摘要
目的:探讨RNA干扰技术对人食管癌EC9706细胞哺乳动物雷帕霉素靶蛋白(mTOR)的表达及细胞生长与凋亡的影响。方法:设计合成mTORsiRNA,采用低、中、高浓度(50、100与150nmol/L)的mTORsiRNA分别转染EC9706细胞24、48与72h,同时设无义对照组(转染无义对照siRNA)、空白对照组(转染空脂质体)及正常对照组(不转染)。采用免疫细胞化学与原位杂交法分别检测各组EC9706细胞中mTOR蛋白与mRNA的表达;应用MTT法检测细胞增殖,计算细胞生长抑制率;应用TUNEL法检测转染24h时细胞凋亡,计算凋亡指数(AI)。结果:转染24、48与72h,6组细胞mTOR蛋白和mRNA的表达差异均有统计学意义(mTOR蛋白:F=24.14,45.78,59.19,P均<0.001;mTORmRNA:F=41.42,69.74,43.71,P均<0.001),细胞生长抑制率差异亦有统计学意义(F=143.85,172.98,155.01,P均<0.001);低、中、高浓度组转染不同时间点间比较,mTOR蛋白(F=42.23,29.46,50.22,P均<0.001)、mTORmRNA(F=6.48,8.50,4.80,P均<0.05)及细胞生长抑制率(F=78.77,76.14,52.28,P均<0.001)差异均有统计学意义。mTORsiRNA转染组EC9706细胞mTOR蛋白与mRNA的表达均低于各对照组,且mTOR蛋白及mRNA的表达随mTORsiRNA浓度的增加及转染时间的延长而减弱(P<0.05)。不同浓度mTORsiRNA转染组EC9706细胞生长抑制率均高于各对照组,且细胞生长抑制率随mTORsiRNA浓度的增加和转染时间的延长而升高(P<0.05)。转染24h,6组细胞AI相比,差异有统计学意义(F=442.96,P<0.001),mTORsiRNA转染组AI均高于各对照组,且高浓度转染组AI高(P<0.05)。结论:mTORsiRNA可有效抑制EC9706细胞mTOR蛋白与mRNA的表达,且能抑制EC9706细胞的增殖并促进其凋亡。
Aim：To explore the effect of mTOR siRNA on the expression of mTOR gene,cell growth and apoptosis in human esophageal carcinoma EC9706 cells.Methods：The oligonucleotide templates of mTOR siRNA were designed firstly.Three different concentrations of mTOR siRNA（50,100 and 150 nmol/L） was used to transfect EC9706 with different time exposure（24,48 and 72 h）,and the control groups were established accordingly by using siRNA with insignificant order,liposome only and no transfection.The level of mRNA and protein of mTOR in the 6 groups was measured by in situ hybridization and immunocytochemistry.MTT method was used to detect the cell proliferation,and TUNEL method was used to detect the apoptosis.Results：There were significant differences in the expressions of mTOR mRNA and protein and the growth inhibition rate of EC9706 cells among the 6 groups at 24,48,and 72 h（protein：F=24.14,45.78,59.19,P0.001;mRNA：F=41.42,69.74,43.71,P0.001;the growth inhibition rate：F=143.85,172.98,155.01,P0.001）.There were significant differences in the expressions of mTOR mRNA and protein and the growth inhibition rate of EC9706 cells in each mTOR siRNA transfection groups at different transfection time（protein：F=42.23,29.46,50.22,P0.001;mRNA：F=6.48,8.50,4.80,P0.05;the growth inhibition rate：F=78.77,76.14,52.28,P0.001）.The expressions of mTOR mRNA and protein in the groups transfected with mTOR siRNA were lower than those of the control groups,which weakened with the increase of siRNA concentration and time exposure（P0.05）.Compared with the control groups,the growth inhibition rate of EC9706 cells transfected with mTOR siRNA for different time exposure was higher（P0.05）,and increased with the increase of siRNA concentration and time exposure（P0.05）.There were significant differences in the apoptosis index among the 6 groups（F=442.96,P0.001）.The apoptosis index of EC9706 cells transfected with different concentrations of mTOR siRNA for 24 h exposure was higher than those of the control groups（P0.05）,and increased with the increase of siRNA concentration.Conclusion：RNA interference of mTOR in EC9706 cell line could effectively knock down the expression of mTOR,which could significantly inhibit the cell proliferation and promote cell apoptosis.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2010年第1期11-15,共5页
Journal of Zhengzhou University(Medical Sciences)
基金
河南省杰出青年科学基金资助项目074100510009
河南省科技攻关计划基金资助项目082102310011
河南省基础与前沿技术研究计划基金资助项目092300410016
郑州市科技创新团队基金项目课题
