Expression Level And Significance Of M~6A Modification Hsa＿circ＿0000817 In CD4~+T Cells Of RA Patients

Research background:Rheumatoid arthritis(RA)is an autoimmune disease characterized by joint swelling,pain,stiffness and progressive destruction of bone.The pathogenesis of RA involves multiple immune cells,cytokines and signaling pathways that lead to abnormal immune responses and tissue damage.Among them,CD4~+T cells play a key role in initiating and maintaining RA.Circular RNAs(circRNA)are a class of endogenous non-coding RNAs produced by reverse splicing of precursor m RNAs.they have multiple functions in regulating gene expression at the transcriptional,post-transcriptional and translational levels.In recent years,there is increasing evidence that circRNA play an important role in RA,affecting the differentiation,activation and function of CD4~+T cells.N6-methyladenine(m~6A)modifications are among the most abundant and conserved internal transcriptional modifications in eukaryotes,and are present in both m RNAs and nc RNAs.m~6A modifications can affect RNA stability,splicing,migration,binding protein affinity,and are involved in the regulation of various physiological processes and disease progression.Notably,recent studies have found that m~6A modifications can play a role in the regulation of circRNA biological functions by affecting circRNA levels and thus in the body’s immune response.However,the distribution characteristics of m~6A-modified circRNAs in CD4~+T cells of RA patients and their effects on CD4~+T function are still unclear.Purpose of the study:The aim of this project is to investigate the differential expression of m~6A-modified circRNAs in CD4~+T cells and their influence in the development of RA by regulating CD4~+T cell function,and to provide new ideas for uncovering the molecular mechanism of RA and finding novel therapeutic targets.Research methods:1.Three RA patients and three healthy controls(healthy control,HC)were selected to detect the expression of m~6A-circular RNA in peripheral blood CD4~+T cells in RA and HC using m~6A-Circular RNA Epitranscriptomic Microarray(8*15K)microarray technology,respectively.2.Three m~6A-circRNAs(hsa＿circ＿0001931,hsa＿circ＿0001313,hsa＿circ＿0000817),which may be associated with the development of RA,were screened based on the detection results of microarray technology.The alternative m~6A-circRNAs were then validated in this study using MeRIP-qPCR.3.RT-qPCR was performed to detect the expression of the above three circRNA and m~6A modifier-related genes in CD4~+T cells from HC and RA patients:total cellular RNA was extracted according to the Trizol kit instructions,and total RNA concentration was determined spectrophotometrically.The total cellular RNA was reverse transcribed to cDNA according to the reverse transcription kit instructions.The relative expression levels of the target genes were determined on an ABI7500 real-time fluorescence quantitative PCR instrument using the SYBR kit to investigate the differential expression of hsa＿circ＿0000817 and m~6A modifier-related genes in RA CD4~+T cells and the correlation between them.4.Flow cytometric detection of Th17 cell ratio in peripheral blood of HC and RA patients:CD4~+T cells were extracted and resuspended,added with cell activator and incubated in a thermostat for 5h,followed by surface labeling of cells with surface antibodies anti-human CD3-APC-Cy7 and anti-human CD4-BB700,and fixed permeabilization.The cells were endogenously labeled with endogenous antibody anti-human IL-17A-PE and washed to remove excess antibody,and the labeled cells were detected and analyzed by flow cytometry.To investigate whether there is a difference in Th17%in peripheral blood of HC and RA patients and the correlation between hsa＿circ＿0000817 and Th17%.5.Analysis of the relationship between hsa＿circ＿0000817 expression and RA disease severity and treatment:Based on the RT-qPCR results of hsa＿circ＿0000817,we analyzed the relationship between hsa＿circ＿0000817 expression levels in peripheral blood CD4~+T of RA patients and gender,age,RA patients’disease activity,clinical manifestations,autoantibody levels(anti-CCP,RF,etc.)and the degree of inflammation(ESR,CRP,IgG,C3,C4,etc.),and treatment prognosis.Study results:1.The results of m~6A-Circular RNA Epitranscriptomic Microarray(8*15K)microarray assay showed that 71 circRNAs were differentially expressed in the percentage of m~6A modifications(5 