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
Effect of TTF-1 promoter regulated miR-7 expression on the apoptosis of human lung cancer 95D cells in vitro

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(P<0.05)；磷酸化Caspase3和磷酸化Caspase9蛋白水平均显著增加(P<0.05)，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(P<0.05)。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 regulated miR-7 expression on the apoptosis of human lung cancer 95D cells in vitro and to explore its significance.Methods A eukaryotic expression vector of TTF-1 was detected by FCM, and the apoptosis of cells was analyzed by TUNEL assay. Furthermore, the expression levels of phos-Casp-3 and phos-Casp-9 protein in 95D cells were detected by Western blot. Meanwhile, the expression of CGGBP1 and EGFR, twotarget molecules of miR-7, were examined by immunofluorescence assay confocal microscopy. Finally, the expression of phosphorylated Akt and promoter regulation miR-7(p-TmiR-7) and control vector p-Cont which had been previous constructed were transiently transfected into the human lung cancer 95D cells in vitro, respectively. TTF-1 promoter regulated miR-7 eukaryotic expression vector (named p-TmiR-7) and control vector p-Cont were transiently transfected into human lung cancer 95D cells in vitro, respectively. The expressions of phosphorylated Akt and Erk protein in each group were assayed by Western blot. Results At 48h after transfection in vitro, the apoptosis ratio of cells in p-T-miR-7 transfection group significantly increased compared with that in p-Cont transfection group (P<0.05). Meanwhile, the expression levels of phos-Casp-3 and phos Casp-9 protein significantly increased (P<0.05). Furthermore, the expressions of EGFR and CGGBP1 protein decreased obviously, accompanied by decreased expressions of phos-Akt and phos-Erk in p-T-miR-7 transfection group (P<0.05). Conclusion TTF-1 promoter regulated miR-7 expression can induce the apoptosis of human lung cancer cells in vitro, which provides some experimental basis for the development of human lung cancer gene targeting therapeutic strategy