MiR-155在类风湿关节炎滑膜成纤维细胞中的表达及功能研究

目的 检测miR-155在类风湿关节炎(RA)患者滑膜成纤维细胞(SFs)中的表达,探讨miR-155对RASFs细胞因子分泌、细胞增殖、侵袭能力的影响及其机制.方法 留取RA患者及骨关节炎(OA)患者各9份膝关节置换术后滑膜组织,分离培养滑膜成纤维细胞,取第3～5代细胞用于实验.①提取总RNA,利用实时定量聚合酶链反应(RT-PCR)方法测定miR-155在RASFs和OASFs中固有性表达水平.②Lipo2000脂质体分别转染化学合成的miR-155类似物,miR-155抑制剂及无关序列小RNA对照.③转染48 h后通过酶联免疫吸附试验(E12SA)检测细胞上清基质金属蛋白酶(MMP)-3的分泌;通过3H掺入法检测细胞增殖;通过细胞侵袭试验(transwell)法检测细胞侵袭能力;通过RT-PCR法检测miR-155下游靶标IKBKE的mRNA表达水平.2组比较采用独立样本t检验,多组比较采用方差分析.结果 ①RA患者SFs中miR-155的表达明显高于OA组[分别为(1.79±1.94)和(0.11±0.17),P<0.05];②miR-155可抑制RASFs分泌MMP-3、细胞增殖和侵袭能力;③RASFs过表达miR-155后IKBKE的mRNA水平明显下调(P<0.05).结论 RASFs miR-155的表达上调,可能与滑膜局部的炎症环境相关;miR-155抑制RASFs增殖和降低侵袭能力可能是RASFs分泌MMP-3减少的原因之一,miR-155抑制RASFs分泌MMP-3可能与miR-1.55下调靶标IKBKE mRNA表达水平相关.

The expression and the function of miR-155 on rheumatoid arthritis synovial fibroblasts

Objective To screen for the miR-155 expression in synovial fibroblasts of rheumatoid arthritis (RASFs) and osteoarthritis (OASFs) and to evaluate the function of miR-155 on RASFs and its possible target mRNAs. Methods The expression levels of miR-155 in RASFs and OASFs were detected by real-time PCR. MiR-155 mimic and miR-155 inhibitor, as well as scrambled control were transfected into cultured RASFs by Lipofectamine 2000. Forty-eight hours later, MMP-3 levels in the cell culture supernatant were detected by ELISA and fibroblast proliferation was assayed by 3H -TdR incorporation test. Fibroblast invasive ability was tested by transwell system. IKBKE which previously identified as actual target of miR-155 was examined by real-time PCR. Comparisons between groups were performed with t test or one-way ANOVA analysis. Results It was shown that miR-155 was up-regulated in RASFs (1.79 ±1.94) and it was higher than that in OASFst (0.11±0.17), P<0.05]. Up-regulation of miR-155 could decrease MMP-3 levels (P<0.05). The proliferation and invasion of RASFs transfected with miR-155 were both evidently suppressed (P<0.05), while reducing the endogenous miR-155 could significantly enhance RASF proliferation (P<0.05). The expression of IKBKE of RASFs transfected with miR-155 was obviously down-regulated compared to those transfected with the scrambled control (P<0.05). Conclusion miR-155 is up-regulated in RASFs which may be a protective factor against the inflammatory effect, at least partially by attenuating the expression of IKBKK.