miR-27a-3p介导类风湿关节炎滑膜成纤维细胞增殖和侵袭的分子机制研究

Study on the molecular mechanism of miR-27a-3p mediating proliferation and invasion of synovial fibroblasts in rheumatoid arthritis

目的探讨微RNA-27a-3p(miR-27a-3p)调控类风湿关节炎滑膜成纤维细胞(RASFs)增殖、侵袭的分子机制。方法体外分离培养正常滑膜成纤维细胞(SFs)和RASFs。实时荧光定量PCR(RT-PCR)检测miR-27a-3p表达;预测miR-27a-3p可能结合的潜在靶基因,并通过荧光素酶报告试验进行验证;通过Lipofectamine 2000将miR-27a-3p抑制剂转染入RASFs;分别采用细胞计数试剂盒8(CCK-8)、Transwell检测miR-27a-3p和靶基因对RASFs增殖、侵袭的影响。结果与SFs比较,miR-27a-3p在RASFs中过表达,miR-27a-3p可通过作用于分泌型卷曲相关蛋白1(SFRP1)的3′非翻译区抑制其表达;SFRP1在RASFs中弱表达,抑制miR-27a-3p表达后,SFRP1表达增加,细胞增殖和侵袭能力明显减弱(P<0.05)。同时抑制SFRP1后,增殖和侵袭能力明显恢复(P<0.05)。结论miR-27a-3p可促进RASFs的增殖与侵袭能力,对类风湿关节炎的发生起着重要作用,其机制可能是通过抑制SFRP1的表达实现的。

Objective To investigate the molecular mechanisms of microRNA-27a-3p(miR-27a-3p)regulating the proliferation and invasion of rheumatoid arthritis synovial fibroblasts(RASFs).Methods Normal synovial fibroblasts(SFs)and RASFs were isolated and cultured in vitro.MiR-27a-3p expression was detected by real-time fluorescence quantitative PCR(RT-PCR).Potential target genes which miR-27a-3p might bind to were predicted and validated by luciferase reporter assay.The miR-27a-3p inhibitor was transfected into RASFs by Lipofectamine 2000.The effects of miR-27a-3p and target genes on the proliferation and invasion of RASFs were examined by Cell Counting Kit 8(CCK-8)and Transwell,respectively.Results Compared with SFs,miR-27a-3p was overexpressed in RASFs,and miR-27a-3p inhibited the expression of secretory frizzled-related protein 1(SFRP1)by acting on the 3′untranslated region of SFRP1.SFRP1 was weakly expressed in RASFs.After inhibition of miR-27a-3p expression,the SFRP1 expression was increased,and the cell proliferation and invasive ability was significantly reduced(P<0.05).The proliferative and invasive ability was significantly restored after simultaneous inhibition of SFRP1(P<0.05).Conclusion MiR-27a-3p can promote the proliferation and invasive ability of RASFs and play an important role in the development of rheumatoid arthritis,which probably through the inhibition of SFRP1 expression.