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目的:研究微小RNA-23a(miR-23a)对人卵泡颗粒细胞中蛋白激酶B(Akt)磷酸化和PTEN表达的影响,探讨miR-23a调控人颗粒细胞凋亡的分子机制。方法:分别将miR-23a前体(pre-miR-23a转染组)、miR-23a抑制剂(miR-23a抑制剂组)和pre-miRNA(阴性对照组)转染至体外培养的人卵泡颗粒细胞。应用实时定量荧光PCR技术检测miR-23a的表达,Western blot法检测Akt、p-Akt、PTEN、半胱天冬氨酸蛋白酶(Caspase-3)等凋亡相关蛋白的表达,并应用Hoechst 33258法检测细胞的凋亡率。结果:(1)与阴性对照组比较(4.05±0.92),转染pre-miR-23a后,miR-23a表达量(19.18±1.69)显著升高,转染miR-23a抑制剂后,miR-23a表达量(1.27±0.15)显著降低,差异均有统计学意义(P<0.05)。(2)转染pre-miR-23a后,Akt和p-Akt相对表达量显著低于阴性对照组(P<0.05);PTEN和Caspase-3裂解片断(Cleaved-caspase-3)相对表达量显著高于阴性对照组(P<0.05)。(3)pre-miR-23a转染组的细胞凋亡率为(17.87±2.20)%,显著高于阴性对照组(4.20±1.23)%(P<0.05)。结论:微小RNA-23a通过对其靶基因的调控参与PTEN/Akt信号通路介导的细胞凋亡,启动Caspase-3依赖的信号传导诱导体外培养人颗粒细胞的凋亡。
Objective: To investigate the effect of microRNA-23a (miR-23a) on the phosphorylation of protein kinase B (Akt) and the expression of PTEN in human granulosa cells and to explore the molecular mechanism by which miR-23a regulates the apoptosis of human granulosa cells. Methods: Human follicles cultured in vitro were transfected with miR-23a precursor (pre-miR-23a transfected group), miR-23a inhibitor (miR-23a inhibitor group) and pre-miRNA Granulosa cells. The expression of miR-23a was detected by real-time fluorescence quantitative PCR, and the expressions of Akt, p-Akt, PTEN and Caspase-3 were detected by Western blot. The Hoechst 33258 Cell apoptosis rate was detected. Results: (1) Compared with the negative control group (4.05 ± 0.92), miR-23a expression was significantly increased after pre-miR-23a transfection (19.18 ± 1.69) 23a expression (1.27 ± 0.15) was significantly lower, the difference was statistically significant (P <0.05). (2) The relative expression of Akt and p-Akt in transfected pre-miR-23a was significantly lower than that in the negative control group (P <0.05); The relative expression of cleaved-caspase-3 in PTEN and Caspase-3 was significant Higher than the negative control group (P <0.05). (3) The apoptosis rate of pre-miR-23a transfected group was (17.87 ± 2.20)%, which was significantly higher than that of the negative control group (4.20 ± 1.23)% (P <0.05). Conclusion: MicroRNAs-23a is involved in PTEN / Akt pathway-mediated apoptosis through the regulation of its target genes, and initiates Caspase-3-dependent signaling to induce apoptosis of human granulosa cells in vitro.