondylitis (AS) is thoughtto be a complex process that involves multiple genetic alterations In this study MSCs derived from both healthy donors and ASpatients were cultured in normal media or media mimicking an inflammatory environment Whole genome expression profilinganalysis of 33351 genes was performed and differentially expressed genes related to AS were analyzed by GO term analysis andKEGG pathway analysis Our results showed that in normal media 676 genes were differentially expressed in AS 354 upregulatedand 322 downregulated while in an inflammatory environment 1767 genes were differentially expressed in AS 1230 upregulatedand 537 downregulated GO analysis showed that these genes were mainly related to cellular processes physiological processesbiological regulation regulation of biological processes and binding In addition by KEGG pathway analysis 14 key genes fromthe MAPK signaling and 8 key genes from the TLR signaling pathway were identified as differentially regulated The results ofqRT-PCR verified the expression variation of the 9 genes mentioned above Our study found that in an inflammatory environmentankylosing spondylitis pathogenesis may be related to activation of the MAPK and TLR signaling pathways

1 Introduction

Ankylosing spondylitis (AS) is a chronic immune-mediatedinflammatory disease characterized by inflammatory backpain and enthesis [1] To date several hypotheses have beenproposed to describe the pathogenic mechanism behind thisdisease including genetic susceptibility of HLA-B27 [2] andERAP1 [3] infection [4] and environmental factors [5]However none of these hypotheses can fully account for thepathogenesis of ankylosing spondylitis Recent evidence hasincreasingly suggested that autoimmune disorders may beinvolved in the onset and development of this disease [6]

It has been recently demonstrated thatmesenchymal stemcells (MSCs) possess an immunosuppressive capability toinhibit Th17 cells and induce Treg subpopulations of CD4+T cells [7 8] In 2011 our research showed that comparedto healthy donors the immunomodulatory ability of MSCs

was reduced in AS patients This was manifested by anincrease in Th17 cells and a reduction in Treg cells in CD4+T cell subgroups after a mixed lymphocyte reaction (MLR)[9] Furthermore our clinical trial has also suggested thatinfusion of MSCs is a feasible safe and promising treatmentforAS patients [10]Therefore we believe that themodulatoryfunction of MSCs can play a significant role in improvingdisease condition or clinical symptoms in AS patients andthat immunoregulatory dysfunction of MSCs may play acritical role in the pathogenesis of AS

Recently several researchers carried out whole genomeexpression profiling analyses comparing AS patients tohealthy donors [11ndash13] These studies found that AS hasa strong association with HLA-B27 and other non-HLAsusceptibility genes such as IL23R and ERAP1 [14 15]However these genetic variations could not fully accountfor the pathogenesis of ankylosing spondylitis For example

Hindawi Publishing CorporationStem Cells InternationalVolume 2014 Article ID 913050 11 pageshttpdxdoiorg1011552014913050

2 Stem Cells International

HLA-B27 accounts for only sim45 of the genetic risk for ASTherefore in this study we focused mainly on the significantrole of MSCs in AS pathogenesis in order to provide a newviewpoint on this inflammatory disease Our study investi-gated whether MSCs grown in vitro from AS patients exhibitgene expression differences in normal culture media or in aninflammatory environment as compared to healthy controlsWe found that mimicking an inflammatory environment canactivate the MAPK and TLR signaling pathways in MSCsderived fromAS patients thereby upregulating inflammatorygene expression This data provides suggestive clues in theexploration of the pathogenic mechanism behind ankylosingspondylitis

2 Materials and Methods

21 Patients and Controls Thepresent study was approved bythe Ethics Committee of the Sun Yat-sen Memorial Hospitalof SunYat-senUniversity GuangzhouChina From June 2012to December 2012 twelve healthy donors (HD 9 men and3 women) with an average age of 221 years and twelve ASpatients (10 men and 2 women) with an average age of 219years were enrolled in this study (Table 1) The diagnosesof AS patients were all performed according to the ASASclassification criteria [16] In addition all patients involvedwere diagnosed for the first time and remained in active stage(all Bath Ankylosing Spondylitis Disease Activity Index ge 4)

22 Isolation and Preparation of MSCs After being informedabout scientific significance possible risks and complicationsand the treatment measures for bone marrow aspirations alldonors and patients signed the informed consent and wereaspirated by our skilled allied health professionals in strictaccordance with the international standardized procedureBone marrow MSCs were isolated from the bone marrowaspirates by density gradient centrifugation as previouslydescribed by our work and that of others [9 17] AfteridentifyingMSC immunophenotype markers by flow cytom-etry passages three to five were used for the experimentsdescribed below Both HD and AS groups were separatedinto two subgroups MSCs cultured in normal media (Norm)and MSCs cultured in an inflammatory environment (Infla)which was created by adding TNF-120572 (10 ngmL) and IFN-120574(10 ngmL) into normal culture media for four hours Fiverepresentatives of cultured MSCs from HD and AS patientswere randomly chosen cultured in both types of media andfurther analyzed Twenty samples were chosen to completewhole genome expression profiles 5 HD-Norm 5 HD-Infla5 AS-Norm and 5 AS-Infla samples

23 Total RNA Extraction and Quality Control 1mL Trizol(Invitrogen Carlsbad USA) was added to lyse the collectedcells Total RNA was extracted using the phenolchloroformmethod and salt was washed away with 70 alcohol Afterair-drying at 15sim30∘C the appropriate amount of RNase-free water was added to dissolve RNA The integrity andconcentration of RNA were assessed to ensure accuracyand reliability Total RNA from each sample was quantified

Table 1 Demographic data and disease characteristics of enrolledpatients and healthy donors

Items AS HDNumbers 12 12SexMen 10 9Women 2 3

Age (years) 229 (20ndash30) 231 (21ndash31)Body weight (Kg) 633 (58ndash65) 635 (60ndash66)Disease duration from diagnosis(months)