up-regulated,66 down-regulated)and 204circRNAs were differentially expressed in the m~6 A modifications in the RA group compared to the HC group(195 up-regulated and 9 down-regulated).2.Me RIP-qPCR validation of microarray results:Based on the microarray assay results,three m~6A-circRNAs(hsa＿circ＿0001931,hsa＿circ＿0001313,hsa＿circ＿0000817)that may be associated with the development of RA were screened in this study.Me RIP-qPCR assays performed on peripheral blood CD4~+T cell specimens collected from 12 RA and 12 HC cases showed that the level of m~6A modification of hsa＿circRNA＿001931 was significantly upregulated in peripheral blood CD4~+T cells from RA patients,and the level of m~6A modification of hsa＿circ＿0001313 was not expressed in peripheral blood CD4~+T cells from RA patients The level of m~6A modification of hsa＿circ＿0000817 was significantly downregulated in peripheral blood CD4~+T cells from RA patients.3.The RT-qPCR technique was applied to further verify whether hsa＿circ＿0001931,hsa＿circ＿0001313,hsa＿circ＿0000817 could help to identify HC and RA patients.53 patients with primary RA,37 patients with repeat RA and 38 patients with HC peripheral blood CD4~+T cells were collected and detected by RT-qPCR The expression levels of the above three circRNAs were detected by RT-qPCR,and statistical analysis revealed that the expression levels of hsa＿circ＿0000817 were significantly higher in peripheral blood CD4~+T cells of RA patients than in HC,and the expression levels of hsa＿circ＿0000817 were significantly higher in CD4~+T cells of primary RA patients than in repeat RA patients,but the expression levels of hsa＿circ＿0001931,hsa＿circ＿0001313 expression levels were not significantly different in peripheral blood CD4~+T cells of HC and RA patients.4.Whether m~6 A modifying enzyme-related genes were differentially expressed on CD4~+T cells in HC and RA patients was examined by RT-qPCR technique.The results showed that the expression levels of ALKBH5 was significantly higher on CD4~+T cells in patients with primary RA than in HC,and analysis of primary and repeat RA patients revealed that ALKBH5 was significantly more expressed on CD4~+T cells in patients with primary RA than in patients with repeat RA,while other m~6 A related genes were not significantly different in expression levels on CD4~+T cells in HC and primary RA patients.On this basis,clinical laboratory indices of RA patients were collected,and analysis revealed a positive correlation between the expression levels of ALKBH5 on CD4~+T cells and rheumatoid factor in patients with primary RA.5.After the above experiment detected the expression levels of m~6A methylation-related genes and hsa＿circ＿0000817 on CD4~+T cells of RA patients,the correlation analysis revealed that the expression levels of hsa＿circ＿0000817 were positively correlated with those of ALKBH5 and YTHDF2,respectively.6.Peripheral blood of HC and RA patients was examined by flow cytometry Th17%,and the results showed that Th17%was significantly higher in peripheral blood of RA patients than in HC,followed by a positive correlation between hsa＿circ＿0000817 expression levels and Th17%in CD4~+T cells of RA patients.Research findings:1.Compared with HC,there was a difference in m~6A modified circRNA expression profile in peripheral blood CD4~+T cells of RA patients,and Me RIP-qPCR confirmed that the m~6A modification level of hsa＿circ＿0000817 was significantly downregulated in peripheral blood CD4~+T cells of RA patients,suggesting the presence of aberrantly expressed m~6A modified circRNA in CD4~+T cells of RA patients.2.hsa＿circ＿0000817 was differentially expressed in RA patients and HC CD4~+T cells and correlated with therapeutic levels,suggesting that hsa＿circ＿0000817may play an important role in the development of RA and may be used as a biomarker or target for RA therapy.3.Compared with HC,ALKBH5 expression was significantly higher in CD4~+T cells from RA patients and positively correlated with the expression level of hsa＿circ＿0000817,suggesting that ALKBH5-mediated hsa＿circ＿0000817 m~6A modification may affect the expression of hsa＿circ＿0000817.4.Th17%was significantly higher in the peripheral blood of RA patients than HC,and the expression level of hsa＿circ＿0000817 was positively correlated with Th17%,suggesting that ALKBH5-mediated m~6A modification of hsa＿circ＿0000817 may influence the development of RA disease by regulating Th17 cellular response.