Expression And Clinical Significance Of MiR-16in Plasma And Synovial Fluid Of Patients With Rheumatoid Arthritis

Objective: Rheumatoid arthritis (RA) is an autoimmune disease primarilycharacterised by chronic inflammation of synovial tissue, the disease is a greatthreat to human health with considerable morbidity and disability. At present,the symptoms and signs of RA are mainly diagnosed by x-ray and bloodmarkers such as rheumatoid factor, anticyclic citrullinated peptideantibody(anti-CCP antibody),etc,but a significant proportion of RA patientsare typically diagnosed only once damage to the joints has already begun,which the window for optimal treatment may have been missed, Thus, it ishoped that a new indicators which can be used in the early diagnosis of RAwill emerge. MicroRNA (miRNAs),a class of endogenous small non-codingRNAs,21~25-nucleotides(nt) in length,which are identified in the lives ofcells, they regulate gene expression at the post-transcriptional level by bindingat sites in the3′untranslated region of their target mRNAs leading totranslational repression or mRNA degradation. Recently studies have shownthat the miRNAs dysregulation (miR-155,miR-146a and miR-203）of synovialtissue or synovial fibroblasts may play an important role in RAetiopathogeneis,the upregulated expression of miR-16,miR-146a, miR-132and miR-155in peripheral blood mononuclear cells（PBMCs）that providegreater sensitivity and specificity in the early diagnosis of RA, furthermore,they demonstrated that the abnormal level of miR-16expression correlatedwith active disease, revealing its great potiental as a novel biomarker forimproving the diagnosis of RA and an effective indicators for the prediction ofdisease development. Currently,the reports about the study of RA are mainlyfocus on the miRNAs in synovial tissue or synovial fibroblasts,but there is noreport associated with miRNAs in plasma or synovial fluid of RApatients,compared to tissue or cell miRNAs, circulating miRNAs such as plasma miRNA,serum miRNA,which are conveniently obtained and easilydetected,they may have a promising clinical application future. The purposeof this study is to observe the expression level of miR-16in plasma andsynovial fluid from patients with RA,and to explore its value in the earlydiagnosis,differentiated diagnosis and prediction of disease severity of RA。Methods: Both plasma and PBMCs were taken from30patients with RAand20healthy controls,synovial fluid was obtained from16patients with RAand10patients with osteoarthritis (OA).The expression levels of miR-16inplasma, synovial fluid and PBMCs were detected by stem-loop quantitativereal-time fluorescent polymerase chain reaction (stem-loop RT-qPCR) withSYBR Green Ⅰ dye, choose U6snRNA as endogenous control.Statisticalanalyses were performed using SPSS18.0for Windows. The Mann-WhitneyU test was used to compare the gene expression between two groups, P valuesless than0.05were considered to be statistically significant. The correlationsbetween the expression level of miR-16and other clinical variables wereanalyzed with Spearson product-moment correlation coefficient,P values lessthan0.05were considered to be statistically significant.Results:1The amplication curves and the dissociation curves of miR-16(target gene）and U6snRNA(endogenous control)were both running well,there were noprimer dimmers, and the products of PCR were specific.2The expression level of miR-16in plasma、synovial fluid and PBMCs of RApatients.The expression level of miR-16was8.7-,9.6-fold,respectively, higher forplasma than for synovial fluid and PBMCs (P <0.01); The expression level ofmiR-16was1.5-fold higher for synovial fluid than for PBMCs,but nostatistical significance（P>0.05）.3The expression level of miR-16in plasma and PBMCs of patients with RAand healthy controls.The expression level of miR-16was0.65-fold lower for RA patients thanfor healthy controls,but no statistical significance (P>0.05);The expression level of miR-16in PBMCs was1.9-fold higher for RA patients than forhealthy controls (P <0.01).4The expression level of miR-16in synovial fluid from RA and OA patients.The expression level of miR-16in synovial fluid was6.25-fold higher forRA patients than for OA patients (P <0.01).5Expression of miR-16and its relationship with clinical variables.The expression level of plasma miR-16was inversely correlated with the28-joint Disease Activity Score(DAS28)(P<0.05),but not withage,gender,course year,Erythrocyte sedimentation rate (ESR) and C-reactiveprotein(CRP)(P>0.05); The expression level of PBMCs miR-16waspositively correlated with DAS28,ESR and CRP (P<0.05),but not withage,gender and course year(P>0.05).The expression level of synovial fluidmiR-16had no correlations with age,gender,course year,DAS28,ESR, andCRP (P>0.05);Conclusion:1In RA patients,plasma and synovial fluid miR-16are distinctly generated,there were no correlations between plasma miRNAs and synovial miRNAs,plasma and synovial fluid miRNAs had distinct profiles.2The upregulated expression of miR-16in synovial fluid of RA patients mayreflect the inflammatory condition of joint space.The expression level ofsynovial fluid miR-16may provide a new way for differentiated diagnosis ofRA and OA and provide a new way for the analysis of RA pathogenesis.3The expression level of plasma miR-16can serve as an effective novelindicators in assessment of the clinical disease activity of RA.