DNA Methylation And RNA Methylation In CD4~+T Cells From Lupus Nephritis

BackgroundSystemic lupus erythematosus(SLE)is an autoimmune disease caused by multiple factors and involving multiple systems.Lupus nephritis(LN)is the most common complication of systemic lupus erythematosus,occurring in approximately 50%of patients with SLE.Many scholars believed that complex diseases such as SLE and LN may be the result of interactions between genes and the environment,which may be largely regulated by epigenetic modifications.Epigenetic modification refers to gene expression modification independent of gene sequence changes,which can respond to environmental signals and regulate gene expression.At present,there are few studies on epigenetic modification of lupus nephritis.Therefore,this study will investigate epigenetic modifications among LN patients,SLE patients without kidney injury and healthy controls from the two perspectives of DNA methylation modification and RNA methylation modification to explore possible epigenetic modifications and biomarkers of LN in Chinese Han population.MethodsThis study is divided into two parts.In the first part we collected SLE patients without renal injury,LN patients,and Health control(HC)(n=8)who met the inclusion criteria between 2019and 2021.We collected 20m L of peripheral blood of enrolled patients for CD4~+T cell flow sorting,then DNA methylation chip sequencing and transcriptome sequencing were performed,respectively.Through bioinformatics analysis,the DNA methylation modification and expression characteristics of SLE and LN were obtained,as well as the sites and genes unique to LN with differential DNA methylation modification and differential expression.Then,bisulfite sequencing and vitro experiments were performed to verify our results.In the second part,we collected SLE patients without kidney injury(divided into inactive SLE group and active SLE group according to the Systemic Lupus Erythematosus Disease Activity Index),LN group,and HC group(n=5)from 2019 to 2021.We collected 20 m L peripheral blood for CD4~+T cells magnetic bead sorting and then RNA methylation chip sequencing was performed.Through bioinformatics analysis,we obtained the m~6A RNA methylation modification and expression characteristics of SLE and LN,as well as the unique sites and genes of LN with differential m~6A RNA methylation modifications and differential expression.Me RIP-q PCR were performed to verity our results and detected potential m~6A methylated sites for gene IFI27,CD36 and TMEM173.ResultsIn the first part,we obtained a total of 1021 differentially methylated DNA positions(DMPs)unique to LN.Combined analysis of DNA methylation modification and gene expression,45DMPs with differential DNA methylation and differential expression were unique in LN,which annotate 34 genes.Basically,same with sequencing results,cg08332381,cg03297029 and cg16797344 could be the potential biomarkers for LN.After the treatment of 5-Azacitidine,the expression of IFI27 and BCL2L14 are increased and the DNA methylation of IFI27 and BCL2L14 are decreased.In the second part,we obtained 572 differentially modified transcripts of m~6A RNA methylation that were unique to LN,which annotated 509 differentially modified genes.Combined analysis of m~6A RNA methylation modification and gene expression,we obtained the unique differential m~6A RNA methylation modification and differentially expressed genes in LN:77 differential transcripts(76 annotation genes)with increased m~6A and expression;107 differential transcripts(102 annotation genes)with increased m6A and down-regulated expression;186 differential transcripts(180 annotation genes)with reduced m~6A and up-regulated expression;31 differential transcripts(30 annotation genes)with reduced m~6A and down-regulated expression.After GO analysis and KEGG analysis,the unique differential genes in LN were mainly concentrated in the regulation of RNA polymerase II promoter and the Hippo signaling pathway.The results of Me RIP-q PCR(n=5)showed that the RNA methylation of SMAD3,BCL2 and IFI27 are almost same with the sequencing results.The possible m~6A modification sites of IFI27,CD36 and TMEM173 were detected by MERIP-q PCR.ConclusionIn this study,we conducted DNA methylation and m~6A RNA methylation studies in SLE patients and LN patients,and we also described the characteristics of DNA methylation and m~6A methylation modifications in SLE patients and LN patients.We got the potential biomarker for LN and the gene may be involved in the progression of LN.