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

作　　者：卢佳[1,2,3] 陈超[1,3] 廖珍媛[1] 崔盼盼[1] 雷良玉 徐华林[1] 田丹[1,3] 郑静[1,3] 徐林[1,2,3] 

机构地区：[1]贵州省遵义医学院免疫学教研室 [2]贵州省普通高等学校特色药物肿瘤防治特色重点实验室 [3]贵州省生物治疗人才基地,贵州遵义563000

出　　处：《西安交通大学学报（医学版）》2017年第2期176-181,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

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)~~

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。Objective To investigate the effect of thyroid transcription factor-1（TTF-1）promoter regulated miR-7 expression on the apoptosis of human lung cancer 95 D cells in vitroand 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 95 D 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 95 D 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 cancer95 D cells in vitro,respectively.The expressions of phosphorylated Akt and Erk protein in each group were assayed by Western blot.Results At 48 h 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-7expression 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.

关 键 词：miR-7 甲状腺转录因子-1(TTF-1) 启动子 基因治疗 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]