miR-382-3p靶向RASA1基因调控骨关节炎软骨细胞增殖和凋亡

目的:探讨mi R-382-3p对骨关节炎软骨细胞增殖和凋亡的影响及其机制。方法:用100 ng/mL的脂多糖(LPS)处理软骨细胞,记为LPS组,以正常培养的软骨细胞作为正常对照(NC)组。mi R-NC、mi R-382-3p、anti-miR-NC、anti-miR-382-3p转染至软骨细胞中,记为mi R-NC组、mi R-382-3p组、anti-miR-NC组、anti-miR-382-3p组;将mi R-NC、mi R-382-3p、si-NC、si-RASA1转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-NC+LPS组、mi R-382-3p+LPS组、si-NC+LPS组、si-RASA1+LPS组;将mi R-382-3p分别与pcDNA-NC、pcDNA-RASA1共转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-382-3p+pcDNA-NC+LPS组、mi R-382-3p+pcDNA-RASA1+LPS组。实时荧光定量PCR(RT-qPCR)检测mi R-382-3p和Ras p21蛋白活化因子1(RASA1)m RNA表达水平;蛋白质印迹(Western blot)法检测RASA1、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡;荧光素酶报告实验检测mi R-382-3p和RASA1的靶向关系。结果:LPS诱导的软骨细胞中mi R-382-3p表达水平显著降低,RASA1表达水平显著升高,CyclinD1表达水平显著降低,Cleaved-caspase-3表达水平显著升高,细胞存活率显著降低,细胞凋亡率显著升高(P0.05)。过表达mi R-382-3p和敲减RASA1,LPS诱导的软骨细胞中CyclinD1表达水平显著升高,Cleaved-caspase-3表达水平显著降低,细胞存活率显著升高,细胞凋亡率显著降低(P0.05)。mi R-382-3p靶向调控RASA1,高表达RASA1部分逆转了mi R-382-3p高表达对LPS处理的软骨细胞增殖和凋亡的影响。结论:过表达mi R-382-3p促进软骨细胞增殖,抑制LPS诱导的软骨细胞凋亡,其机制可能与RASA1有关。

miR-382-3p Targets RASA1 Gene to Regulate Osteoarthritis Chondrocyte Proliferation and Apoptosis

ABSTRACT Objective: To investigate the effect of miR-382-3p on the proliferation and apoptosis of chondrocytes in osteoarthritis and its mechanism. Methods: Chondrocytes were treated with 100 ng/mL lipopolysaccharide (LPS) and recorded as LPS group, normally cultured chondrocytes were used as normal control (NC) group. miR-NC, miR-382-3p, anti-miR-NC, anti-miR-382-3p were transfected into chondrocytes, and recorded as miR-NC group, miR-382-3p group, anti-miR-NC group and anti-miR-382-3p group; miR-NC, miR-382-3p, si-NC, si-RASA1 were transfected into chondrocytes and then treated with 100 ng/mL LPS, and recorded as miR-NC+ LPS group, miR-382-3p+LPS group, si-NC+LPS group, si-RASA1+LPS group; co-transfect miR-382-3p with pcDNA-NC and pcDNA-RASA1 into chondrocytes, respectively, and then then treated with 100 ng/mL LPS, recorded as miR-382-3p+pcDNA-NC+LPS group and miR-382-3p + pcDNA-RASA1+LPS group. Real-time quantitative PCR (RT-qPCR) was used to detect miR-382-3p and Ras p21 protein activator 1 (RASA1) mRNA expressions; Western blot was used to detect RASA1, CyclinD1 and cleaved cysteine-containing aspartate-specific proteases-3 (Cleaved-caspase-3) protein expression; Tetramethylazozolium colorimetry (MTT) to detect cell survival rate; flow cytometry to detect apoptosis; luciferase report experiments to detect the targeting relationship between miR-382-3p and RASA1. Results: The expression of miR-382-3p in chondrocytes induced by LPS was significantly reduced, the expression of RASA1 was significantly increased, the expression of CyclinD1 was significantly reduced, the expression of Cleaved-caspase-3 was significantly increased, the cell survival rate was significantly reduced, and the apoptosis rate was significantly increased (P<0.05). Overexpression of miR-382-3p and knockdown of RASA1, CyclinD1 expression was significantly increased in chondrocytes induced by LPS, Cleaved-caspase-3 expression was significantly reduced, cell survival rate was significantly increased, apoptosis rate was significantly reduced (P<0.05). miR-382-3p targets RASA1, and overexpression of RASA1 partially reverses the effect of overexpression of miR-382-3p on the proliferation and apoptosis of chondrocytes treated with LPS. Conclusion: Overexpression of miR-382-3p promotes chondrocyte proliferation and inhibits LPS-induced chondrocyte apoptosis; the mechanism may be related to RASA1.