339(28ndash396) 0

HLA-B27 11 (917) 0History of extra-axial involvement(number of patients)Uveitis 0 0Psoriasis 0 0Dactylitis 2 (167) 0Inflammatory bowel disease 1 (83) 0Enthesitis 5 (417) 0

using a NanoDrop 1000 and RNA integrity was assessed bystandard denaturing agarose gel electrophoresis About 5 120583gtotal RNA from each sample was used for labeling and arrayhybridization following these steps (1) Reverse transcriptionby Invitrogen Superscript ds-cDNA synthesis kit (2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit(3) array hybridization using the NimbleGen HybridizationSystem followed by washing with the NimbleGen washbuffer kit (4) array scanning using the Axon GenePix 4000Bmicroarray scanner (Molecular Devices Corporation)

24 Microarray Scanning andData Analysis Scanned imageswere then imported into NimbleScan software (version 25)for grid alignment and expression data analysis Data werenormalized by quantile normalization and the robust multi-chip average (RMA) algorithm included in the NimbleScansoftware Probe level files and gene level files were generatedafter normalizationThe twenty gene level files were importedinto Agilent GeneSpring GX software (version 115) forfurther analysis After GeneSpring quantile normalizationdifferentially expressed genes were identified using VolcanoPlot filtering KEGG pathway analysis and GO analysis wereapplied to determine the roles of these differentially expressedgenes in biological pathways or GO terms Finally the posthoc Bonferroni test was used to correct for multiplicity sincea large number of false positive 119875 values were observed 119875values lt001 were considered significant

25 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) After MSCs were cultured in inflammatory envi-ronment mentioned above total RNA was extracted usingTrizol reagent (Life Technologies Inc USA) Synthesis ofcDNA (RT-PCR) was carried out using the SuperScriptH IIIFirst-Strand Synthesis System (Invitrogen) according to themanufacturerrsquos instructions The quantitative real-time PCR

Stem Cells International 3

(qRT-PCR) analyses were performed in a Light Cycler 480Real-Time PCR System (Roche Basel) using a SYBR PremixEx Taq II kit (Takara Otsu) The program for quantitative-PCR assay was 95∘C for 30 s followed by 40 cycles of 95∘C for5 s and 60∘C for 20 s The mean mRNA levels were calculatedfrom triplicate analyses of each sample and melting curveanalysis was performed to confirm the specific amplificationof the targets The relative amount or fold change of thetarget genes was normalized according to the expression ofthe housekeeping gene 120573-actin Parts of the primer sequencesused in the real-time PCR assay are as follows

120573-actin 51015840-CATGTACGTTGCTATCCAGGC-31015840(Forward) and 51015840-CTCCTTAATGTCACGCAC-GAT-31015840 (Reverse)GNA12 51015840-CCGCGAGTTCGACCAGAAG-31015840 (For-ward) and 51015840-TGATGCCAGAATCCCTCCAGA-31015840(Reverse)DUSP1 51015840-AGTACCCCACTCTACGATCAGG-31015840(Forward) and 51015840-GAAGCGTGATACGCACTGC-31015840(Reverse)MAPK11 51015840-CTGAACAACATCGTCAAGTGCC-31015840(Forward) and 51015840-CATAGCCGGTCATCTCCTCG-31015840 (Reverse)RAC1 51015840-ATGTCCGTGCAAAGTGGTATC-31015840 (For-ward) and 51015840-CTCGGATCGCTTCGTCAAACA-31015840(Reverse)MAPK3 51015840-CTACACGCAGTTGCAGTACAT-31015840(Forward) and 51015840-CAGCAGGATCTGGATCTCCC-31015840 (Reverse)TRAF3 51015840-CAGACTAACCCGCCGCTAAAG-31015840(Forward) and 51015840-GATGCTCTCTTGACACGC-TGT-31015840 (Reverse)IFNAR1 51015840-AACAGGAGCGATGAGTCTGTC-31015840(Forward) and 51015840-TGCGAAATGGTGTAAATG-AGTCA-31015840 (Reverse)IL6 51015840-ACTCACCTCTTCAGAACGAATTG-31015840(Forward) and 51015840-CCATCTTTGGAAGGTTCA-GGTTG-31015840 (Reverse)IFNA1 51015840-GCCTCGCCCTTTGCTTTACT-31015840 (For-ward) and 51015840-CTGTGGGTCTCAGGGAGATCA-31015840(Reverse)

3 Results

31 Identification of MSC Immunophenotype MSCs wereobtained from bone marrow aspirates of both healthy vol-unteers and AS patients MSCs were cultured and purifiedto homogeneity according to the culture method describedin our previous work [9] Figure 1(a) shows adherent MSCsgrowing like intersecting spindle bundles In additionimmunophenotyping was performed by flow cytometry Dotplots showing all (ungated) cells as well as gated MSCsare displayed in Figure 1(b) MSCs constitutively expressedCD29 CD44 and CD105 but not CD34 CD45 or HLA-DR(Figure 1(c))

32 Microarray Hybridization and Data Analysis The exper-imental system was stable and fluorescent signal intensitywas strong and homogenous When cultured under inflam-matory conditions the number of differentially expressedgenes in MSCs from AS patients was increased (Figure 2(a))There were 676 differentially expressed genes in the AS-Norm group of which 354 were upregulated and 322 weredownregulated as compared to the HD-Norm group 1767genes were differentially expressed in the AS-Infla group1230 upregulated and 537 downregulated as compared tothe HD-Infla group We used GO (gene ontology) to analyzethese differentially expressed genes within three domains(Figure 2(b))

In the biological processes domain we found 79 sig-nificant functional description nodes for group AS-Normwhich were mainly related to intracellular signaling cascadespositive regulation of cellular metabolic processes regulationof molecular function positive regulation of macromoleculemetabolic processes positive regulation of cellular biosyn-thetic processes and positive regulation of metabolic pro-cesses There were 134 functional description nodes (119875 lt001) in group AS-Infla versus group BD-Infla which weremainly related to mRNA metabolic processes nucleobasenucleoside nucleotide and nucleic acid metabolic processescell cycle mitotic cell cycle gene expression and nitrogencompound metabolic processes (Table 2)

