miR-375在骨关节炎软骨中的表达及对软骨细胞增殖和凋亡的影响

Expression of miR-375 in osteoarthritis cartilage and its effect on proliferation and apoptosis of the chondrocyte

目的探讨miR-375在骨关节炎软骨中的表达及对软骨细胞增殖和凋亡的影响。方法选择60例行膝关节置换术患者的骨关节炎软骨组织标本,另选择20例正常软骨组织作为对照。采用酶两步消化法获取并原代培养骨关节软骨细胞,利用Lipofectamine 2000分别将miR-375 inhibitor和miR-375 NC转染至软骨细胞,并分为miR-375 inhibitor组、阴性对照组和空白对照组。qRT-PCR法检测软骨组织及骨关节软骨细胞中miR-375的表达水平;MTT法检测各组细胞的增殖活力;流式细胞仪检测各组细胞凋亡率;qRT-PCR法和Western blot法分别检测各组细胞中Bcl-2、Cleaved Caspase-3 mRNA及蛋白的表达水平。结果miR-375的表达水平在骨关节炎软骨组织中要明显高于正常软骨组织(P<0.05)。与空白对照组和阴性对照组比较,miR-375 inhibitor组miR-375表达水平明显降低(P<0.05);各时段的增殖能力减弱、凋亡率增加(P<0.05);Bcl-2 mRNA及蛋白表达水平均降低(P<0.05),Cleaved Caspase-3 mRNA及蛋白表达水平均明显增高(P<0.05)。结论miR-375在骨关节炎软骨组织中高表达,降低miR-375的表达能抑制骨关节软骨细胞的增殖能力,促进其凋亡,其作用机制可能与影响Bcl-2、Cleaved Caspase-3表达水平有关。

Objective To investigate the expression of miR-375 in osteoarthritis cartilage and its effect on proliferation and apoptosis of the chondrocyte.Methods Osteoarthritis cartilage tissue specimens from 60 patients undergoing knee arthroplasty were selected,and normal cartilage tissue specimens of 20 body donors were selected as control,osteoarthritis chondrocytes were obtained and primary cultured by enzymatic two-step digestion method,Lipofectamine 2000 was used to transfect miR-375 inhibitor and miR-375 NC into chondrocytes,respectively,and the cells were divided into miR-375 inhibitor group,negative control group and blank control group.The expression levels of miR-375 in cartilage tissue and osteoarticular chondrocytes were detected by qRT-PCR method,the proliferation activity of cells in each group was detected by MTT assay,the apoptosis rate was detected by flow cytometry,the expression levels of Bcl-2,Cleaved Caspase-3 mRNA and protein in each group were detected by qRT-PCR and Western blot method.Results The expression level of miR-375 in osteoarthritis cartilage tissues was significantly higher than that in normal cartilage tissues(P<0.05).Compared with the blank control group and the negative control group,the expression level of miR-375 in the miR-375 inhibitor group was significantly decreased(P<0.05),the proliferation capacity of each period was significantly reduced and the apoptotic rate was significantly increased(P<0.05);the expression levels of Bcl-2 mRNA and protein were decreased(P<0.05),and the expression levels of Cleaved Caspase-3 mRNA and protein were significantly increased(P<0.05).Conclusions miR-375 is overexpressed in human osteoarthritis cartilage,reducing the expression of miR-375 can inhibit the proliferation capacity of osteoarticular chondrocytes,and promote their apoptosis,the mechanism of action may be related to the influencing of the expression levels of Bcl-2 and Cleaved Caspase-3.