Study On Roles Of N6-methyladenosine(m6A) Modification In Pathogenesis Of Systemic Lupus Erythematosus

BackgroundSystemic lupus erythematosus(SLE)is a complex chronic autoimmune disease,mediated by autoimmunity and characterized by abnormal production of multiple autoantibodies,multi-system and multi-organ involvement,and diversified clinical manifestations.The prognosis is poor,quality of life is low,fatality rate is high,and incidence rate is rising in patients with SLE,as well as this disease mostly affects women of childbearing age.However,the pathogenesis of SLE is still unknown.Studies on genetic factors have made great efforts to reveal the pathogenesis of SLE,but it is still not enough to explain complex clinical manifestations and treatment problems.In recent years,epigenetic modifications have provided a new perspective for exploring the pathological mechanisms of SLE,as well as the diagnosis,treatment and prevention of this disease.Many epigenetic mechanisms occur in SLE,such as DNA methylation modification mediating lymphocytes development,non-coding RNAs regulating immune cells,chromatin remodeling,histone modification,and etc.These epigenetic modifications cause heritable changes in gene function without changing any DNA sequence,and ultimately lead to changes in the phenotypes of disease.As with DNA modification and protein modification,disruption of N6-methyladenosine(m6A)modification,the mechanism that regulates post-transcriptional gene expression at the RNA level,may lead to abnormal RNA expression.The m6A modification is the methylation of the nitrogen atom at the sixth position of adenine in RNA,and can regulate its gene expression.This modification exists widely in human m RNAs,and it is catalyzed by specific methyltransferases and can be removed under the catalysis of demethylases.After the recognition proteins read the information contained in m6A modification,the modification information is converted into a functional signal.Under the combined action of these m6A methylases,the dynamic changes of methylation and demethylation of RNA happen,thereby regulating various biological functions.Most of m6A modification sites locate in important functional regions such as transcription initiation,internal coding regions,and 3'-UTR.Studies have shown that m6A modification was involved in many biological processes such as cell metabolism regulation,embryonic stem cell differentiation,circadian rhythm,and cell apoptosis.More and more evidence suggested that m6A modification was an important regulator of T cell homeostasis and immune response after bacterial or viral infection in the regulatory mechanism of immune response and inflammation regulatory network.In SLE,ALKBH5 has been reported to be an inflammatory response marker,and YTHDF2was related to SLE disease activity.However,it is still necessary to explore the mechanisms of m6A modification in SLE.m6A modification may play important roles in disease process,and even disease treatment and prognosis,and it is expected to become new intervention targets for the treatment and prevention of diseases.However,studies on m6A modification in SLE are limited,and a further research is needed.ObjectivesFirst,to establish m6A modification profile and expression profile in m RNAs of patients with SLE by methylated RNA immunoprecipitation combined with high-throughput sequencing(Me RIP-seq),then to screen and validate m6A related-m RNAs.After that,to investigate the function of differentially expressed m RNAs,and to reveal the roles of m6A modification of m RNAs in the disease process of SLE.Furthermore,this study aims to find m6A methylases in expression profile of m RNAs in patients with SLE,and validate m6A methyltransferases.Then,the functions of differentially expressed methyltransferases are detected to reveal the roles and mechanisms of m6A methyltransferases in SLE disease.MethodsThe case-control design was performed in this study,and incluing the following four phases:Phase 1:The first stage was to establish the m6A modification profile and expression profile of m RNAs in patients with SLEThe m6A methylation modification profile and expression profile of m RNAs were established by Me RIP-seq in peripheral blood mononuclear cells(PBMCs)from three newly-onset