Correlation Analysis Of The Expression Of TWEAK-related Genes In Peripheral Blood Of Rheumatoid Arthritis And Its Influencing Factors

Objectives:Rheumatoid arthritis（RA）is a chronic autoimmune disease that often involves multiple peripheral joints.Its pathogenesis is yet unclear,the role of epigenetic mechanism DNA methylation in the occurrence and development of RA has increasingly become a research topic of interest.Tumor necrosis factor-like weak inducer of apoptosis（TWEAK）and its related genes Fn14 and LIGHT may be related to its pathogenesis.This study detected TWEAK,Fn14 and LIGHT gene mRNA expression levels,TWEAK gene serum protein concentration and DNA methylation level in peripheral blood and analyzed their relationship with disease activity,autoantibodies and clinical indicators of RA.It aims to explore the relationship between TWEAK-related genes and the pathogenesis of RA,in order to provide new ideas and methods for the clinical diagnosis and treatment of RA.Methods:Consecutive RA patients and healthy volunteers who were hospitalized in the Department of Rheumatology and Immunology of the First Affiliated Hospital of Kunming Medical University from October 2019 to January 2021 were Selected as the research participants.（1）Determination of the mRNA expression levels of TWEAK,Fn14,and LIGHT genes in the peripheral blood of subjects was done using Real-Time PCR;（2）Serum TWEAK protein concentration of subjects was detected using enzyme linked immunosorbent assay（ELISA）;（3）MassArray method was used to detect the DNA methylation level of TWEAK gene in the peripheral blood of subjects;（4）RA patients were grouped according to disease activity and presence of antibody positivity;data on various clinical indicators of RA patients were collected.Gene mRNA expression,protein concentration and methylation level were analyzed for its correlation with disease activity,antibodies and clinical indicators of RA.Results:In total,132 RA patients and 90 healthy volunteers were included.There was no statistical difference in age and gender between the two groups.1.Level of TWEAK and Fn14 gene mRNA expression in the peripheral blood of the RA group is lower than that of the normal control group（P<0.05）;and the expression of TWEAK gene is related to disease activity.Level of TWEAK gene mRNA expression is lower in the high disease activity group when compared to that in low and medium disease activity groups（P<0.05）;The mRNA expression level of LIGHT gene is higher in RA patients than that in the normal control group（P<0.05）;and it is related to autoantibodies.Level of LIGHT gene mRNA expression in the anti-CCP antibody positive group is higher than that in the antibody negative group（P<0.05）.2.The overall methylation level of genes like TWEAK,CpG₁1,CpG₁7.18.19.20,and CpG₄0.41.42 are higher in RA patients than those in the normal control group（P<0.05）;also,genetic methylation levels are related to disease activity.The methylation level of CpG₅5.56 is higher in the high disease activity group than that in the low disease activity group（P<0.05）.3.There is no significant difference between serum TWEAK protein concentration and normal control group（P>0.05）,but it was positively correlated with mRNA expression level（r=0.482,P<0.001）.4.The expression of TWEAK,Fn14,and LIGHT genes are correlated with clinical indicators.The mRNA expression level of TWEAK gene is positively correlated with albumin and immunoglobulin G（IgG）and negatively correlated with neutrophil count（NEUT）,C-reactive protein（CRP）,rheumatoid factor IgA（RF IgA）,RF IgM（P<0.05）.Similarly,the mRNA expression level of Fn14 gene is positively correlated with total protein,globulin,and IgG（P<0.05）.And,the mRNA expression level of LIGHT gene is positively correlated with IgG,IgM,IgA,erythrocyte sedimentation rate（ESR）,interleukin-10（IL-10）,and tumor necrosis factor-α（TNF-α）,and negatively correlated with RF IgM（P<0.05）.Level of methylation at CpG₃9,CpG₅5.56 and DAS28 score（r=0.353,P=0.026;r=0.319,P=0.045）are positively correlated.Level of methylation at CpG₁1 site is negatively correlated with NEUT（P<0.05）;the methylation level at CpG₁7.18.19.20 site was negatively correlated with IgM（P<0.05）.5.Multiple linear regression analysis found that IgG,CRP,RF IgM（P<0.05）are significant influencing factors of TWEAK gene mRNA expression level;IgG（P<0.05）is a significant influencing factor of Fn14 gene mRNA expression level;and NEUT,absolute value of basophils,IgG,RF IgM（P<0.05）are significant influencing factors of LIGHT gene mRNA expression level.Conclusion:TWEAK,Fn14,and LIGHT genes are all involved in the pathogenesis of RA.Hypermethylation of TWEAK gene may be one of the epigenetic mechanisms regulating low expression of mRNA.The low expression of TWEAK and Fn14 gene mRNA and the high expression of LIGHT gene mRNA are closely related to the onset and progression of RA.Additionally,clinical indicators such as IgG and RF IgM are significantly related to gene expression,whereas TWEAK gene mRNA expression is related to disease activity,and can be used as one of the important indicators for clinical monitoring and evaluation of RA.