过表达microRNA-29a上调锌脂蛋白91对大鼠肾上腺嗜铬细胞瘤细胞凋亡的影响

Overexpression of microRNA-29a up-regulates zinc finger protein 91 expression and regulates cell apoptosis in PC12

摘要:

目的  建立高感染效率、稳定过表达microRNA-29a（miR-29a）的大鼠肾上腺嗜铬细胞瘤细胞（PC12），探索miR-29a对PC12细胞凋亡的影响。方法  将miR-29a前体表达载体、空白对照载体进行慢病毒包装，用病毒上清感染PC12并进行抗性筛选。实时荧光定量聚合酶链反应（RT-qPCR）检测miR-29a的表达，流式细胞术检测miR-29a稳定过表达组、空质粒对照组以及未感染组PC12细胞的凋亡情况，利用TargetScan 6.0数据库预测miR-29a的关键靶点，RT-qPCR以及Western blot检测预测的miR-29a下游靶基因mRNA以及蛋白表达的变化。结果  经抗性筛选后获得稳定表达miR-29a的PC12细胞系，荧光显微镜下观察各组PC12细胞的感染效率均达80%以上，miR-29a过表达组凋亡率为（5.1±0.92）%，空质粒对照组为（1.832±0.26）%，未感染组为（1.667±0.185）%，miR-29a过表达促进PC12细胞凋亡（P <0.01），空质粒对照组与未感染组细胞凋亡率比较，差异无统计学意义；RT-qPCR检测miR-29a过表达时锌指蛋白91（ZFP91）的表达量升高，miR-29a过表达组ZFP91 mRNA的表达量是空质粒对照组的1.835倍（P =0.000），空质粒对照组与未感染组ZFP91 mRNA的表达量比较差异无统计学意义；Western blot检测的ZFP91蛋白表达量与空质粒对照组比较也升高。结论  在PC12中miR-29a过表达促进PC12细胞凋亡，过表达miR-29a也促进ZFP91表达，其机制仍待进一步阐明。

Abstract:

Objective To establish a rat adrenal pheochromocytoma cell line (PC12) with highly-infective efficiency and stable overexpression of miR-29a, and to explore the role of miR-29a in apoptosis of PC12. Methods The miR-29a precursors expressing viral vectors (Lvx-miR-29a-eGFP) and control vector (Lvx-eGFP-control) were packaged to Lentivirus respectively, and PC12 cells were infected by Lentivirus and underwent resistance screening. Flow cytometry (FCM) was used to detect the cell apoptosis, and then real time quantitative PCR (RT-qPCR) and Western blot were used to measure the levels of targets gene mRNA and protein of miR-29a. Results PC12 cell lines with high infection efficiency and stable overexpression of miR-29a were established, the EGFP-positive cell percentage was over 80% in the infected PC12 cells. miR-29a overexpression significantly increased cell apoptosis rate in PC12 when campared to the eGFP-control group and non-infection group (P < 0.01), and significantly up-regulated the mRNA and protein levels of zinc finger protein 91 (ZFP91) in the PC12. Conclusions Our study demonstrated overexpression of miR-29a can effectively up-regulate ZFP91 expression and promote apoptosis of PC12. Its mechanism remains to be further elucidated.