| Objective: The purpose of this study is to analyze the difference of expression profiles of circularRNA(circRNAs)in periphe RAl blood mononuclear cells(PBMCs)of Rheumatoid arthritis(RA)patients with wind-cold-dampness syndrome,and to explore the application value of circRNAs in the diagnosis of ra with wind-cold-dampness syndrome.Methods:(1)Twenty patients(group T)who were diagnosed as RA by the Department of Rheumatology and Immunology,Affiliated Hospital of Jiangxi University of Chinese Medicine and 20 healthy subjects(group C)who were differentiated into the syndrome of wind-cold-dampness resistance in traditional Chinese medicine were selected as the research subjects,and four research subjects in each of the two groups were randomly selected by the random number method.PBMCs were extracted from peripheral blood by collecting the peripheral blood,and the circRNAs differentially expressed in the PBMCs of the two groups were detected by Arraystar circRNA gene chip.According to the screening conditions(FC > 1.5,P value < 0.05),circRNA with significantly imbalanced expression was screened for source chromosomal localization and source region lookup.(2)The circRNA target gene prediction database(CIRCRNA Inter ACT OM and Circ Bank Database)is used to predict the binding sites of CIRCRNA and miRNA;GO analysis and KEGG pathway enrichment analysis were performed on parental genes expressing dysfunctional circRNA using the DAVID and KOBAS databases;(3)On the condition that FC value > 2.0,P value < 0.05 and original signal value being high value,three differentially expressed CIRCRNAs among the CIRCRNAs with significant differential expression were selected,and the real-time fluorescent quantitative PCR(RT-q PCR)technology was applied to verify whether the expressions of the three CIRCRNAs in patients with RA wind-cold-dampness resistance syndrome were consistent with the expression results of microarray chips,with patients in groups T and C as the research subjects;(4)circRNA that was consistent with the results of microarray during RT-q PCR verification was selected and analyzed by receiver operating characteristic curve(ROC)to explore its diagnostic value in RA due to wind-cold-dampness stagnation.Spearman was used to analyze the correlation between circRNA expression and inflammatory indicators of RA.Finally,bioinformatics analysis was performed on the genes involved in the circRNA.Results:(1)A total of 11088 circRNA were detected on the circRNA microarray,and 399 differentially expressed circRNA were screened out,of which,149 circRNA expressions were up-regulated and 250 CIRCRNA expressions were down-regulated.(2)The 399 differentially expressed circRNA were mainly located on chromosome 1(48),followed by chromosomes 19(32),11(28),2(27),3(26),and 7(26).Among all the differentially expressed circRNA,no distribution was found on chromosome Y;Meanwhile,of the 399 differentially expressed circRNA s,322 circRNA s were from exons,43 from introns,22 from the justice overlap region,4 from intergenic,and 8 from antisense strand.No circRNA s were found in antisense strand or intergenic.(3)The results of GO analysis indicated that the parental gene with dysfunctional circRNA expression was manifested as protein binding,membrane binding,organic ring compound binding,etc.in biological processes.In terms of cell components,they are expressed in cells,membrane-bound organelles,and cytoplasm.In terms of molecular function,they are manifested as cellular protein metabolism,organic nitrogen complex metabolism,and negative regulation of biological processes.The results of KEGG analysis indicated that the differentially expressed circRNA parental genes mainly involved in ubiquitin-mediated proteolysis,actin cytoskeletal regulation,fcγR-mediated phagocytosis,bacterial invasion of epithelial cells,human cytomegalovirus infection,ribosome,mineral absorption,protein digestion and absorption,ECM receptor interaction,endocrine and other factor-regulated calcium re-absorption,Salmonella infection,glycosylphosphatidylinositol(GPI)-anchor biosynthesis and other pathways,and thus participated in the occurrence and development of RA.(4)RT-qPCR verification results showed that in group T and group C,the expression of hsa＿circRNA＿009012 was significantly up-regulated(P < 0.01),and the expression of hsa＿circRNA＿101328 was significantly down-regulated(P < 0.01).There was no significant difference in the expression level of hsa＿circRNA＿050898 between group T and group C(P >0.05).Two circRNAs with identical RT-q PCR verification results and microarray chip results were selected to increase the sample size for verification(n=40).The results showed that the expression of hsa＿circRNA＿101328 in group T was significantly down-regulated as compared with that in group C(P < 0.01).There was no significant difference in the expression of hsa＿circRNA＿009012 in group T as compared with that in group C(P > 0.05).(5)The area under the receiver operating characteristic curve(AUC)of HSA＿CIRCRNA＿101328 in the diagnosis of RA due to wind-cold-dampness stagnation syndrome was 0.957.The correlation analysis between hsa＿circRNA＿101328 and inflammatory indexes of RA showed that hsa＿circRNA＿101328 had a significant negative correlation with CRP(P < 0.01)and no correlation with RF,CCP and ESR(P > 0.05).Conclusion:(1)In this study,the expression profiles of circRNA in RA patients with wind-cold-damp stagnation syndrome were mined,and the differentially expressed circRNA was screened out.(2)It has been discovered in the present study that the differentially expressed circRNA may affect the occurrence and development of RA wind-cold-dampness stagnation syndrome by participating in the pathways of infection and human immune regulation,which will provide basic data reference for further accurate differentiation of "syndrome".(3)hsa＿circRNA＿101328 can be used as a clinical diagnostic biomarker of potential RA wind-cold-dampness resistance syndrome,and hsa＿circRNA＿101328 reflects the activity of RA disease. |
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