摘要
目的探讨微小RNA(miRNA,miR)-4287对人软骨细胞聚蛋白多糖酶-2(ADAMTS5)的调控作用及其机制。方法从膝关节软骨组织中分离得到软骨细胞,白细胞介素(IL)-1β诱导软骨炎症模型,过表达或抑制细胞中miR-4287水平,实时荧光定量聚合酶链反应(FQ-PCR)及蛋白质印迹法(Western blot)分析miR-4287和ADAMTS5 mRNA及蛋白表达,双荧光素酶报告基因方法验证miR-4287与ADAMTS5 mRNA 3’非翻译区(UTR)的结合效应。结果miR-4287在人骨关节炎(OA)软骨组织中表达较正常软骨组织减少(0.354±0.167比1.074±0.507;t=3.010,P<0.05),而ADAMTS5 mRNA表达升高(21.019±7.044比1.902±2.081;t=-5.820,P<0.01)。与未刺激组比较,IL-1β刺激软骨细胞12 h后,miR-4287表达降低(0.454±0.095比1.010±0.166;t=11.410,P<0.01),而ADAMTS5 mRNA表达增多(5.067±0.994比1.006±0.137;t=7.306,P<0.05)。转染miR-4287模拟物48 h后,软骨细胞内ADAMTS5 RNA(0.620±0.366比1.023±0.269;t=6.047,P<0.05)和蛋白表达均降低;而转染miR-4287抑制剂后,软骨细胞内ADAMTS5 RNA(2.502±0.625比1.018±0.226;t=5.637,P<0.05)和蛋白表达均升高。结合位点为野生型时,过表达miR-4287能降低报告载体的荧光素酶活性(0.789±0.088比1.018±0.226;t=2.806,P<0.05);结合位点突变后,过表达miR-4287能则不能改变报告载体的荧光素酶活性(1.000±0.006比1.014±0.065;t=0.364,P>0.05)。结论miR-4287是人软骨细胞ADAMTS5表达的直接调控因子,可能在软骨基质代谢稳态维持方面具有重要的作用。
Objective This study was undertaken to determine whether aggrecanase-2(ADAMTS5)can be regulated by microRNA(miRNA,miR)-4287.Methods Chondrocytes were separated from knee cartilage and induced with interleukin-1 beta(IL-1β).The expression of miR-4287 and ADAMTS5 was quantified by real time fluorescence quantitive polymerase chain reaction(FQ-PCR)by SYBR Green assay and Western blotting.Luciferase reporter assay was used to validate the direct interaction between miR-4287 and putative site in the 3’untranslated region(3’UTR)of ADAMTS5 mRNA.Results Compared with normal cartilages,the expression of miR-4287 was significantly lower(0.354±0.16 vs.1.074±0.507;t=3.010,P<0.05)and the expression of ADAMTS5 mRNA was higher(21.019±7.044 vs.1.902±2.081;t=-5.820,P<0.01)in osteoarthritic knee articular cartilages.Simulated with IL-1βled to a reduction in miR-4287 expression(0.454±0.095 vs.1.010±0.166;t=11.410,P<0.01)and upregulation in ADAMTS5 expression(5.067±0.994 vs.1.006±0.137;t=7.306,P<0.05).ADAMTS5 expression was suppressed in mRNA(0.620±0.366 vs.1.023±0.269;t=6.047,P<0.05)and protein by transfection with miR-4287 mimics and induced in mRNA(2.502±0.62 vs.1.018±0.226;t=5.637,P<0.05)and protein by transfection with miR-4287 inhibitor on the contrary.MiR-4287 overexpression directly suppressed the luciferase activity of a reporter construct containing the 3’UTR of ADAMTS5 mRNA(0.789±0.088 vs.1.018±0.226;t=2.806,P<0.05).However,treatment with miR-4287 mimics failed to influence the luciferase activity of this reporter construct(1.000±0.006 vs.1.014±0.065;t=0.3643,P>0.05).Conclusion MiR-4287 is a direct regulator of ADAMTS5 expression in human chondrocytes that may play an important role in the regulation of human cartilage homeostasis.
作者
孙红
张志奇
毛谷平
黄志宇
余宝禧
张程云
傅明
Sun Hong;Zhang Zhiqi;Mao Guping;Huang Zhiyu;Yu Baoxi;Zhang Chengyun;Fu Ming(Department of Orthopedics,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,China;Department of Joint Surgery,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第5期906-908,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(81572171).