In the cellular component domain 8 significant func-tional description nodes were found for the AS-Norm groupcompared to the BD-Norm group which weremainly locatedin the extracellular region cyclin-dependent protein kinaseholoenzyme complex extracellular region part keratin fila-ment neuron projection cytoplasmic microtubule extracel-lular space and apical plasma membrane In contrast com-pared to group BD-Infla there were 44 significant functionaldescription nodes for the AS-Infla group which were mainlylocated in the intracellular part intracellular membrane-bounded organelle membrane-bounded organelle intracel-lular organelle lumen and organelle lumen (Table 3)

In the molecular function domain when comparedto group BD-Norm there were 12 significant functionaldescription nodes for the AS-Norm group such as pro-tein tyrosineserinethreonine phosphatase activity growthfactor activity fibroblast growth factor receptor bindingand receptor signaling protein activity Simultaneously 20significant functional description nodes (119875 lt 001) werefound for group AS-Infla versus group BD-Infla whichincluded protein binding nucleic acid binding structure-specific DNA binding RNA binding and hexosaminidaseactivity (Table 3)

33 Screening of Signaling Pathways Related to AS KEGGpathway analysis was used to discover key signaling pathwaysand relationships between differentially expressed genes Inthe inflammatory environment the two highest scores ofindicators in the AS group were hsa04010 mitogen-activatedprotein kinase (MAPK) signaling pathway and hsa04620toll-like receptor (TLR) signaling pathway (Table 4) Inthe MAPK pathway there were 14 differentially expressed


(a)

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Figure 1 Immunophenotype and differentiation of MSCs (a) Under the 40x microscope spindle-shaped MSCs grow like intersectingbundles (b) Dot plot data showing all (nongated) cells as well as gated hMSC (P1) (c) MSCs were harvested labeled with antibodies againstimmunophenotype and analyzed by flow cytometry MSCs expressed CD29 CD44 and CD105 but not CD34 CD45 and HLA-DR

genes (119875 lt 005) DUSP1 GNA12 RAC1 MRAS ELK1ATF4MAPK3 CDC25B STMN1MAX TGFBR2MAPK11CACNB3 FGF8 and RASA2 (Table 5) In the TLR pathwaythere were 8 key genes with a significant difference inexpression (119875 lt 005) RAC1 TRAF3 MAPK3 IFNAR1

TICAM1 IL6 MAPK11 and IFNA1 (Table 5) However therewere only two differentially expressed genes (CACNA2D3and PPM1B) in theMAPK pathway and one key gene (TLR6)in the TLR pathway in the AS group cultured in normalmedia both with an insignificant KEGG analysis score


14

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Group AS-InflaGroup AS-Norm

(a)

GO other items

GO0009987 cellularprocess

GO0007582 physiologicalprocess

GO0065007 biologicalregulation

GO0050789 regulation ofbiological process

GO0005488 binding

GO0030528 transcriptionregulator activity

GO0030234 enzymeregulator activity

GO0002376 immunesystem process

GO0032991macromolecular complexGO0048519 negative

regulation of biological processGO0060089 molecular

transducer activityGO0005215 transporter

activityGO0044422 organelle partGO0051234 establishment

of localizationGO0048518 positive

regulation of biological processGO0051179 localization

GO0043226 organelleGO0050896 response to

stimulusGO0032502

developmental processGO0032501 multicellular

organismal processGO0003824 catalytic

activityGO0008152 metabolismGO0044464 cell part

GO0005623 cell

(b)

Figure 2 Whole genomic expression analysis of MSCs in AS patients (a) shows the scatter plot of differentially expressed genes of MSCsfrom both healthy donors and AS patients respectively in both normal and inflammatory culture environment The values of 119909 and 119910 axesin the scatter plot are the normalized signal values of each sample (log2 scaled) The green lines are fold change lines (the default fold changevalue given is 20) Genes above the top green line and below the bottom green line indicated more than 20-fold change of genes betweentwo compared arrays (b) shows the pie chart of gene ontology (GO) analysis of results of the differentially expressed genes of MSCs betweengroup AS-Infla and group HD-Infla Among the 1590 genes 892 (5610) participated in biological process 461 (2899) played a role inmolecular function and 237 (1491) associated with cellular component


Table 2 GO analysis of ankylosing spondylitis with different biological processes related to gene

Category GO ID Term Count of genesPercent ofcount ofgenes

Foldenrichment 119875 value

GOTERM BP GO0002376 Immune system process 50 1269 17 000016GOTERM BP GO0002520 Immune system development 19 482 203 000285GOTERM BP GO0003006 Reproductive developmental process 14 355 205 000898GOTERM BP GO0006325 Chromatin organization 21 533 195 000276GOTERM BP GO0006816 Calcium ion transport 13 33 271 000107GOTERM BP GO0006950 Response to stress 64 1624 135 000765GOTERM BP GO0006996 Organelle organization 54 1371 14 000669GOTERM BP GO0007154 Cell communication 71 1802 159 000005GOTERM BP GO0007165 Signal transduction 108 2741 124 000756GOTERM BP GO0007242 Intracellular signaling cascade 73 1853 17 0GOTERM BP GO0007243 Protein kinase cascade 25 635 171 000663GOTERM BP GO0007275 Multicellular organismal development 100 2538 128 000422GOTERM BP GO0009891 Positive regulation of biosynthetic process 37 939 202 000004GOTERM BP GO0009893 Positive regulation of metabolic process 46 1168 187 000003GOTERM BP GO0009966 Regulation of signal transduction 46 1168 185 000004GOTERM BP GO0009967 Positive regulation of signal transduction 20 508 232 000044GOTERM BP GO0010212 Response to ionizing radiation 6 152 38 00048

GOTERM BP GO0010557 Positive regulation of macromoleculebiosynthetic process 34 863 198 000011

GOTERM BP GO0010604 Positive regulation of macromoleculemetabolic process 44 1117 192 000002

