Effect of miR⁃29c overexpression on the proliferation，apoptosis and differentiation of P19 embryonal carcinoma cells
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摘要:
目的：探讨microRNA-29c（miR-29c）过表达对P19细胞增殖、凋亡和分化的影响及机制。方法：体外传代培养P19细胞，利用细胞转染技术构建miR-29c过表达细胞（mimic组）。CCK-8试剂盒检测细胞增殖，流式细胞技术检测细胞周期；Hoechst染色结合流式细胞技术检测细胞凋亡情况，定量PCR和蛋白质免疫印迹（Western blot）检测凋亡相关因子Bax/Bcl-2表达情况；二甲亚砜（DMSO）诱导P19细胞分化，定量PCR检测心肌特异性标志基因肌球蛋白重链（α-myosin heavy chain，αMHC），转录因子GATA4和心肌增强因子2c（myocyte enhancer factor 2c，Mef2c）表达情况，明确miR-29c过表达对P19细胞分化的影响；生物信息学分析结合荧光素酶报告实验和Western blot明确miR-29c的靶基因。结果：与对照组相比，miR-29c过表达组细胞增殖速率较慢，细胞周期S期比例降低；miR-29c过表达组细胞凋亡率增高，Bax表达水平增高，而Bcl-2表达无明显差异；P19细胞分化第6天和第8天miR-29c过表达组αMHC、GATA4和Mef2c的表达水平显著高于对照组；Akt3被证实为miR-29c的靶基因。结论：miR-29c过表达可能是通过调控Akt3来抑制P19细胞增殖、促进P19细胞的凋亡和分化。
Abstract:
Objective：To explore the effect of miR-29c overexpression on P19 cell proliferation，apoptosis and differentiation. Methods：P19 cells were cultured and transfected with miR-29c mimics. Proliferation was examined with CCK-8 kit and cell cycle was examined with flow cytometey. Apoptosis rate was checked with Hoechst staining and flow cytometry. The mRNA and protein expression of Bax/Bcl-2 was tested with qPCR and Western blot. P19 cells were induced to differentiate with DMSO and the mRNA expression level of αMHC，GATA4，Mef2c was checked with methods of qPCR. We used bioinformatic analysis，lucierase assays and Western blot to find target gene of miR-29c. Results：Compared with the control group，cells in the miR-29c overexpression group showed a lower proliferation rate and lower S cycle percentage. Apoptosis rate of the miR-29c overexpression group was higher than that of the control group. Expression of Bax of the miR-29c overexpression group was significantly higher than that of the control group，while there was no difference seen in Bcl-2 expression. About The mRNA expression level of αMHC，GATA4，Mef2c，the miR-29c overexpression group was significantly higher than those of the control group at day 6 and day 8. Akt3 was proved to be a target gene of miR-29c. Conclusion：Overexpression of miR-29c can inhibits proliferation and promotes apoptosis and differentiation in P19 embryonal carcinoma cells with Akt3.
