Study On The Role Of Long Non-coding RNA Promoter Region Methylation In Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus(SLE)is a chronic inflammatory autoimmune disease,which tends to occur in women of childbearing age,and the incidence rate of male and female is about 1:9.Although existing researches have revealed some characteristics of human lupus,treatments for SLE still lag behind other autoimmune diseases.SLE is highly complex and heterogeneous,and its pathogenesis is not fully understood.Long non-coding RNA(lncRNA)refers to non-coding RNA with a length of more than 200 nucleotides,which plays an important role in immune cell differentiation and activation through interaction with DNA,RNA and proteins.More and more evidence suggests that lncRNA can be used as a new treatment target and disease biological marker in SLE.Previous studies have shown abnormal expression of a large number of lncRNAs in SLE,which are related to the clinical characteristics and disease activity of SLE.However,current studies on the occurrence and development of lncRNA in SLE are still limited,and the cause of abnormal expression of lncRNA in SLE is also unknown.Promoter region DNA methylation can regulate RNA transcription and further participate in the pathogenesis of a variety of diseases,and is expected to be a biomarker for disease diagnosis and prognosis.However,there are few reports on promoter region methylation regulating lncRNA in SLE and other autoimmune diseases.In fact,lncRNA promoter region methylation has been reported to be involved in the pathogenesis of other diseases by regulating lcRNA expression.T lymphocytes play a central role in cell-mediated immune responses,and multiple defects have been observed in T cells in SLE patients.Therefore,this study speculated that the methylation of lncRNA promoter region may be involved in the disease process of SLE by regulating lncRNA expression.This study attempted to find lncRNA regulated by DNA methylation modification and closely related to the pathogenesis of SLE,and to study its function and mechanism of action.ObjectiveThe purpose of this study was to establish the lncRNA expression profile and DNA methylation profile of T cells in SLE patients by using human whole transcriptome sequencing and 850K-methylation chip technology.Comprehensive analysis was performed to screen out lncRNA related to DNA methylation modification,and verified the expression level of lncRNA in a large sample population experiment.The association between differentially expressed lncRNA and clinical characteristics of SLE cases and receiver operating characteristic curve(ROC)of each lncRNA were also discussed.Finally,the differentially expressed candidate lncRNAs related to DNA methylation modification were verified for cell function.This study preliminarily revealed the related mechanisms of SLE disease process from the perspective of upstream promoter region and downstream coding genes of lncRNA.It is expected to discover new SLE specific molecular biomarkers and therapeutic targets,and provide important scientific basis for screening and disease prevention and control of SLE high-risk population.MethodsPhase 1:In this study,3 pairs of RNA and DNA of T lymphocytes of SLE new cases and healthy controls were detected by human whole transcriptome sequencing technology and 850K-methylation chip,and mRNA expression profile,non-coding RNA expression profile(including lncRNA expression profile)and DNA methylation profile were established.LncRNA with abnormal expression that may be related to DNA methylation modification was preliminarily screened.Phase 2:qRT-PCR was used to validate the 8 candidate lncRNAs in preliminary screening in 101 SLE cases and 117 healthy control T cells,and the association between the above 8 lncRNAs and clinical characteristics of SLE was analyzed.ROC curve was used to evaluate the diagnostic value of the above differentially expressed lncRNAs in SLE.Phase 3:The Jurkat(Clone E6-1)cell line was constructed,and the effects of methylation level changes in promoter region on lncRNA expression and T cell function were explored using 5-AZa-2 '-Deoxycytidine(5-AZa-DC),a DNA methylation inhibitor of methylation.Phase 4:In vitro cell function experiments,the stable interference of MALAT-1 and lnc-SERPINB9-1 genes in T cells were achieved by lentivirus interference vector and Jurkat(Clone E6-1)cells,respectively.For MALAT-1 gene,this study used Cell Counting Kit-8(CCK-8)and flow cytometry to detect the effects of MALAT-1 gene interference on cell proliferation,apoptosis and cell cycle.The effects of MALAT-1 gene on secretion of inflammatory cytokines were determined by enzyme linked immunosorbent assay(ELISA).The interaction mechanisms of MALAT-1,miR-2355-3p and SCIMP were predicted and verified by bioinformatics analysis and double luciferase gene reporter assay.Western blotting(WB)was used to investigate the effect of MALAT-1 gene on the expression of SCIMP.For lnc-SERPINB9-1 gene,the effects of lnc-SERPINB9-1 gene on cell proliferation and apoptosis were detected by CCK-8 and Annexin-V/PI double staining.ResultsIn this study,human whole transcriptome sequencing was used to identify 1486 differentially expressed lncRNAs in T cells of SLE patients and healthy controls,of which 800 lncRNAs