GOTERM BP GO0010627 Regulation of protein kinase cascade 15 381 207 000632GOTERM BP GO0010628 Positive regulation of gene expression 32 812 211 000006GOTERM BP GO0010646 Regulation of cell communication 50 1269 175 000008GOTERM BP GO0010647 Positive regulation of cell communication 21 533 225 000048

GOTERM BP GO0010740 Positive regulation of protein kinasecascade 13 33 255 000185

GOTERM BP GO0015674 Di- trivalent inorganic cation transport 13 33 227 000501GOTERM BP GO0016568 Chromatin modification 18 457 226 000114GOTERM BP GO0018108 Peptidyl-tyrosine phosphorylation 9 228 321 000197GOTERM BP GO0018212 Peptidyl-tyrosine modification 9 228 315 000225Part of GO analysis of biological processes is supplied here more data can be found in additional files

(119875 gt 005) The results of qRT-PCR verified the expressionvariation of such genes mentioned above including GNA12DUSP1 MAPK11 RAC1 MAPK3 TRAF3 IFNAR1 IL6 andIFNA1 (Figure 3)

4 Discussion

Ankylosing spondylitis is a type of chronic inflammatoryarthritis Our previous work demonstrated its pathogenesismay be associated with immunomodulatory dysfunction ofMSCs in AS patients [9] The pathogenesis of dysfunctionalimmunoregulation of MSCs in AS is thought to be a complexprocess involving multiple genetic alterations In this studywe focused our interest on the difference inMSCs betweenASpatients and healthy donors and carried out whole genome

expression profiling Our study showed that there were 676genes differentially expressed between the AS-Norm groupand HD-Norm group 354 upregulated and 322 downregu-lated 1767 genes were differentially expressed between theAS-Infla group andHD-Infla group 1230 upregulated and 537downregulated Furthermore we found that when culturedin an inflammatory environment MSCs from AS patientssuggested an activation of the MAPK and TLR signalingpathways

By GO analysis differentially expressed genes involved inbiological processes were mainly related to mRNAmetabolicprocesses nucleobase nucleoside nucleotide and nucleicacid metabolic processes cell cycle mitotic cell cycle geneexpression and nitrogen compound metabolic processes Inaddition differentially expressed genes involved mainly inmolecular functions were mainly related to protein binding


Table 3 GO analysis of differentially expressed genes associated with the cellular location and molecular function



GOTERM CC GO0000307 Cyclin-dependent protein kinaseHoloenzyme complex 4 092 1235 000022

GOTERM CC GO0005576 Extracellular region 82 1876 152 000006GOTERM CC GO0005615 Extracellular space 29 664 16 000908GOTERM CC GO0005881 Cytoplasmic microtubule 3 069 695 000842GOTERM CC GO0016324 Apical plasma membrane 10 229 239 000945GOTERM CC GO0043005 Neuron projection 18 412 2 000423GOTERM CC GO0044421 Extracellular region part 41 938 161 000181GOTERM CC GO0045095 Keratin filament 8 183 329 000297GOTERM MF GO0000287 Magnesium ion binding 22 52 173 0009GOTERM MF GO0004725 Protein tyrosine phosphatase activity 8 189 286 0007GOTERM MF GO0005057 Receptor signaling protein activity 11 26 251 00047GOTERM MF GO0005102 Receptor binding 37 875 155 00055GOTERM MF GO0005104 Fibroblast growth factor receptor binding 4 095 695 00023

GOTERM MF GO0005201 Extracellular matrix structuralconstituent 7 165 304 00082

GOTERM MF GO0005262 Calcium channel activity 7 165 332 00051GOTERM MF GO0008083 Growth factor activity 12 284 272 00016

GOTERM MF GO0008138 Protein tyrosineserinethreoninephosphatase activity 6 142 486 00014

GOTERM MF GO0008168 Methyltransferase activity 11 26 227 00097GOTERM MF GO0008276 Protein methyltransferase activity 6 142 377 0005GOTERM MF GO0042054 Histone methyltransferase activity 5 118 405 00075

Table 4 Ankylosing spondylitis KEGG pathway analysis result

Groupcompared KEGG pathway ID and name Count of

genesPercent of count

of genes Gene name 119875 value

Normalenvironment

Hsa04010 MAPK signalingpathway 2 030 CACNA2D3 PPM1B 012000

Hsa04620 toll-like receptorsignaling pathway 1 015 TLR6 020000

Inflammatoryenvironment

Hsa04010 MAPK signalingpathway 14 079

GNA12 DUSP1 MAPK11 RAC1MRAS ELK1 ATF4 MAPK3

CDC25B STMN1 MAX TGFBR2CACNB3 FGF8 and RASA2

000000

Hsa04620 toll-like receptorsignaling pathway 8 045 RAC1 TRAF3 MAPK3 IFNAR1

TICAM1 IL6 MAPK11 and IFNA1 000002

nucleic acid binding structure-specific DNA binding RNAbinding and hexosaminidase activity Furthermore KEGGpathway analysis showed that MAPK and TLR signalingpathways were significantly associated with MSCs from ASpatients in an inflammatory environment

Some early research has confirmed that TNF induces aresponse in MSCs by activating MAPK-dependent mecha-nisms [18] In addition whole blood transcriptional profilingof AS patients has also revealed inflammatory candidategenes including MAPK [19] We have to consider that underinflammatory conditions the MAPK signaling pathway may

play a key role in MSCs of AS patients In this study 14MAPK signaling genes were differentially expressed includ-ing GNA12 DUSP1 MAPK11 RAC1 MRAS ELK1 ATF4MAPK3 CDC25B STMN1 MAX TGFBR2 CACNB3FGF8 and RASA2 (119875 lt 001) Some of these geneshave been reported to have a relationship with the immunesystem or even with inflammatory autoimmune diseases Forexample GNA12 has been demonstrated to play a key rolein barrier function in the development of ulcerative colitis[20] DUSP1 (also referred to as MKP1) has been shown to bea critical negative regulator of inflammatory cytokines [21]


