miR-31对骨关节炎软骨细胞增殖、凋亡及炎症因子表达的影响

Effects of miR-31 on proliferation,apoptosis and inflammatory factors of osteoarthritis chondrocytes

目的:探讨miR-31靶向IL-34对骨关节炎(OA)软骨细胞增殖、凋亡及炎症因子分泌的影响和分子机制。方法:采用10 ng/L的IL-1β处理软骨细胞构建OA细胞模型。荧光定量PCR(RT-qPCR)检测软骨细胞中miR-31和IL-34 mRNA的表达;MTT实验检测细胞活力;流式细胞术检测细胞凋亡;Western blot检测P21和半胱氨酸天冬氨酸蛋白酶3(Caspase-3)的表达;ELISA法检测TNF-α和IL-6的表达。将miR-31模拟物转染软骨细胞,IL-1β处理后,采用上述方法检测细胞的存活、凋亡以及炎症因子表达情况。双荧光素酶报告基因实验验证miR-31对IL-34的靶向调控作用。将miR-31模拟物和IL-34过表达载体共转染软骨细胞,IL-1β处理后,采用上述方法检测细胞的存活、凋亡以及炎症因子表达情况。结果:IL-1β处理后,软骨细胞中miR-31的表达显著降低,IL-34 mRNA的表达显著升高,细胞存活率显著降低,凋亡率显著升高,P21和Caspase-3蛋白的表达显著升高,TNF-α和IL-6的表达显著升高(P<0.05);过表达miR-31后,IL-1β处理的软骨细胞存活率显著升高,凋亡率显著降低,P21和Caspase-3蛋白的表达显著降低,TNF-α和IL-6的表达显著降低(P<0.05)。miR-31靶向IL-34并负调控其表达。与过表达miR-31比较,同时过表达miR-31和IL-34后IL-1β处理的软骨细胞存活率显著降低,凋亡率显著升高,P21和Caspase-3蛋白的表达显著升高,TNF-α和IL-6的表达显著升高(P<0.05)。结论:miR-31通过靶向IL-34可促进OA软骨细胞增殖,抑制细胞凋亡和炎症因子分泌。

Objective:To investigate the effect and molecular mechanism of miR-31 on the proliferation,apoptosis and inflam⁃matory factor secretion of osteoarthritis chondrocytes by targeting IL-34.Methods:Chondrocytes were treated with IL-1β(10 ng/L)to construct an osteoarthritis cell model.real-time quantitative PCR detected the expression of miR-31 and IL-34 mRNA in chondrocytes;MTT assay detected cell viability;flow cytometry detected apoptosis;Western blot detected the expression of P21 and Caspase-3 pro⁃teins;ELISA kit detected the expression of TNF-αand IL-6.After miR-31 mimics were transfected into chondrocytes and treated with IL-1β,the cell survival,apoptosis and expression of inflammatory factors were detected by the above method.Double luciferase report⁃er gene experiments verified the targeted regulation of miR-31 on IL-34.The miR-31 mimic and IL-34 overexpression vector were cotransfected into chondrocytes,after IL-1βtreatment,the above methods were used to detect cell survival,apoptosis and expression of inflammatory factors.Results:After IL-1βtreatment,the expression of miR-31 in chondrocytes was significantly reduced,the expres⁃sion of IL-34 mRNA was significantly increased,the cell survival rate was significantly reduced,the apoptosis rate was significantly increased,and the expression of P21 and Caspase-3 proteins was significantly increased,the expression of TNF-αand IL-6 was signif⁃icantly increased(P<0.05).After miR-31 was overexpressed,the survival rate of chondrocytes treated with IL-1βwas significantly in⁃creased,and the apoptosis rate was significantly reduced,the expression of P21 and Caspase-3 proteins were significantly reduced,and the expression of TNF-αand IL-6 was significantly reduced(P<0.05).miR-31 targets IL-34 and negatively regulates its expres⁃sion.Compared with overexpressing miR-31,the survival rate of chondrocytes treated with IL-1βafter overexpressing miR-31 and IL-34 was significantly decreased,the apoptosis rate was significantly increased,the expression of P21 and caspase-3 protein was signifi⁃cantly increased,and the expression of TNF-αand IL-6 was significantly increased(P<0.05).Conclusion:miR-31 could promote the proliferation,and inhibit apoptosis and inflammatory factor secretion of osteoarthritis chondrocytes by targeting IL-34.