目的 研究microRNA-22在骨关节炎中对软骨细胞衰老的影响。方法 原代培养人骨关节炎软骨细胞;软骨细胞转染microRNA-22模拟物、抑制物,通过MTT实验检测microRNA-22对于软骨细胞增殖活性的影响,通过半乳糖苷酶染色实验检测microRNA-22对于软骨细胞衰老的影响,通过Western blot检测microRNA-22对于靶基因SIRT1,以及细胞衰老标志物P16蛋白表达的影响。结果 (1)与对照组软骨细胞相比,转染microRNA-22模拟物可显著抑制软骨细胞增殖速率,转染microRNA-22抑制物可促进软骨细胞增殖(P＜0.01);(2)与对照组软骨细胞相比,转染microRNA-22模拟物可显著增加半乳糖苷酶染色阳性软骨细胞比例,促进软骨细胞衰老,转染microRNA-22抑制物可降低半乳糖苷酶染色阳性软骨细胞比例,减缓软骨细胞衰老(P＜0.01);(3)与对照组软骨细胞相比,转染microRNA-22模拟物可显著抑制SIRT1蛋白表达水平,同时促进P16蛋白表达,转染microRNA-22抑制物可促进SIRT1蛋白表达,抑制P16蛋白表达水平(P＜0.01)。结论 在骨关节炎软骨细胞中,microRNA-22可抑制软骨细胞增殖,并通过抑制SIRT1蛋白表达、促进P16蛋白表达,促进软骨细胞衰老,从而在骨性关节炎的发生与进展中扮演了重要角色。
Abstract
Objective To investigate the effects of microRNA-22 on chondrocyte senescence in osteoarthritis. Methods Primary chondrocytes were collected from OA cartilage and divided into miR-22 mimics group, miR-22 inhibitor group and Negative Control (NC) group, which were transfected with miR-22 mimics, miR-22 inhibitor and negative control respectively. MTT assay was used to detect the effects of microRNA-22 on cell proliferation of chondrocyte; senescence-associated β-galactosidase (SA-β-Gal) staining was performed to evaluate the effects of microRNA-22 on senescence of chondrocyte. Western blot was used to detect the expression level of SIRT1 and P16. Results Compared with Negative Control (NC) group, the proliferation rate of chondrocytes was significantly inhibited in miR-22 mimics group and upregulated in miR-22 inhibitor group. The ratio of SA-β-Gal-positive chondrocytes group was elevated in miR-22 mimics and downregulated in miR-22 inhibitor group. After chondrocytes were transfected with miR-22 mimics, the expression level of P16 was promoted while SIRT1 was inhibited. On the contrary, the expression level of P16 was decreased while SIRT1 was elevated after chondrocytes were transfected with miR-22 inhibitor. Conclusions For OA, microRNA-22 could inhibit chondrocyte proliferation, as well as promote chondrocyte senescence by regulating SIRT1 and P16, so microRNA-22 could be a novel target of OA treatment.
关键词
microRNA-22 /
衰老 /
骨关节炎 /
SIRT1 /
P16 /
软骨细胞
Key words
microRNA-22 /
senescence /
osteoarthritis /
SIRT1 /
P16 /
chondrocyte
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基金
海南省自然科学基金青年基金项目(819QN379)
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