Table 5 Key gene list of signaling pathways related to AS

Signalingpathway

NCBI geneID

Genename

Fold changeabsolute 119875 value Regulation Description

MAPK 1843 DUSP1 389 002027 Up Dual specificity phosphatase 1

MAPK 2768 GNA12 370 002885 Up Guanine nucleotide binding protein (G protein)alpha 12

MAPK 5879 RAC1 315 000019 Up RAS-related C3 botulinum toxin substrate 1 (rhofamily small GTP binding protein Rac1)

MAPK 22808 MRAS 314 000491 Up Muscle RAS oncogene homologMAPK 2002 ELK1 280 001115 Up ELK1 member of ETS oncogene familyMAPK 5595 MAPK3 273 001417 Up Mitogen-activated protein kinase 3MAPK 994 CDC25B 266 003507 Up Cell division cycle 25B

MAPK 468 ATF4 250 004995 Up Activating transcription factor 4 (tax-responsiveenhancer element B67)

MAPK 3925 STMN1 247 001463 Up Stathmin 1oncoprotein 18MAPK 4149 MAX 231 000410 Up MYC associated factor X

MAPK 7048 TGFBR2 218 003642 Up Transforming growth factor beta receptor II(7080 kDa)

MAPK 5600 MAPK11 212 004901 Up Mitogen-activated protein kinase 11

MAPK 784 CACNB3 204 003028 Up Calcium channel voltage-dependent beta 3subunit

MAPK 2253 FGF8 349 003611 Down Fibroblast growth factor 8 (androgen-induced)MAPK 5922 RASA2 283 001406 Down RAS p21 protein activator 2TLR 7187 TRAF3 286 003732 Up TNF receptor-associated factor 3TLR 3454 IFNAR1 237 001055 Up Interferon (alpha beta and omega) receptor 1TLR 148022 TICAM1 223 002105 Up Toll-like receptor adaptor molecule 1TLR 3569 IL6 218 001526 Up Interleukin 6 (interferon beta 2)

TLR 5879 RAC1 315 000019 Up RAS-related C3 botulinum toxin substrate 1 (rhofamily small GTP binding protein Rac1)

TLR 5595 MAPK3 273 001417 Up Mitogen-activated protein kinase 3TLR 5600 MAPK11 212 004901 Up Mitogen-activated protein kinase 11TLR 3439 IFNA1 262 003022 Down Interferon alpha 1

Increased expression of DUSP1 in group AS-Infla may be theresult of systemic inflammation in AS patients MoreoverRAC1 activation can be directly induced by IL17A [22] andRAC1 inhibitory peptide has been found to suppress Tcell activation and autoantibody production in autoimmunedisease [23] MAPK11 (also referred to as p38BETA) is oneof the four members of the p38MAPK family (p38alphap38beta p38gamma and p38delta) A large body of evi-dence now indicates that p38MAPK activity is critical fornormal immune and inflammatory responsesThep38MAPKpathway is a key regulator of proinflammatory cytokinebiosynthesis at both the transcriptional and translational lev-els making components of this pathway attractive potentialtargets for the treatment of autoimmune and inflammatorydiseases [24] Furthermore p38MAPK inhibitors have beenverified to be efficacious in animal models of rheumatoidarthritis such as collagen-induced arthritis in the mouse andadjuvant-induced arthritis in the rat [25 26] Our study hasreported that the above-mentioned genes were significantlyupregulated in MSCs from AS patients suggesting a rolein the pathogenesis of ankylosing spondylitis However

the relationship between these differentially expressed genesand ankylosing spondylitis has not been previously reported

It is well known that upregulation of TLR depends onMAPK signaling [27] Our present study found that eightkey genes from the TLR signaling pathway (including RAC1TRAF3 MAPK3 IFNAR1 TICAM1 IL6 MAPK11 andIFNA1) were significantly differentially expressed (119875 lt 001)Some previous studies have reported that IL6may participatein the pathogenesis of rheumatoid arthritis (RA) and severalclinical trials have been carried out evaluating safety and effi-cacy of Tocilizumabmdashan anti-IL6 antibodymdashin RA patients[28] Increased IL6 expression in AS patients observed inour study may provide a helpful clue in illustrating itspathogenesis In addition tumor necrosis factor receptor-associated factor 3 (TRAF3) is a negative regulator of IL17receptor (IL17R) signaling TRAF3 suppresses IL17-inducedNF-120581B and mitogen-activated protein kinase activation andthe subsequent production of inflammatory cytokines andchemokines [29] Increased expression of TRAF3 observedin group AS-Infla may be due to persistent inflammationin AS patients Moreover Kole et al demonstrated that


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Figure 3 Verification of differentially expressed genes ofMSCs in AS patients cultured in inflammatory environment RNAwas isolated fromMSCs of HD (119899 = 5) and AS patients (119899 = 5) after MSCs were cultured in inflammatory environment mimicked by TNF-120572 (10 ngmL) andIFN-120574 (10 ngmL) Comparison of the levels of differentially expressed genes in MSCs from both groups was based on quantitative real-timepolymerase chain reaction (qRT-PCR) Beta-actin was used as an internal control The results showed that when grown in inflammatoryculture medium 9 of 22 differentially expressed genes from whole genomic expression analysis in AS patients were truly differentiallyexpressed These genes include GNA12 DUSP1 MAPK11 RAC1 MAPK3 TRAF3 IFNAR1 IL6 and IFNA1 The symbol ldquolowastrdquo represents119875 lt 005

IFN-1 plays an essential role in producing anti-inflammatorycytokines and maintaining intestinal T cell homeostasis byboth limiting effector T cell expansion and promoting Tregstability [30] Using a knockout mouse model De Weerdet al found that lipopolysaccharide-induced sepsis was ame-liorated in Ifnar1minusminus mice but not Ifnar2minusminus mice whichsuggests IFNAR1-IFN-120573 signaling plays a pathological role ininflammatory disease [31] This study found that IFNA1 wassignificantly reduced while IFNAR1 was enhanced in MSCsderived from AS patients cultured in an inflammatory envi-ronment which accounts for the increased proinflammatoryability and reduced anti-inflammatory status of AS patients