SLE patients and three age-and sex-matched controls.GO enrichment analysis,KEGG enrichment analysis,and differential expression and differential peak correlation analysis of m RNAs were carried out,and four m6A related-m RNAs(IFIT5,CSF3R,SP140 and IFIH1)were screened for further validation.Furthermore,the expression profile of m RNAs was screened to find out m6A methylases that have been reported so far,and m6A methyltransferases(METTL3,METTL14,METTL16 and WTAP)were selected for further exploration.Phase 2:The second stage was to validate the expression of m6A-related m RNAs and methyltransferases in PBMCs from patients with SLE and controlsThe gene expression levels of four m6A related-m RNAs(IFIT5,CSF3R,SP140and IFIH1)and four m6A methyltransferases(METTL3,METTL14,METTL16 and WTAP)were validated by quantitative Real-time Polymerase Chain Reaction(q RT-PCR)in PBMCs from 108 SLE patients and 110 controls.Next,combined with clinical information,association analyses of differentially expressed genes were conducted with clinical manifestations,laboratory parameters,and medications of patients with SLE.And then,the protein levels of differentially expressed IFIT5 and CSF3R and METTL3were examined by western blot(WB)in PBMCs from 40 SLE patients and 40 controls.Phase 3:The third stage was to validate the expression of m6A-related m RNAs and METTL3 in T cells from patients with SLE and controlsThe gene expression and protein levels of IFIT5,CSF3R and METTL3 were examined by q RT-PCR and WB in T cells from independently collected 32 SLE patients and 32 controls.Combined with clinical information,the correlations of IFIT5,CSF3R and METTL3 expression were analyzed with clinical characteristics and medications.Phase 4:The fourth stage was to verify the influence of METTL3 on T cellsPLKD-CMV-EGFP-2A-PURO-U6-sh RNA was used to construct IFIT5-sh RNA Jurkat cells;PLKD-CMV-EGFP-2A-PURO-U6-sh RNA was used to construct METTL3-sh RNA Jurkat cells;METTL3 overexpression Jurkat cells was constructed with p SLenti-EF1-EGFP-F2A-PURO-CMV-METTL3-WPRE.After Annexin V-PE and7-amino-actinomycin D double staining,the influences on apoptosis of IFIT5knockdown,METTL3 knockdown,and METTL3 overexpression were examined with flow cytometric analysis.Otherwise,the influences of IFIT5 knockdown,METTL3knockdown and METTL3 overexpression on cell cycles at 0 h,6 h,12 h and 24 h were evaluated with EDU in flow cytometric.Results(1)Analyses of m RNA expression profile and m6A modification profile in SLEIn this study,the m RNA expression profile and m6A modification profile of SLE were established by Me RIP-seq,and it was found that the m6A level in SLE was lower than that in controls.There were 6,293 genes of peaks detected in both SLE and controls,and 2,137 different peaks distributed in 1,943 genes.These peaks were concentrated at the junction of 3'UTR and CDS.Classical Motifs(RRACH,R=A/G,H=A,T,C)were also oberserved in SLE.Four m6A related-m RNAs(IFIT5,CSF3R,SP140 and IFIH1)were screened for verification by differential expression and differential peak correlation analysis.In addition,a total of 20 m6A methylases have been identified in SLE,among which m6A methyltransferases(METTL3,METTL14,METTL16 and WTAP)were selected for further validation.(2)m6A-related m RNAs and m6A methytransferases expression in PBMC from patients with SLE and controlsValidation in PBMCs of a relatively large sample population found that the expression and protein levels of CSF3R and IFIT5 were statistically different in SLE patients compared with controls,in which the expression level and protein level of CSF3R were both down-regulated(both P<0.05),while the expression level and protein level of IFIT5 were up-regulated(both P<0.05)in patients with SLE.Correlation analyses with clinical information showed that the expression level of IFIT5 was increased in SLE patients with leukopenia(Z=-2.557,P=0.011),decreased in patients with positive anti-SSB antibody(Z=-2.357,P=0.018),and increased in patients taking hydroxychloroquine(Z=-2.913,P=0.004).Moreover,CSF3R expression was negatively correlated with ESR level in patients with SLE(r_s=-0.217,P=0.042).Compared with controls,the expression levels of four m6A methyltransferases(METTL3,METTL14,METTL16,and WTAP)were down-regulated in SLE