Association Study Of Methylase For N6-Methyladenosine With Ankylosing Spondylitis

ObjectiveThe aim of this study was to investigate the expression levels of N6-methyladenosine(m6A)methyltransferase(METTL3,MTEEL14,METTL16,WTAP,VIRMA and RBM15),demethylase(FTO and ALKBH5)and m6A binding protein(YTHDF1 and YTHDF2)in peripheral blood mononuclear cells(PBMCs)of patients with ankylosing spondylitis(AS).The methylation level of m6A RNA in total RNA was quantitatively detected by colorimetric method.To explore the correlation between m6A methylase and clinical characteristics and disease activity index of AS.To determine whether the level of m6A methylase is related to the occurrence and development of AS;The diagnostic value of m6A methylase for AS was preliminarily analyzed.MethodsIn this study,a case-control study design was adopted,and peripheral venous blood samples were collected from 49 AS patients and 52 healthy controls matched in age and sex.PBMCs were extracted from peripheral blood samples,and then total RNA was extracted from PBMCs,which was then reverse-transcribed into c DNA.Finally,the expression level of m6A methylase mRNA in PBMCs of 49 AS patients and 52 healthy controls was detected by real-time quantitative PCR.In addition,total RNA was randomly selected from 5 AS patients and 5 healthy controls,and m6A content in total RNA was detected by Epi Quik?m6A RNA methylation quantitative kit(colorimetric).Results(1)A total of 101 people were recruited for this study,including 49 patients with AS and52 healthy controls.In the 49 AS case group,the mean age was 34.96±9.77,with 37males and 12 females.Among 52 healthy control subjects,the mean age was 36.90±10.63,with 36 males and 16 females.There was no significant difference in age(t=0.956,P=0.342)and sex distribution(χ2=0.496,P=0.481)between the two groups.(2)Compared with the healthy control group,the mRNA expression levels of methyltransferase(METTL3,MTEEL14,METTL16,WTAP,VIRMA and RBM15),demethylase(FTO and ALKBH5)and m6A binding protein(YTHDF1 and YTHDF2)were significantly decreased in AS patients,and the difference were statistically significant(P<0.05).The correlation analysis of transcription levels of m6A methylase showed that:the mRNA levels of m6A methyltransferase related factors in PBMCs were positively correlated with the mRNA levels of demethylase related factors and m6A binding protein related factors(P<0.05),and the mRNA levels of m6A demethylase related factors in PBMCs were positively correlated with the mRNA levels of m6A binding protein related factors in patients with AS(P<0.05).(3)Compared with healthy controls,m6A RNA methylation levels in AS were significantly reduced compared with healthy controls(P<0.01).(4)Spearman correlation analysis showed that ESR was only negatively correlated with METTL14 expression level in AS patients(r_s=-0.426,P=0.002).CRP was negatively correlated with the expression levels of METTL3,METTL14,WTAP,VIRMA,RBM15,ALKBH5 and FTO.ASADS were negatively correlated with the expression levels of METTL3,METTL14,WTAP,VIRMA,RBM15,ALKBH5 and FTO.No statistical correlation was found between the expression level of other factors,BASFI,BASDAI and the course of disease(P>0.05).(5)Subgroup analysis showed that:based on BASDAI subgroup,the expression level of METTL14 and YTHDF2 were statistically significant between BASDAI<4 group and BASDAI≥4 group(P<0.05);Only YTHDF1 expression level was significantly difference between treated and untreated groups based on treatment status(P<0.05).However,in subgroup analysis of different gender,HLA-B27 status,ASADS score and biologics use,no statistically significant differences were found in the expression levels of all m6A methylation modification genes(P<0.05).(6)Binary Logistic regression analysis showed that the decreased mRNA expression of YTHDF2 and male may be risk factors for AS.(7)ROC curve results showed that RBM15(AUC=0.854,95%CI=0.774-0.932),FTO(AUC=0.816,95%CI=0.735-0.903)and YTHDF2(AUC=0.886,95%CI=0.824-0.951)had high diagnostic performance.ConclusionThe study showed that the m6A RNA methylation level in peripheral blood of patients with AS was significantly reduced compared with the control group.Further research found that the mRNA expression levels of methyltransferase(METTL3,MTEEL14,METTL16,WTAP,VIRMA and RBM15),demethylase(FTO and ALKBH5)and m6A binding protein(YTHDF1 and YTHDF2)were significantly decreased in AS patients compared with the healthy control group in this study.The expression of m6A modified methylase in peripheral blood is related to disease activity.Importantly,logistic regression analysis showed that decreased mRNA of YTHDF2 and male were risk factors for AS.RBM15,FTO and YTHDF2 have reference value for the diagnosis of AS disease.The results of this study provide a theoretical basis for the pathogenesis of AS patients,the related diagnosis and the development of new therapeutic targets.