5 Conclusions

Our present study confirmed that the pathogenesis of ASstems from multiple gene interactions and key signalingpathway activation Our study found that an inflammatory

environment may activate the MAPK and TLR signalingpathways in MSCs derived from AS patients and causesupregulation of inflammatory gene expressionThis data pro-vides suggestive clues in exploring the pathogenesis of anky-losing spondylitis Finally further verification and validationof these differentially expressed genes in the pathogenesis ofAS will be needed in future research

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yuxi Li and Peng Wang contributed equally to this work


Acknowledgments

This work is supported by the National Natural Sci-ence Foundation of China (81271951 81401850 81101392)Guangdong Natural Science foundation (S2012010009654S2012010008927 10151008901000076) and the GuangzhouScience and Technology Foundation (2014J4100167) Theauthors thank Mingliang Ren for laboratory and technicalsupport In addition the authors are grateful to all colleaguesin the Centre for Biotherapy for supplying cells and coordi-nation

References

[1] J Braun and J Sieper ldquoAnkylosing spondylitisrdquoThe Lancet vol369 no 9570 pp 1379ndash1390 2007

[2] R A Colbert T M Tran and G Layh-Schmitt ldquoHLA-B27misfolding and ankylosing spondylitisrdquoMolecular Immunologyvol 57 no 1 pp 44ndash51 2014

[3] S Keidel L Chen J Pointon and P Wordsworth ldquoERAP1 andankylosing spondylitisrdquo Current Opinion in Immunology vol25 no 1 pp 97ndash102 2013

[4] T Rashid and A Ebringer ldquoAnkylosing spondylitis is linked toKlebsiellamdashthe evidencerdquo Clinical Rheumatology vol 26 no 6pp 858ndash864 2007

[5] J D Taurog J A Richardson J T Croft et al ldquoThe germfreestate prevents development of gut and joint inflammatory dis-ease in HLA-B27 transgenic ratsrdquo The Journal of ExperimentalMedicine vol 180 no 6 pp 2359ndash2364 1994

[6] J F Zambrano-Zaragoza J M Agraz-Cibrian C Gonzalez-Reyes M D J Duran-Avelar and N Vibanco-Perez ldquoAnky-losing spondylitis from cells to genesrdquo International Journal ofInflammation vol 2013 Article ID 501653 16 pages 2013

[7] P Luz-Crawford M Kurte J Bravo-Alegrıa et al ldquoMesenchy-mal stem cells generate a CD4+CD25+Foxp3+ regulatory T cellpopulation during the differentiation process of Th1 and Th17cellsrdquo StemCell ResearchampTherapy vol 4 no 3 article 65 2013

[8] S Ghannam J Pene and G Torcy-Moquet ldquoMesenchymalstem cells inhibit human Th17 cell differentiation and functionand induce a T regulatory cell phenotyperdquo Journal of Immunol-ogy vol 185 no 1 pp 302ndash312 2010

[9] Y Wu M Ren R Yang et al ldquoReduced immunomodulationpotential of bone marrow-derived mesenchymal stem cellsinduced CCR4+CCR6+ThTreg cell subset imbalance in anky-losing spondylitisrdquoArthritis Research andTherapy vol 13 no 1article R29 2011

[10] P Wang Y Li L Huang et al ldquoEffects and safety of allogenicmesenchymal stem cells intravenous infusion in active ankylos-ing spondylitis patients who failed NSAIDs a 20 week clinicaltrialrdquo Cell transplantation vol 23 no 10 pp 1293ndash1303 2014

[11] C Zhang and L Shen ldquoFunctional modules analysis basedon protein-protein network analysis in ankylosing spondylitisrdquoEuropean Review for Medical and Pharmacological Sciences vol16 no 13 pp 1821ndash1827 2012

[12] L Xu Q Sun S Jiang J Li C He and W Xu ldquoChanges ingene expression profiles of the hip joint ligament of patientswith ankylosing spondylitis revealed by DNA chiprdquo ClinicalRheumatology vol 31 no 10 pp 1479ndash1491 2012

[13] H Ma D Xu and Q Fu ldquoIdentification of ankylosingspondylitis-associated genes by expression profilingrdquo Interna-tional Journal of Molecular Medicine vol 30 no 3 pp 693ndash6962012

[14] P R Burton D G Clayton L R Cardon et al ldquoAssociationscan of 14500 nonsynonymous SNPs in four diseases identifiesautoimmunity variantsrdquo Nature Genetics vol 39 pp 1329ndash13372007

[15] M A Brown ldquoGenetics and the pathogenesis of ankylosingspondylitisrdquoCurrent Opinion in Rheumatology vol 21 no 4 pp318ndash323 2009

[16] J Sieper M Rudwaleit X Baraliakos et al ldquoThe Assessmentof SpondyloArthritis international Society (ASAS) handbooka guide to assess spondyloarthritisrdquo Annals of the RheumaticDiseases vol 68 supplement 2 pp ii1ndashii44 2009

[17] D C Colter R Class C M DiGirolamo and D J ProckopldquoRapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrowrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 97 no 7 pp 3213ndash3218 2000

[18] M Wang P R Crisostomo C Herring K K Meldrum andD R Meldrum ldquoHuman progenitor cells from bone marrowor adipose tissue produce VEGF HGF and IGF-I in responseto TNF by a p38 MAPK-dependent mechanismrdquoThe AmericanJournal of PhysiologymdashRegulatory Integrative and ComparativePhysiology vol 291 no 4 pp R880ndashR884 2006

[19] FM Pimentel-Santos D LigeiroMMatos et al ldquoWhole bloodtranscriptional profiling in ankylosing spondylitis identifiesnovel candidate genes that might contribute to the inflamma-tory and tissue-destructive disease aspectsrdquo Arthritis ResearchandTherapy vol 13 no 2 article R57 2011

