miR-138靶向GRK6调控骨关节炎软骨细胞增殖和凋亡的研究  

Experimental study on the action mechanism of miR-138 regulating the proliferation and apoptosis of chondrocyte in osteoarthritis via targeting GRK6

作　　者：樊萍[1] 冯秀媛[1] 胡楠 蒲丹[1] 吕晓虹[1] 孙怡宁[1] 何岚[1] FAN Ping;FENG Xiuyuan;HU Nan(Department of Rheumatology and Immunology,The First Affiliated Hospital of Xi’an Jiaotong University,Shaanxi, Xi’an 710061,China)

机构地区：[1]西安交通大学第一附属医院风湿免疫科,西安市710061

出　　处：《河北医药》2021年第1期10-15,共6页Hebei Medical Journal

基　　金：陕西省自然科学基础研究计划项目(编号:2019JM-560)。

摘　　要：目的探讨微小RNA-138(miR-138)靶向G蛋白偶联受体激酶6(GRK6)对骨关节炎(OA)软骨细胞增殖和凋亡的影响。方法分离培养人原代OA软骨细胞及对应正常软骨细胞,采用qRT-PCR与Western blot检测miR-138、GRK6 mRNA及蛋白在软骨细胞中的表达。OA软骨细胞分别转染miR-138 mimics、si-GRK6,通过MTT法、流式细胞术评估OA软骨细胞的增殖与凋亡情况。Western blot检测软骨细胞中Bcl-2、Bax蛋白表达。采用双荧光素酶与Western blot验证miR-138与GRK6之间的靶向关系。GRK6过表达验证miR-138对OA软骨细胞增殖与凋亡的作用机制。结果miR-138在OA软骨细胞中呈低表达(P<0.05),而GRK6呈高表达(P<0.05);双荧光素酶与Western blot证实miR-138与GRK6之间存在靶向关系;miR-138过表达与沉默GRK6表达的OA软骨细胞中,细胞活力明显增强(P<0.05),细胞凋亡率降低(P<0.05),Bcl-2的表达水平上调(P<0.05),Bax的表达水平下调(P<0.05);GRK6过表达可逆转miR-138过表达对人OA软骨细胞增殖及凋亡的作用。结论miR-138可通过抑制GRK6的表达而增强OA软骨细胞增殖,抑制细胞凋亡。Objective To investigate the effects of microRNA-138(miR-138)targeting G protein-coupled receptor kinase 6(GRK6)on proliferation and apoptosis of osteoarthritis(OA)chondrocytes in vitro.Methods The human primary OA chondrocytes and corresponding normal cells were isolated and cultured in vitro,and the expression levels of miR-138,GRK6 mRNA and protein in chondrocytes were detected by qRT-PCR and Western Blot.Moreover the OA chondrocytes were transfected by miR-138 mimics and si-GRK6,respectively.The proliferation and apoptosis of OA chondrocytes were detected by MTT assay and flow cytometry.Western Blot was used to detect the expression levels of Bcl-2 and Bax in chondrocytes.The targeting relationship between miR-138 and GRK6 was verified by dual luciferase and Western Blot,and the overexpression of GRK6 was used to verify the action mechanism of miR-138 on proliferation and apoptosis of OA chondrocytes.Results The expression of miR-138 was down-regulated in OA chondrocytes(P<0.05),however,the GRK6 was highly expressed(P<0.05).Dual luciferase and Western Blot confirmed that there was a targeting relationship between miR-138 and GRK6.In OA chondrocytes with overexpressing miR-138 and silencing GRK6 expression,the cell viability was significantly enhanced(P<0.05),and the expression levels of Bcl-2 were up-regulated(P<0.05),but the apoptosis rate was decreased(P<0.05),however,the expression levels of Bax were up-regulated(P<0.05).Moreover the overexpression of GRK6 could reverse the effects of miR-138 overexpression on proliferation and apoptosis of human OA chondrocytes.Conclusion The miR-138 can enhance the proliferation of osteoarthritic chondrocytes,and can inhibit the cell apoptosis by inhibiting the expression of GRK6.

关 键 词：骨关节炎 软骨细胞 微小RNA-138 G蛋白偶联受体激酶6 凋亡 

分 类 号：R681[医药卫生—骨科学]