were up-regulated and 686 lncRNAs were down-regulated in SLE patients.In T cells of SLE patients and healthy controls,the methylation of 1780 differentially methylated positions(DMP)were deceted significantly different by 850K-methylation chip,of which 1460 DMP sites were hypomethylated and 320 DMP sites were hypermethylated in SLE patients.Eight lncRNAs(lnc-EPSTI1-4,MIR4435-2HG,lnc-STYK1-2,lnc-SERPINB9-1,MALAT-1,TSPOAP1-AS1,ZMIZ1-AS1,lnc-ZNF138-2)that may be related to DNA methylation modification were screened out basing on human whole transcriptomic sequencing and 850K-methylation chip results.QRT-PCR results of T cells in 101 SLE patients and 117 healthy controls showed that the expression levels of the above eight lncRNAs were statistically different in SLE patients compared with the control group(all P<0.05).Among them,the expression of lnc-EPSTI1-4,MIR4435-2HG,lnc-STYK1-2,lnc-SERPINB9-1 and ZMIZ1-AS1(all P<0.05)increased in SLE patients.The expression of MALAT-1,TSPOAP1-AS1 and lnc-ZNF138-2(all P<0.05)decreased in T cells of SLE patients.The above lncRNAs were associated with the laboratory indicators of SLE disease activity and clinical manifestations and clinical medication,suggesting the important role of these lncRNAs in SLE disease process.Mann-whitney U nonparametric test was used to analyze the association between lncRNA expression levels in peripheral blood T cells of SLE patients and clinical medication.Statistically significant difference was found in the expression level of lnc-ZNF138-2 between SLE patients being treated with immunosuppressants and those without immunosuppressant(Z=-2.119,P=0.034),and no significant difference was observed between the expression of other lncRNAs and clinical treatments(P>0.05).ROC curve was used to analyze the validity of above lncRNA expression levels in T cells of SLE patients as diagnostic indicators of SLE.We found that the AUC of lnc-STYK1-2 and MALAT-1 were over 0.70,indicating certain diagnostic value.MALAT-1 had the largest area under the curve,reaching 0.7886 with a sensitivity of 66.34%and specificity of 83.76%.After DNA methylation inhibitor 5-Aza-dC interfered with Jurkat(Clone E6-1)cells,the expression levels of lnc-EPSTI1-4 MALAT-1 and lnc-SERPINB9-1 were significantly up-regulated compared with the control group(all P<0.05),and the methylation levels of target fragments in the three lncRNAs promoter regions decreased,but the differences were not statistically significant which indicated that the expression levels of lnc-EPSTI1-4,MALAT-1 and lnc-SERPINB9-1 may be regulated by methylation level in promoter region,but the specific regulatory mechanism was unclear,which deserved further exploration.In addition,cell function experiments showed that 5-Aza-dC could inhibit T cells proliferation and promote T cells apoptosis.In vitro cell function test results showed that compared with the negative control group,the proliferation ability of MALAT-1 gene interference group was significantly increased at 24 h,48 h and 72 h(all P<0.05).The proportion of apoptosis in MALAT-1 gene interference group decreased(P<0.05).In MALAT-1 gene interference group,G1 phase was prolonged and G2 phase was shortened(P<0.05),there was no significant difference in S phase(P>0.05).The expression levels of IL-6,IL-17 and TNF-? in MALAT-1 gene interference group were increased(P<0.05),and IFN-? expression level was decreased;Compared with the negative control group,miR-2355-3p expression level was significantly increased and SCIMP expression level was significantly decreased(P<0.05)in the MALAT-1 gene interference group.WB results showed that compared with the negative control group,SCIMP protein expression level in MALAT-1 gene interference group decreased,but there was no statistical significance between the two groups(P=0.061).The results of the double luciferase gene reporter experiment suggested that miR-2355-3p could target to MALAT-1 and SCIMP.The cell function experiment of lnc-SERPINB9-1 showed that compared with the negative control group,the cell proliferation ability of lnc-SERPINB9-1 gene interference group was significantly decreased at 24 h,48 h and 72 h(all P<0.001).The proportion of apoptosis increased in the lnc-SERPINB9-1 gene interference group(P<0.05).Conclusions(1)Multiple lncRNA expressions were abnormal in T cells of SLE patients,and extensive DNA hypommethylation was observed in T cells of lupus patients.lnc-EPSTI1-4,MIR4435-2HG,lnc-STYK1-2,lnc-SERPINB9-1,ZMIZ1-AS1 MALAT-1,TSPOAP1-ASland lnc-ZNF138-2 were abnormally expressed in T cells of SLE patients.(2)Methylation inhibitors can increase the expression levels of lnc-EPSTI1-4 MALAT-1 and lnc-SERPINB9-1 in Jurkat cells.The expression of these three lncRNAs may be related to the methylation level of lncRNA promoter region,but the specific regulatory mechanism was not clear.The relationship between lncRNA expression and SLE deserves further study.(3)MALAT-1 interference can promote T cells proliferation,inhibit T cells apoptosis,affect T cells cycle,and up-regulate the expression levels of inflammatory factors IL-16,IL-17 and TNF-?.MALAT-1 may regulate the expression level of SCIMP as a ceRNA via sponging miR-2355-3p.(4)The interference of lnc-SERPINB9-1 reduced T cells proliferation and promoted T cells apoptosis.