[20] C W Lees J C Barrett M Parkes and J Satsangi ldquoNew IBDgenetics common pathways with other diseasesrdquo Gut vol 60no 12 pp 1739ndash1753 2011

[21] Q Zhao X Wang L D Nelin et al ldquoMAP kinase phosphatase1 controls innate immune responses and suppresses endotoxicshockrdquo Journal of ExperimentalMedicine vol 203 no 1 pp 131ndash140 2006

[22] E M Moran M Connolly W Gao J McCormick U Fearonand D J Veale ldquoInterleukin-17A induction of angiogenesiscell migration and cytoskeletal rearrangementrdquo Arthritis andRheumatism vol 63 no 11 pp 3263ndash3273 2011

[23] J R F Abreu W Dontje S Krausz et al ldquoA Rac1 inhibitorypeptide suppresses antibody production and paw swelling inthe murine collagen-induced arthritis model of rheumatoidarthritisrdquo Arthritis Research and Therapy vol 12 no 1 articleR2 2010

[24] A Cuenda and S Rousseau ldquop38 MAP-Kinases pathway reg-ulation function and role in human diseasesrdquo Biochimica etBiophysica Acta Molecular Cell Research vol 1773 no 8 pp1358ndash1375 2007

[25] A M Badger J N Bradbeer B Votta J C Lee J L Adamsand D E Griswold ldquoPharmacological profile of SB 203580 aselective inhibitor of cytokine suppressive binding proteinp38kinase in animal models of arthritis bone resorption endo-toxin shock and immune functionrdquo The Journal of Pharmacol-ogy andExperimentalTherapeutics vol 279 no 3 pp 1453ndash14611996

[26] A M Badger D E Griswold R Kapadia et al ldquoDisease-modifying activity of SB 242235 a selective inhibitor ofp38 mitogen-activated protein kinase in rat adjuvant-induced


arthritisrdquo Arthritis and Rheumatism vol 43 no 1 pp 175ndash1832000

[27] J-H Chang J-Y Park and S-K Kim ldquoDependence on p38MAPK signalling in the up-regulation of TLR2 TLR4 andTLR9gene expression in Trichomonas vaginalis-treated HeLa cellsrdquoImmunology vol 118 no 2 pp 164ndash170 2006

[28] I Al-Shakarchi N J Gullick and D L Scott ldquoCurrent perspec-tives on tocilizumab for the treatment of rheumatoid arthritisa reviewrdquo Patient Preference and Adherence vol 7 pp 653ndash6662013

[29] S Zhu W Pan P Shi et al ldquoModulation of experimentalautoimmune encephalomyelitis through TRAF3-mediated sup-pression of interleukin 17 receptor signalingrdquo The Journal ofExperimental Medicine vol 207 no 12 pp 2647ndash2662 2010

[30] A Kole J He A Rivollier et al ldquoType I IFNs regulate effectorand regulatory T cell accumulation and anti-inflammatorycytokine production duringT cell-mediated colitisrdquoThe Journalof Immunology vol 191 no 5 pp 2771ndash2779 2013

[31] N ADeWeerd J P Vivian T KNguyen et al ldquoStructural basisof a unique interferon-120573 signaling axismediated via the receptorIFNAR1rdquo Nature Immunology vol 14 no 9 pp 901ndash907 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


HLA-B27 accounts for only sim45 of the genetic risk for ASTherefore in this study we focused mainly on the significantrole of MSCs in AS pathogenesis in order to provide a newviewpoint on this inflammatory disease Our study investi-gated whether MSCs grown in vitro from AS patients exhibitgene expression differences in normal culture media or in aninflammatory environment as compared to healthy controlsWe found that mimicking an inflammatory environment canactivate the MAPK and TLR signaling pathways in MSCsderived fromAS patients thereby upregulating inflammatorygene expression This data provides suggestive clues in theexploration of the pathogenic mechanism behind ankylosingspondylitis

2 Materials and Methods

21 Patients and Controls Thepresent study was approved bythe Ethics Committee of the Sun Yat-sen Memorial Hospitalof SunYat-senUniversity GuangzhouChina From June 2012to December 2012 twelve healthy donors (HD 9 men and3 women) with an average age of 221 years and twelve ASpatients (10 men and 2 women) with an average age of 219years were enrolled in this study (Table 1) The diagnosesof AS patients were all performed according to the ASASclassification criteria [16] In addition all patients involvedwere diagnosed for the first time and remained in active stage(all Bath Ankylosing Spondylitis Disease Activity Index ge 4)

22 Isolation and Preparation of MSCs After being informedabout scientific significance possible risks and complicationsand the treatment measures for bone marrow aspirations alldonors and patients signed the informed consent and wereaspirated by our skilled allied health professionals in strictaccordance with the international standardized procedureBone marrow MSCs were isolated from the bone marrowaspirates by density gradient centrifugation as previouslydescribed by our work and that of others [9 17] AfteridentifyingMSC immunophenotype markers by flow cytom-etry passages three to five were used for the experimentsdescribed below Both HD and AS groups were separatedinto two subgroups MSCs cultured in normal media (Norm)and MSCs cultured in an inflammatory environment (Infla)which was created by adding TNF-120572 (10 ngmL) and IFN-120574(10 ngmL) into normal culture media for four hours Fiverepresentatives of cultured MSCs from HD and AS patientswere randomly chosen cultured in both types of media andfurther analyzed Twenty samples were chosen to completewhole genome expression profiles 5 HD-Norm 5 HD-Infla5 AS-Norm and 5 AS-Infla samples

23 Total RNA Extraction and Quality Control 1mL Trizol(Invitrogen Carlsbad USA) was added to lyse the collectedcells Total RNA was extracted using the phenolchloroformmethod and salt was washed away with 70 alcohol Afterair-drying at 15sim30∘C the appropriate amount of RNase-free water was added to dissolve RNA The integrity andconcentration of RNA were assessed to ensure accuracyand reliability Total RNA from each sample was quantified

