过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。

Research of MicroRNA-21 Regulates Autophagia of Human Degenerative Nucleus Pulposus Cells via PTEN/PI3K/AKT Signaling Pathway

ABSTRACT Objective: To investigate the effect of overexpression of miR-21 on autophagy of human degenerative nucleus pulposus cells via PTEN/PI3K/AKT pathway. Methods: The stable overexpression of miR-21 mimic in human degenerated nucleus pulposus cells was constructed, and the meaningless sequence was transfected as miR-21 mimic control group. The transfection efficiency was detected by RT-qPCR. The autophagy vesicles were observed by MDC fluorescence staining. Western Blot was used to detect the expression of autophagy related proteins LC3 and P62, as well as the expression levels of PTEN, PI3K and AKT, key proteins in the PTEN/PI3K/AKT signaling pathway. Results: RT-qPCR showed that transfection of miR-21 mimic was successful and efficient, which was significantly different from that of miR-21 mimic control group and blank cell control group (P<0.05). The results of MDC staining observed by Fluorescence microscopy, showed that autophagosomes were hardly found in cells of miR-21 mimic group, while miR-21 mimic control group and blank control group had more autophagosomes, which were significantly different from the former(P<0.05). The ratio of LC3-II/LC3-I expression in miR-21 mimic group cells was significantly lower than that in miR-21 mimic control group and blank control group (P<0.05). While the expression of P62 in miR-21 mimic group was significantly higher than that in miR-21 mimic control group and blank cell control group, which had statistical significance (P<0.05). MiR-21 mimic group had a low expression level of PTEN protein, which was statistically significant compared with the other two groups (P<0.05). Phosphorylated PI3K (p-pi3k) and AKT(p-akt) in miR-21 mimic group were higher than that in miR-21 mimic control group and blank cell control group, the difference was statistically significant(P<0.05). Conclusion: MiR-21 can promote the phosphorylation of PI3K and AKT by targeting silencing PTEN, which can activate the PTEN/PI3K/AKT signal pathway, ultimately inhibit the autophagy of the degenerative nucleus pulposus cells of human intervertebral disc.