TTF-1启动子调控miR-7表达对人肺癌95D细胞体外凋亡的影响

发布时间：2018-03-26 20:38

本文选题：miR-　切入点：甲状腺转录因子-(TTF-)　出处：《西安交通大学学报(医学版)》2017年02期

【摘要】：目的观察甲状腺转录因子-1(TTF-1)启动子调控miR-7表达对人肺癌细胞体外凋亡的影响,并探讨其意义。方法将前期构建的TTF-1启动子调控miR-7真核表达载体(命名为p-T-miR-7)与对照载体p-cont分别体外瞬时转染人肺癌95D细胞,采用FCM法检测95D细胞凋亡比例;TUNEL法检测95D细胞凋亡情况;Western blot检测各组中磷酸化Caspase3和磷酸化Caspase9的蛋白的表达;共聚焦显微技术检测miR-7靶分子CGGBP1和EGFR蛋白的表达;最后,采用Western blot检测各组中磷酸化Akt和Erk蛋白的表达。结果体外转染48h后,与p-cont转染组相比,转染p-T-miR-7组细胞凋亡比例明显增加(P0.05);磷酸化Caspase3和磷酸化Caspase9蛋白水平均显著增加(P0.05),同时EGFR和CGGBP1蛋白表达均明显降低,且细胞中磷酸化Akt和Erk蛋白水平亦显著降低(P0.05)。结论 TTF-1启动子调控miR-7表达可导致人肺癌细胞的凋亡。这为后续基于miR-7的人肺癌基因靶向治疗策略的开发提供了前期实验依据。
[Abstract]:Objective to investigate the effect of thyroid transcription factor-1 TTF-1) promoter on apoptosis of human lung cancer cells in vitro by regulating the expression of miR-7. Methods the eukaryotic expression vector of miR-7 regulated by TTF-1 promoter (named p-T-miR-7) and the control vector p-cont were transiently transfected into human lung cancer 95D cells in vitro. The apoptosis of 95D cells was detected by FCM and Tunel. The expression of phosphorylated Caspase3 and phosphorylated Caspase9 in each group was detected by Western blot. The expression of CGGBP1 and EGFR was detected by confocal microscopy. Western blot was used to detect the expression of phosphorylated Akt and Erk protein in each group. Results after transfection in vitro for 48 hours, the expression of phosphorylated Akt and Erk protein was compared with that of p-cont transfection group. The ratio of apoptosis and phosphorylated Caspase3 and phosphorylated Caspase9 protein were significantly increased, while the expression of EGFR and CGGBP1 protein were significantly decreased in p-T-miR-7 transfected group. The level of phosphorylated Akt and Erk protein also decreased significantly. Conclusion the regulation of miR-7 expression by TTF-1 promoter can lead to apoptosis of human lung cancer cells, which provides a preliminary experimental basis for the further development of targeted therapy strategy for human lung cancer gene based on miR-7.
【作者单位】：贵州省遵义医学院免疫学教研室;贵州省普通高等学校特色药物肿瘤防治特色重点实验室;贵州省生物治疗人才基地;
【基金】：国家自然科学基金资助项目(No.31370918) 贵州省高层次人才项目(黔科合人才(2016)4031号) 贵州省教育厅特色重点实验室建设项目(黔教合KY字[2014]212)~~
【分类号】：R734.2

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