Table 1 Demographic data and disease characteristics of enrolledpatients and healthy donors

Items AS HDNumbers 12 12SexMen 10 9Women 2 3

Age (years) 229 (20ndash30) 231 (21ndash31)Body weight (Kg) 633 (58ndash65) 635 (60ndash66)Disease duration from diagnosis(months)

339(28ndash396) 0

HLA-B27 11 (917) 0History of extra-axial involvement(number of patients)Uveitis 0 0Psoriasis 0 0Dactylitis 2 (167) 0Inflammatory bowel disease 1 (83) 0Enthesitis 5 (417) 0

using a NanoDrop 1000 and RNA integrity was assessed bystandard denaturing agarose gel electrophoresis About 5 120583gtotal RNA from each sample was used for labeling and arrayhybridization following these steps (1) Reverse transcriptionby Invitrogen Superscript ds-cDNA synthesis kit (2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit(3) array hybridization using the NimbleGen HybridizationSystem followed by washing with the NimbleGen washbuffer kit (4) array scanning using the Axon GenePix 4000Bmicroarray scanner (Molecular Devices Corporation)

24 Microarray Scanning andData Analysis Scanned imageswere then imported into NimbleScan software (version 25)for grid alignment and expression data analysis Data werenormalized by quantile normalization and the robust multi-chip average (RMA) algorithm included in the NimbleScansoftware Probe level files and gene level files were generatedafter normalizationThe twenty gene level files were importedinto Agilent GeneSpring GX software (version 115) forfurther analysis After GeneSpring quantile normalizationdifferentially expressed genes were identified using VolcanoPlot filtering KEGG pathway analysis and GO analysis wereapplied to determine the roles of these differentially expressedgenes in biological pathways or GO terms Finally the posthoc Bonferroni test was used to correct for multiplicity sincea large number of false positive 119875 values were observed 119875values lt001 were considered significant

25 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) After MSCs were cultured in inflammatory envi-ronment mentioned above total RNA was extracted usingTrizol reagent (Life Technologies Inc USA) Synthesis ofcDNA (RT-PCR) was carried out using the SuperScriptH IIIFirst-Strand Synthesis System (Invitrogen) according to themanufacturerrsquos instructions The quantitative real-time PCR




3 Results








(a)

250K

250K

200K

200K

150K

150K

100K

100K

50K

50K0

0

SSC-

A

P1

FSC-A

(b)

Cou

nt

CD45

0

0 102

103

104

105

HLA-DR

Cou

nt0

0 102

103

104

105

Cou

nt

0

0 102

103

104

105

CD34

CD29

Cou

nt

0

0 102

103

104

105

CD44

Cou

nt

0

0 102

103

104

105

CD105

Cou

nt

0

0 102

103

104

105

(c)





14

12

12

10

8

6

6 8 10 14

14

12

12

10

8

6

6 8 10 14

Gro

up H

D-I

nfla

Gro

up H

D-N

orm


(a)

GO other items





GO0005488 binding











stimulusGO0032502




GO0005623 cell

(b)













4 Discussion


















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




3 Results








(a)

250K

250K

200K

200K

150K

150K

100K

100K

50K

50K0

0

SSC-

A

P1

FSC-A

(b)

Cou

nt

CD45

0

0 102

103

104

105

HLA-DR

Cou

nt0

0 102

103

104

105

Cou

nt

0

0 102

103

104

105

CD34

CD29

Cou

nt

0

0 102

103

104

105

CD44

Cou

nt

0

0 102

103

104

105

CD105

Cou

nt

0

0 102

103

104

105

(c)





14

12

12

10

8

6

6 8 10 14

14

12

12

10

8

6

6 8 10 14

Gro

up H

D-I

nfla

Gro

up H

D-N

orm


(a)

GO other items





GO0005488 binding











stimulusGO0032502




GO0005623 cell

(b)













4 Discussion


















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)

250K

250K

200K

200K

150K

150K

100K

100K

50K

50K0

0

SSC-

A

P1

FSC-A

(b)

Cou

nt

CD45

0

0 102

103

104

105

HLA-DR

Cou

nt0

0 102

103

104

105

Cou

nt

0

0 102

103

104

105

CD34

CD29

Cou

nt

0

0 102

103

104

105

CD44

Cou

nt

0

0 102

103

104

105

CD105

Cou

nt

0

0 102

103

104

105

(c)





14

12

12

10

8

6

6 8 10 14

14

12

12

10

8

6

6 8 10 14

Gro

up H

D-I

nfla

Gro

up H

D-N

orm


(a)

GO other items





GO0005488 binding











stimulusGO0032502




GO0005623 cell

(b)













4 Discussion


















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


14

12

12

10

8

6

6 8 10 14

14

12

12

10

8

6

6 8 10 14

Gro

up H

D-I

nfla

Gro

up H

D-N

orm


(a)

GO other items





GO0005488 binding











stimulusGO0032502




GO0005623 cell

(b)













4 Discussion


















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












4 Discussion


















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Normalenvironment







000000








Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Signalingpathway

NCBI geneID

Genename


















6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


6

5

4

3

2

1

0

GNA12

lowast

Relat

ive m

RNA

expr

essio

n

HD AS

6

5

4

3

2

1

0

DUSP1lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK11lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

TRAF3lowast

Relat

ive m

RNA

expr

essio

n

HD AS

5

4

3

2

1

0

MAPK3

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

6

5

4

3

2

1

0

RAC1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IFNAR1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

5

4

3

2

1

0

IL6

Relat

ive m

RNA

expr

essio

n

HD AS

lowast

3

2

1

0

IFNA1

Relat

ive m

RNA

expr

essio

n

HD AS

lowast



5 Conclusions








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Whole Genome Expression Profiling and Signal Pathway …downloads.hindawi.com/journals/sci/2014/913050.pdf · 2019-07-31 · Research Article Whole Genome Expression