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  

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

作　　者：于萍[1] 龙丽[2] 王世瑶[2] 李茹 张晓萍[3] 刘燕鹰[2] 崔刘福[1] 栗占国[2] 

机构地区：[1]华北煤炭医学院附属开滦医院风湿免疫科,唐山063000 [2]北京大学人民医院风湿免疫科 [3]山东潍坊医学院附属医院风湿免疫科

出　　处：《中华风湿病学杂志》2010年第7期460-463,共4页Chinese Journal of Rheumatology

基　　金：国家重点基础研究发展计划（973计划）项目（2010CB529100）;国家自然科学基金（30801021）

摘　　要：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（0A）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3N5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48h后通过酶联免疫吸附试验（ELISA）检测细胞上清基质金属蛋白酶（MMP）-3的分泌；通过，H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RASFsmiR-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-155下调靶标IKBKEmRNA表达水平相关。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 supematant 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 OASFs [ （ 0. 11 ±0.17 ）, P〈0.053. 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 IKBKE.

关 键 词：微小RNAS 关节炎 类风湿 滑膜 成纤维细胞 MIR-155 

分 类 号：R593.22[医药卫生—内科学]