微小RNA-27b-3p与基质金属蛋白酶13在人软骨细胞的对应关系  

Relationship of microRNA-27 b-3 p and metalloproteinase 13 in human chondrocytes

作　　者：李兴 李震 肖方骏 翁家贤 潘建科 何沛恒[3] 苏海涛 Li Xing;Li Zhen;Xiao Fangjun;Weng Jiaxian;Pan Jianke;He Peiheng;Su Haitao(Department of Orthopedic Surgery,The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510000,China;Guangzhou University of Chinese Medicine,Guangzhou 510000,China;Department of Joint Surgery,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510000,China)

机构地区：[1]广州中医药大学第二附属医院骨伤科,510000 [2]广州中医药大学,510000 [3]中山大学附属第一医院关节外科,广州510000

出　　处：《中华关节外科杂志（电子版）》2022年第4期431-440,共10页Chinese Journal of Joint Surgery(Electronic Edition)

基　　金：广东省自然科学基金(2020A151501919);国家自然科学基金(82004386);中国博士后科学基金(2019M662876)。

摘　　要：目的:探讨微小RNA-27b-3p(miR-27b-3p)与基质金属蛋白酶-13(MMP-13)在人软骨细胞表达及其靶向对应关系。方法:运用蛋白质印迹法(WB)与实时定量PCR技术(qRT-PCR)明确miR-27b-3p与MMP13在正常和骨关节炎(OA)人软骨细胞的表达。利用不同浓度的白介素(IL)1β干预原代人软骨细胞24 h,或利用不同时间点的IL-1β(10 ng/ml)干预原代人软骨细胞。利用原位杂交、转染及双荧光素酶报告技术确定miR-27b-3p与MMP13的靶向对应关系;结合运用核转录因子-κB(NF-κB)和丝裂原活化蛋白激酶(MAPK)信号通路抑制剂评估其作用机制。两组资料比较采用独立样本t检验,多组资料比较采用单因素方差分析,LSD法多重比较检验。结果:WB、qRT-PCR和原位杂交检测结果显示,与正常软骨相比,OA软骨中miR-27b-3p表达降低(t=5.07,P<0.01),MMP13表达升高(t=-6.31,P<0.01)。IL-1β干扰后的结果显示miR-27b-3p表达降低(F=129.54,P<0.05),MMP-13表达升高(F=394.50,P<0.05)。通过TargetScan数据库和荧光素酶报告基因检测结果分析,野生型-MMP13组荧光素酶活性降低(F=55.27,P<0.001),突变型-MMP-13荧光素酶活性变化没有统计学意义(P=0.654)。利用特异性MAPK信号抑制剂和NF-kB抑制剂干预IL-1β诱导软骨细胞模型结果提示,与对照组相比,抑制剂组的MMP13表达水平降低(F=28.43,P<0.001),miR-27b-3p表达水平增高(F=35.04,P<0.001)。结论:miR-27b-3p在OA软骨细胞呈现低表达,并负向调控MMP13的表达,其作用机制可能是通过NF-κB和MAPK信号通路,这结果提示这miR-27b-3p可能作为OA诊断与治疗的潜在靶点。Objective To investigate the expression of microRNA(miR)-27b-3p and matrix metalloproteinase(MMP)-13 in human chondrocytes and their targeting relationship.Methods Western blot(WB)and quantitative real time PCR(qRT-PCR)were used to determine the expression of miR-27b-3p and MMP13 in chondrocytes from normal human and osteoarthritis(OA).Primary human chondrocytes were treated with interleukin(IL)-10 at different concentrations for 24 h,and with IL-10(10 ng/ml)at different time points.The targeting relationship of miR-27b-3p and MMP13 were determined by in situ hybridization,transfection and dual luciferase reporter;inhibitors of nuclear factor-kappa B(NF-kB)and mitogen-activated protein kinase(MAPK)signaling pathways were used to evaluate the mechanism of miR-27b-3p.The data of two groups were compared by independent sample t test,and multiple groups of data were compared by oneway AN OVA and LSD multiple comparison test.Results The results of WB,qRT-PCR and in situ hybridization showed that compared with normal cartilage,the expression of miR-27b-3p in OA cartilage decreased(t=5.QI,P<0.01),and the expression of MMP13 increased(t=-6.31,P<0.01).After treated by IL-10,the expression of miR-27b-3p was low(F=129.54,P<0.05)while the expression of MMP-13 was high(F=394.50,P<0.05).Through TargetScan database and luciferase reporter gene detection,the results showed that the relative luciferase activity carrying MMP13 wild-type 3Z-UTR decreased(F=55.27,P<0.001).There was no statistically significant change in the luciferase activity of the mutant MMP-13 plasmid(P=0.654).Specific MAPK signaling inhibitors and NF-kB inhibitors were applied to intervene in the chondrocyte model induced by IL-10.The results showed that compared with the control group,the expression level of MMP13 in the inhibitor group was significantly reduced(F=28.43,P<0.001),and the expression level of miR-27b-3p was increased(F=35.04,P<0.001).Conclusion miR-27b-3p is low expressed in OA chondrocytes,and it negatively regulates the expression of MMP13,and

关 键 词：骨关节炎 微RNAS 基质金属蛋白酶类 NF-κB 丝裂原激活蛋白激酶类 

分 类 号：R681.3[医药卫生—骨科学]