成软骨相关miR-4287调控人软骨细胞聚蛋白多糖酶-1表达的研究  

Effect of chondrogenesis related miR-4287 on expression of aggrecanase-1 in human chondrocytes

作　　者：孙红[1,2] 张志奇 黄志宇[1] 毛谷平[1] 余宝禧 张程云 傅明 

机构地区：[1]中山大学附属第一医院关节外科,广州510080 [2]贵州医科大学附属医院骨科,贵阳550004

出　　处：《中国修复重建外科杂志》2017年第12期1468-1473,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81572171);广东省自然科学基金资助项目(2014A03031385)~~

摘　　要：目的探讨成软骨相关miR-4287对人软骨细胞聚蛋白多糖酶-1(a disintegrin and metalloproteinase with thrombospondin motif 4,ADAMTS4)调控作用及其机制。方法取自愿捐赠的膝关节正常及骨关节炎软骨组织,采用实时荧光定量PCR检测miR-4287和ADAMTS4 mRNA表达量;然后分离培养软骨细胞,取第1代骨关节炎细胞,给予IL-1β处理,观察其对软骨细胞miR-4287和ADAMTS4 mRNA表达的影响;分别给予MAPK信号通路抑制剂SP600125以及NF-κB信号通路抑制剂SN50预处理后联合IL-1β刺激,观察IL-1β介导的信号通路对软骨细胞miR-4287和ADAMTS4 mRNA表达的影响;分别转染miR-4287模拟物及其阴性对照、miR-4287抑制物及其阴性对照,观察miR-4287调控软骨细胞ADAMTS4 mRNA及蛋白的表达。荧光素酶报告实验验证miR-4287与ADAMTS4 mRNA 3’非翻译区(untranslated region,UTR)的直接结合效应。结果与正常软骨组织比较,骨关节炎软骨组织miR-4287相对表达量下降,ADAMTS4 mRNA相对表达量上升,比较差异有统计学意义(P<0.05)。IL-1β下调软骨细胞miR-4287表达、上调ADAMTS4 mRNA表达,与未经IL-1β处理的软骨细胞相比差异均有统计学意义(P<0.05)。经IL-1β介导的信号通路抑制剂预处理后,软骨细胞miR-4287相对表达量上升,ADAMTS4 mRNA相对表达量降低,与未经信号通路抑制剂预处理细胞相比,差异均有统计学意义(P<0.05)。转染miR-4287模拟物后,软骨细胞内ADAMTS4 mRNA及蛋白表达均降低(P<0.05);而转染miR-4287抑制物后,软骨细胞内ADAMTS4 mRNA及蛋白表达均升高(P<0.05)。无论结合位点为野生型或突变型,过表达miR-4287均不能改变报告载体的荧光素酶活性(P>0.05)。结论成软骨相关miR-4287可能是一种与软骨退变相关的miRNA。miR-4287能调控人软骨细胞ADAMTS4表达,但不是通过靶向结合mRNA 3’UTR的方式发挥作用,其具体机制有待进一步研究。Objective To investigate the effect and mechanism of miR-4287, a chondrogenesis associated microRNA, regulated the expression of aggrecanase- 1 （a disintegrin and metalloproteinase with thrombospondin motif 4, ADAMTS4） in human chondrocytes. Methods First, the voluntarily donated normal and osteoarthritic knee articular cartilages were used to detect the expressions of miR-4287 and ADAMTS4 mRNA by real-time fluorescence quantitative PCR. Then, chondrocytes were isolated from knee articular cartilages. The effect of IL-1β on the expression of miR-4287 and ADAMTS4 mRNA was validated by the first generation of osteoarthritic chondrocytes. To confirm the influence of IL-1 β signal pathways on the expression of miR-4287 and ADAMTS4 mRNA, osteoarthritic chondrocytes were pretreated with MAPK signal pathway inhibitor SP600125, NF-κB pathway inhibitor SN50, and finally stimulated with IL-1 β. Chondro-cytes were transfected with miR-4287 mimics and mimics negative control, inhibitors and inhibitors negative control respectively to value the effect of miR-4287 on ADAMTS4 expression. Luciferase reporter assay was used to verify the direct interaction between miR-4287 and putative site in the 3-untranslated region （3＇UTR） of ADAMTS4 mRNA. Results Compared with normal knee articular cartilages, the miR-4287 expression was markedly diminished and conversely ADAMTS4 mRNA expression was raised in osteoarthritis cartilages （P〈0.05）. Stimulation with IL-1β led to a reduction in miR-4287 expression and upregulation in ADAMTS4 mRNA expression, showing significant difference when compared with the untreated groups （P〈0.05）. Pretreatment with IL-1 β signal pathway inhibitors induced miR-4287 expression and attenuated ADAMTS4 mRNA expression in human chondrocytes, which were significantly different from that of unstimulated cells （P〈0.05）. ADAMTS4 mRNA and protein were suppressed by transfection with miR-4287 mimics （P〈0.05） and elevated by transfection with miR-4287 inhibitors （P〈0.05）. As

关 键 词：骨关节炎 miR-4287 IL-1Β 聚蛋白多糖酶-1 软骨细胞 

分 类 号：R684.3[医药卫生—骨科学]