Genome-wide Methylation Regulatory Network Construction And Functional Mechanism Research In Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a complex autoimmune disease with chronic articular synovial inflammation as a characteristic pathological change.So far,the pathogenesis of RA is not entirely clear.Many researchers believe that at least 50%risk of RA is fromgenetic factors.Genetics researches conducted by candidate gene association studies and genome-wide association studiesraised in recent years have been accumulating evidence of the importance of genetic factors in RA.Therefore,clarifying the molecular genetic mechanism is advantageous to the prevention and therapy of RA.Unfortunately,the combined RA heritability explained by the identified genetic loci is still very small,illustrating that there are a lot of genetic factor waiting to be found.DNA methylation is a hotspot in epigenetics study.Epigenetics is a genetic phenomenon beyond Mendel law,that is,stable heritable traits of the genome that do not involve a change in the nucleotide sequence.DNA methylation is a process by which methyl groups are added to the DNA molecule under the catalysis of the DNA methyltransferase.It plays an important role in maintaining the stability of tissue specific expression spectrum,the regulation of chromatin,imprinting,X chromosome inactivation,gene expression,suppression of the expression of virus and foreign genes.DNA methylation is closely related to many complex diseases or life processes.In recent years,studies showed that DNA methylation may also be involved in pathogenesis of RA.With the support of modern high-throughput biochip technology,scientists can overcome the limitations of previous candidate gene studies.Using a non-hypothetical research strategy,they can conduct genome-wide methylation studies of RA to extensively screen differential methylation loci(DML)and to find many more candidate genes,chromosome areas and signaling pathways,thus provide more clues for biomarkers screening,pathogenesis exploring and new therapies development.At present,the human genome research has entered the stage of functional genomics.Moreand more researchers realize the importance of integrated study from genomics,epigenome,transcriptome and proteomics.The advantages of systemic research strategy lie in the effective use of multi-level biological information to verify or to find specific susceptibility genes of complex diseases.By further clarifying the biological function of genes/genome,it would provide us new ideas for prevention and treatment of complex diseases.This study used double omics data of genome-wide methylation and expression to construct regulatory networkof differential methylation genes(DMGs)in RA.The differential methylation of DMGs and their differential expression between RA and heath control were respectively validated in two independent samples.We also selecteda key gene from the regulation network to explore its possible mechanisms of pathogenic role in RA by conducting a series of molecular functional tests in cell cycle,cell proliferation and the expression of inflammatory factors.Chapter One Screening RA specific genome-wide Methylation loci and the regulatory network of DMG constructionObjective:To detect the comprehensive genome-wide methylation sites and build regulatory network of differential methylation genes(DMG)in RA.Methods:The peripheral blood mononuclear cells of 25 RA patients and 18 healthy controls were used to detect genome-wide methylation status and gene expression by 450K Infinium Methylation arrays and IncRNA/mRNA expression BeadChip,respectively.On the basis of correlation analysis of methylation and gene expression,we built the regulatory network of DMGby application of bioinformatics analysis such as STRING,CYTOSCAPE and the Causal Inference Test.Results:450K methylation array identified 1046 differential methylation loci(DML)in RA patients,among which there were 730 DML in gene region and they belong to 598 unique genes.The methylation level of 107 DML(corrrespongding to 91 unique genes)was significantly correlated with gene expression(P<0.05).An interferon inducible gene interaction network was constructed in which MX1?IFI44L?DTX3L?PARP9 were the key differential methylated genes.Conclusions:There are distinctive genome-wide methylation pattern in RA patients compared with health controls.DNA methylation is a potential mediator of genetic risk in RA.The interferon-inducible-gene interaction network with PARP9,MX1,IFI44L and DTX3L as the key genesmight play an important role in RA pathology.Chapter Two Verification of RA-related differential methylation and differential expression in independent samplesObjective:To verify the differential methylation and differential expression of some genes between RA patients and health controls in independent samples.Methods:Ten candidate genes,PARP9?IFI44L?MX1?ISG15?FAM8A1?STK17A?BLK?CNPYI?CBX7?SLC7A14,were selected,most of which were in the RA-related regulatory network found by our chip tests.The DNA methylation status in objective region and the mRNA levels of these genes were tested in two independent samples(RA:Control=25:27 for differential methylation verification,RA:Control=35:35 for differential gene expression verification)using MethyltargetTM technology and RT-PCR,respectively.Results:The differential methylation locus/loci of three genes(BLK?MX1?PARP9),especially for three loci of PARP9,were verified in independent sample.BLK?STK17A and PARP9 were the differential methylation genes between RA and health control according the average methylation level in test region.The results of RT-PCR showed that the mRNA level of six genes,BLK?CBX7?IFI44L?MX1?PARP9?STK17A,was significantly different between RA and controls,and,the up-regulation or down-regulation of BLK?IFI44L?MX1?PARP9 in RA patients were the same with chip tests.Conclusions:The differential methylation and differential expression of some selected genes were respectively verified in two independent samples,indicating the reliability of array results.The regulatory network of differential methylation genes,in which the IF144L?MX1?PARP9 were key genes,might indeed play an important role in RA pathology.Chapter Three The mechanisms of PARP9 DNA methylation in RAObjective:To explore the possible pathological mechanisms of PARP9 DNA methylation in RA.Methods:Firstly,we treated Jurkat cells with 5-Aza for 72h,then cultured the cells without 5-Aza for 72h.We tested the methylation level of specific loci and gene expression level of PARP9 at different time-point(with 5-Aza 72h,without 5-Aza24h,48h and 72h),using methyltargetTM and RT-PCR,respectively.Then,we generated PARP9 over-expressed(OE)and shRNA-disturbed stable(shRNA)Jurkat cell strains to explore the influence of different PARP9 expression to cell cycle(flow cytometry),cell proliferation(CCK8)and the expression of RA-related inflammatory cytokinesIL-1??IL-2?IL-4?IL-6?IL8?TNF? and IFN?(RT-PCR).Results:In Jurkat cells,the methylation level of two loci of PARP9(POS.105/MAPINFO122281940 and POS.163/MAPINFO 122281882)showed significantly negative correlation with gene expression.The proliferation of OE Jurkat cell was significantly faster than that of OE-negative controls(P<0.05),the proliferation of shRNA Jurkat cell was significantly slower than that of shRNA-negative controls(P<0.05).Compared with negative control,the percentage of G0/G1 and S cells waslower than that of G2/M cell in OE Jurkat cells,but the percentage of G0/G1 cells was higher than that of S or G2/M cell in shRNA Jurkat cells.The expression level of IL-2 was positively regulated while IL-4 and IFN?y was negatively regulated by PARP9.The expression level of IL-8 and TNF-a seemed no difference under the conditions of PARP9 over-expression or low-expression.There were no effect on expression of IL-1? and IL-6 when PARP9 over expressed,but the lower expression of PARP9 significantly reduced the expression of IL-1? and IL-6.Conclusions:The abnormal methylation status of specific sites in PARP9 may mediate pathogenic effects in RA by changing gene expression and further influencing T cell cycle,cell proliferation and the expression of some important inflammatory cytokines.