miR-1275/SIRT2信号轴在骨关节炎软骨细胞增殖凋亡中的作用

The role of miR-1275/SIRT2 signal axis in the proliferation and apoptosis of osteoarthritis chondrocytes

目的:探讨微小RNA(miR)-1275对骨关节炎(OA)软骨细胞增殖凋亡的影响及其机制。方法:采用实时荧光定量PCR(qPCR)检测人正常软骨细胞和OA软骨细胞中miR-1275的表达情况。将体外培养的OA软骨细胞分为模拟物对照组、模拟物组、抑制剂对照组和抑制剂组,将miR-1275模拟物、miR-1275抑制剂及其阴性对照转染至OA软骨细胞,采用qPCR检测miR-1275的表达水平,噻唑蓝(MTT)法检测细胞增殖能力,流式细胞术检测细胞凋亡率,Western blotting检测沉默信息调节因子2(SIRT2)、Bax、Cleaved Caspase-3蛋白的表达,生物信息学软件预测、双荧光素酶报告基因实验检验miR-1275和SIRT2的靶向关系。结果:与人正常软骨细胞比较,人OA软骨细胞中miR-1275表达水平明显升高(P<0.05)。与模拟物对照组比较,模拟物组细胞中miR-1275表达水平和细胞凋亡率、Bax、CleavedCaspase-3蛋白水平明显升高,细胞增殖活力与SIRT2蛋白水平明显降低(P<0.05);而抑制剂组细胞中miR-1275表达水平和细胞凋亡率、Bax、Cleaved Caspase-3蛋白水平明显低于抑制剂对照组,细胞增殖活力与中SIRT2蛋白水平明显高于抑制剂对照组(P<0.05)。双荧光素酶报告基因实验显示SIRT2是miR-1275的靶基因。结论:miR-1275在人OA软骨细胞中高表达,并发挥着抑制软骨细胞增殖和促进细胞凋亡的作用,其作用机制可能与靶向调控SIRT2表达有关。

Objective:To investigate the effect and mechanism of microRNA(miR)-1275 on proliferation and apoptosis of osteoarthritis(OA)chondrocytes.Methods:Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of miR-1275 in human normal chondrocytes and OA chondrocytes.OA chondrocytes cultured in vitro were divided into analog control group,analog group,inhibitor control group and inhibitor group.MiR-1275 analogies,miR-1275 inhibitors and their negative controls were transfected into OA chondrocytes.The expression of miR-1275 was detected by qPCR.The ability of cell proliferation was detected by MTT method,and the rate of apoptosis was detected by flow cytometry.The expression of silencing information regulator 2(SIRT2),Bax and Cleaved Caspase-3 protein was detected by Western blotting.The targeting relationship between miR-1275 and SIRT2 was detected by bioinformatics software and double luciferase reporter gene assay.Results:Compared with normal human chondrocytes,the expression of miR-1275 in human OA chondrocytes was significantly increased(P<0.05).Compared with the analog control group,the expression level of miR-1275,the rate of apoptosis,the level of Bax and Cleaved Caspase-3 protein in the analog group were significantly increased,while the cell proliferation activity and SIRT2 protein level were significantly decreased(P<0.05).However,the expression level of miR-1275,apoptosis rate,Bax and Cleaved Caspase-3 protein levels in the inhibitor group were significantly lower than those in the inhibitor control group,while the cell proliferation activity and medium SIRT2 protein level in the inhibitor group were significantly higher than those in the inhibitor control group(P<0.05).Double luciferase reporter gene assay showed that SIRT2 was the target gene of miR-1275.Conclusion:MiR-1275 is highly expressed in human OA chondrocytes and has aneffecton inhibiting chondrocyte proliferation and promoting cell apoptosis.The mechanism may be related to the targeted regulation of SIRT2 expression.