沉默miR-126对关节软骨细胞增殖、凋亡和炎症反应的影响及其机制

Effects of silencing miR-126 on proliferation,apoptosis,and inflammatory response of osteoarticular chondrocytes

目的探讨沉默miR-126对关节软骨细胞增殖、凋亡和炎症反应的影响及其机制。方法体外传代培养关节软骨细胞HC-a。取传3代、对数生长期、生长状态良好的HC-a细胞,随机分为对照组、IL-1β组、IL-1β+miR-NC组、IL-1β+miR-126 inhibitor组,IL-1β+miR-NC组和IL-1β+miR-126 inhibitor组分别转染miR-NC和miR-126 inhibitor;除对照组外,其余三组均予10 ng/mL IL-1β诱导骨关节炎细胞模型。收集各组细胞,采用RT-qPCR法检测miR-126表达,采用CCK-8法检测细胞增殖活性,采用流式细胞术检测细胞凋亡率,采用ELISA法检测培养上清液炎症因子TNF-α、IL-6、IFN-γ,采用Western blotting法检测SIRT1、Caspase-3蛋白表达。结果与对照组比较,IL-1β组miR-126相对表达量升高,细胞增殖活性下降,细胞凋亡率升高,培养上清液TNF-α、IL-6、IFN-γ水平升高,SIRT1蛋白相对表达量降低,Caspase-3蛋白相对表达量升高(P均<0.05);与IL-1β+miR-NC组比较,IL-1β+miR-126 inhibitor组miR-126相对表达量降低,细胞增殖活性升高,细胞凋亡率降低,培养上清液TNF-α、IL-6、IFN-γ水平下降,SIRT1蛋白相对表达量升高,Caspase-3蛋白相对表达量降低(P均<0.05);而IL-1β组和IL-1β+miR-NC组上述指标比较差异均无统计学意义(P均>0.05)。结论沉默miR-126能够促进关节软骨细胞增殖并抑制其凋亡和炎症反应,其机制可能与调控SIRT1蛋白表达有关。

Objective To investigate the effects of silencing miR-126 on proliferation,apoptosis,and inflammatory response of osteoarticular chondrocytes and their mechanism.Methods Human osteoarticular chondrocytes HC-a were cultured in vitro.HC-a cells in the third generation,which were in the logarithmic growth phase and had good growth sta‑tus,were randomly divided into the control group,IL-1βgroup,IL-1β+miR-NC group,and IL-1β+miR-126 inhibitor group,respectively.Cells in the IL-1β+miR-NC group and IL-1β+miR-126 inhibitor group were transfected with miR-NC and miR-126 inhibitor,respectively.Except for the control group,the other three groups were treated with 10 ng/mL IL-1βinduction to construct the cell models of osteoarthritis(OA).The cells of each group were collected,and the expres‑sion of miR-126 was detected by RT-qPCR,cell proliferation activity was detected by CCK-8,apoptosis rate was detected by flow cytometry,and inflammatory factors TNF-α,IL-6 and IFN-γin the culture supernatant were detected by ELISA.The expression levels of SIRT1 and Caspase-3 proteins were detected by Western blotting.Results Compared with the control group,the relative expression of miR-126 increased,the cell proliferation activity decreased,apoptosis rate increased,the levels of TNF-α,IL-6 and IFN-γin culture supernatance increased,the relative expression of SIRT1 pro‑tein decreased,and the relative expression of Caspase-3 protein increased in the IL-1βgroup(all P<0.05).Compared with the IL-1β+miR-NC group,the relative expression decreased,cell proliferation activity increased,apoptosis rate decreased,the levels of TNF-α,IL-6 and IFN-γin culture supernatant decreased,the relative expression of SIRT1 pro‑tein increased,and the relative expression of Caspase-3 protein decreased in the miR-126 inhibitor group(all P<0.05).There were no significant differences in the above indexes between IL-1βgroup and IL-1β+miR-NC group(all P>0.05).Conclusion Silencing miR-126 can promote the proliferation of OA cells and inhibit apoptosis and inflammation,which may be related to the regulation of SIRT1 protein expression.