Study On The Role Of N6-methyladenosine （m6A） Modificated Long Noncoding RNA In Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus（SLE）is a multifaceted autoimmune disease characterized by the production of autoantibodies and the aberrant immune response.The onset of the disease is insidious,the disease is constantly delayed,and prognosis is poor.However,the pathogenesis of SLE is still unknow.In recent years,epigenetic modification has provided new ideas for the pathogenesis of SLE.Among them,N6-methyladenosine（m6A）modification and long non-coding RNA（lnc RNA）have been confirmed to be involved in SLE disease progression by affecting the biological function of immune cells.At the same time,m6 A modification could be involved in gene regulation of lnc RNA through various mechanisms,and involved in the processes of vascular inflammation,abdominal aortic aneurysm,colorectal cancer and other disease.However,the effect of m6 A modification related lnc RNA on SLE is very limited and needs to be further studied.ObjectiveTo screen N6-methyladenosine（m6A）related long noncoding RNAs（lnc RNAs）based on Methylated RNA Immunoprecipitation sequencing（Me RIP-seq）data.The differentially expressed lnc RNAs in T cells of SLE were validated,and the effect of lnc RNAs on T cell stability was validated.MethodsIn this case-control design study,lnc RNAs with different m6 A modification levels were screened by using established Me RIP-seq data.Subsequent experiments were divided into two parts.Part 1: To validate the expression of m6A-related lnc RNAs in T cellsThe gene expression of m6A-related lnc RNAs were examined in T cells from SLE patients（n=70）and controls（n=82）by RTq-PCR.The associations of lnc RNAs expression levels with clinical manifestations,disease activity,antibody indicators and medication of SLE patients were analyzed.Then LINC00342 was selected for cell functional verification.Part 2: The effect of LINC00342 on T cells function in vitroLINC00342 overexpression Jurkat cells were constructed by using pc SLenti-p ALINC00342-CMV-SFH-EGFP-P2A-Puro-WPRE virus.The cell proliferation in LINC00342 overexpressed group and negative control group were quantified by using CCK-8（Cell Counting Kit-8）.After Annexin V labeled with Allophycocyanin（APC）and7-amino-Actinomycin D（7-AAD）double staining,the apoptosis of LINC00342 overexpression group and negative control group was detected with flow cytometric analysis.ResultsIn this study,after analyzed Me RIP-seq data,a total of 7 lnc RNAs with different m6 A modification levels in SLE were preliminarily screened（LINC01578,C1orf132,XIST,PSMB8-AS1,SNH68,LINC00342 and EBLN3P）.Compared with the control group,the expression levels of LINC01578,C1orf132,XIST,PSMB8-AS1,SNH68,LINC00342 and EBLN3 P were down-regulated in SLE patients（all P<0.05）.After grouping the clinical information of patients,we found that LINC00342 expression was down-regulated in patients with systemic lupus erythematosus disease activity index（SLEDAI）> 4,abnormal erythrocyte sedimentation rate（ESR）and thrombocytopenia（all P<0.05）.The expression level of XIST decreased in SLEDAI > 4（Z=-2.316,P=0.021）group and ESR abnormality（Z=-3.349,P=0.001）group.LINC01578,C1orf132,PSMB8-AS1 and SNH68 were down-regulated in patients with abnormally elevated ESR（all P<0.05）.The gene expression level of EBLN3 P decreased in patients with thrombocytopenia（Z=-2.095,P=0.036）,and EBLN3 P was upregulated in patients with hormones（Z=-2.223,P=0.026）.Cell experiments results showed that the apoptosis rate of LINC00342 overexpression group was lower than the controls（t=-9.520,P=0.001）,but no difference in proliferation ability.ConclusionsIn this study,it was found that m6 A modification levels of LINC01578,C1orf132,XIST,PSMB8-AS1,SNH68,LINC00342 and EBLN3 P changed in SLE patients and all down-regulated in T cells.Further,LINC00342 expression was down-regulated in patients with SLEDAI > 4,abnormal ESR and thrombocytopenia.Meanwhile,the apoptosis rate was decreased in LINC00342 over-expressing Jurkat cells.It is suggested that m6A-related lnc RNAs may be involved in the progression of SLE disease.LINC00342 may worsen the progression of SLE by regulating apoptosis of T cell,but the specific mechanism remains to be further studied.