山奈酚通过调控miR-21/SOX9对骨关节炎软骨细胞增殖、凋亡的影响

Effects of Kaempferol on the Proliferation and Apoptosis of Osteoarthritis Chondrocytes by Regulating MiR-21/SOX9

目的:探讨山奈酚(KAE)对骨关节炎软骨细胞增殖、凋亡的影响及其作用机制。方法:将C28/I2细胞分为对照组(正常培养的C28/I2细胞)、模型组(100 ng·ml^(-1)LPS)、山奈酚低(KAE-L组,25μmol·L^(-1)KAE+100 ng·ml^(-1)LPS)、中(KAE-M组,50μmol·L^(-1)KAE+100 ng·ml^(-1)LPS)、高(KAE-H组,100μmol·L^(-1)KAE+100 ng·ml^(-1)LPS)剂量组。利用LipofectamineTM2000转染试剂盒对C28/I2细胞进行转染,并分为:mimics NC组(转染mimics NC+100 ng·ml^(-1)LPS)、miR-21 mimics组(转染miR-21 mimics+100 ng·ml^(-1)LPS)、inhibitor NC组(转染inhibitor NC+100 ng·ml^(-1)LPS)、miR-21 inhibitor组(转染miR-21 inhibitor+100 ng·ml^(-1)LPS)、pcDNA组(转染pcDNA+100 ng·ml^(-1)LPS)、pcDNA-SOX9组(转染pcDNA-SOX9+100 ng·ml^(-1)LPS)。另取部分mimics NC组、miR-21 mimics组C28/I2细胞,用100μmol·L^(-1)KAE处理,记作KAE+mimics NC组、KAE+miR-21 mimics组。采用MTT法检测细胞活力;流式细胞术检测细胞凋亡;qRT-PCR检测细胞中miR-21表达;Western Blot检测细胞中性别决定区Y框蛋白9(SOX9)蛋白表达;双荧光素酶报告基因实验验证miR-21与SOX9靶向关系。结果:与对照组比较,模型组C28/I2细胞中SOX9蛋白表达、细胞活力显著降低,miR-21表达、细胞凋亡率显著升高(P<0.05);与模型组比较,KAE各剂量组C28/I2细胞中SOX9蛋白表达、细胞活力显著升高,miR-21表达、细胞凋亡率显著降低(P<0.05);miR-21可靶向负调控SOX9表达;抑制miR-21或上调SOX9均可促进LPS诱导的C28/I2细胞增殖,抑制细胞凋亡;miR-21过表达逆转了KAE(100μmol·L^(-1))对LPS诱导的C28/I2细胞增殖、凋亡的作用。结论:KAE可能通过下调miR-21表达,上调SOX9表达促进LPS诱导的C28/I2细胞增殖,抑制细胞凋亡。

Objective:To investigate the effects and mechanism of kaempferol(KAE)on the proliferation and apoptosis of osteoarthritis chondrocytes.Methods:C28/I2 cells were divided into control group(normally cultured C28/I2 cells),model group(100 ng·ml^(-1)LPS),kaempferol low(KAE-L group,25μmol·L^(-1)KAE+100 ng·ml^(-1)LPS),middle(KAE-M group,50μmol·L^(-1)KAE+100 ng·ml^(-1)LPS)and high(KAE-H group,100μmol·L^(-1)KAE+100 ng·ml^(-1)LPS)dose groups.C28/I2 cells were transfected with LipofectamineTM2000 transfection kit and divided into mimics NC group(transfected with mimics NC+100 ng·ml^(-1)LPS),miR-21 mimics group(transfected with miR-21 mimics+100 ng·ml^(-1)LPS),inhibitor NC group(transfected with inhibitor NC+100 ng·ml^(-1)LPS),miR-21 inhibitor group(transfected with miR-21 inhibitor+100 ng·ml^(-1)LPS),pcDNA group(transfected with pcDNA+100 ng·ml^(-1)LPS),and pcDNA-SOX9 group(transfected with pcDNA-SOX9+100 ng·ml^(-1)LPS).In addition,some of the C28/I2 cells in the mimics NC group and miR-21 mimics group were withdrawn and treated with 100μmol·L^(-1)KAE,which were recorded as the KAE+mimics NC group and KAE+miR-21 mimics group.MTT method was used to detect cell viability;flow cytometry was used to detect cell apoptosis;qRT-PCR was used to detect the expression of miR-21 in cells;Western blot was used to detect the expression of sex determining region Y-box protein 9(SOX9)protein in cells;dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-21 and SOX9.Results:Compared with those in the control group,the expression of SOX9 protein and the cell viability in C28/I2 cells of the model group were significantly reduced,and the expression of miR-21 and the apoptosis rate were significantly increased(P<0.05);compared with those in the model group,the expression of SOX9 protein and the cell viability in C28/I2 cells of KAE-L,KAE-M,and KAE-H groups were significantly increased,and the expression of miR-21 and the apoptosis rate were significantly decreased(P<0.05);miR-21 could target and negatively regulate SOX9 expression;inhibition of miR-21 or up-regulation of SOX9 could promote LPS-induced C28/I2 cell proliferation and inhibit cell apoptosis;the overexpression of miR-21 reversed the effects of KAE(100μmol/L)on the proliferation and apoptosis of C28/I2 cells induced by LPS.Conclusion:KAE may promote LPS-induced C28/I2 cell proliferation and inhibit cell apoptosis by down-regulating the expression of miR-21 and up-regulating the expression of SOX9.