patients(all P<0.05).In addition,METTL16 expression was decreased in SLE patients with proteinuria(Z=-1.966,P=0.049).WTAP expression was increased in patients with leukopenia(Z=-2.049,P=0.040)and positive anti-nucleosome antibody(Anu A)(Z=-1.992,P=0.046).METTL3 expression was negatively correlated with CRP in SLE patients(r_s=-0.223,P=0.036).The expression levels of METTL3 and WTAP were correlated with glucocorticoids taking by SLE patients(all P<0.05),and METTL14 and METTL16expression levels were statistically correlated with the use of hydroxychloroquine in SLE patients(all P<0.05).To further excavate the function of methyltransferases,METTL3 was selected for a further study.The result of WB analysis showed that protein level of METTL3 in PBMC from SLE patients were lower than those of controls.(3)m6A-related m RNAs and METTL3 expression in T cells from patients with SLE and controlsAnother independent T cell verification results showed that the expression level and protein level of IFIT5 were still higher in SLE patients compared with the control(both P<0.05);and the protein level of CSF3R was also down-regulated in SLE patients.The results of association analyses with clinical information showed that CSF3R expression was associated with leukopenia in SLE patients(Z=-2.014,P=0.044),and negatively correlated with complement C3 level(r_s=-0.400,P=0.028).Besides,IFIT5expression was increased in SLE patients with pyuria(Z=-2.590,P=0.010),and positively correlated with disease activity of SLE patients(r_s=0.388,P=0.028).Compared with the controls,the expression level and protein level of METTL3 in T cells were still lower in patients with SLE(both P<0.05).Correlation analyses with clinical information showed that METTL3 expression in T cells of patients with SLE was statistically correlated with anti-ds DNA antibody,as well as was decreased when the patients'anti-ds DNA antibody was increased(Z=-2.219,P=0.027)and anti-SSB/La antibody was positive(t=3.181,P=0.007).Moreover,METTL3 expression was negatively correlated with complement C3 and C4 in patients with SLE(C3,r_s=0.496,P=0.004;C4,r_s=0.430,P=0.014).(4)Influence of aberrantly expressed m6A related m RNAs and METTL3 on T cellsCell experiment results showed that lentivirus infection efficiency of Jurkat cells reached 90%.Compared with the negative controls,the apoptosis rate of IFIT5-sh RNA Jurkat cells was increased(13.6%vs 24.0%),and the cell proliferation ability of IFIT5-sh RNA Jurkat cells was decreased.METTL3 knockdown accelerated the apoptosis of Jurkat cells,especially the early apoptosis(21.2%vs 44.0%),and the proliferation of Jurkat cells was significantly inhibited,while the apoptosis rate of Jurkat cells was decreased(18.6%vs 9.56%)and the cell proliferation ability was enhanced when METTL3 was overexpression.Conclusions(1)In this study,it was found that m6A level was lower in patients with SLE compared with controls.The m6A peaks mainly concentrated on the vicinity of termination codon,meanwhile classical Motifs were also found in SLE.This study tentatively suggested that m6A modification may be involved in the disease process of SLE.(2)CSF3R and IFIT5 were abnormal expressed in SLE.Moreover,the apoptosis rate was increased and the cell proliferation ability was decreased in IFIT5-sh RNA Jurkat cells.It was preliminarily found that m RNAs modified by m6A methylation may affect the stability of T cells,but the specific regulatory mechanism still needs a further study.(3)This study also found that there were many m6A methylases in SLE,including methyltransferases,demmethylases,and recognition proteins,thus m6A modification might dynamically regulate the diseases process of SLE.Methyltransferases expression was down-regulated in SLE,especially METTL3 was not only associated with antibodies of SLE,but also was associated with CRP and other clinical indicators as well as medications in patients with SLE.It was found that the down-regulation of METTL3 expression may promote T cells apoptosis and inhibit T cells proliferation with the interference and overexpression of METTL3 experiment,which may affect the progression of SLE.Thus,The roles of METLL3 and other differentially expressed m6A methylases in SLE disease and its mechanism deserve further investigation.