RNA干扰hTERT基因对人乳腺癌细胞株MCF-7细胞周期及凋亡影响的研究
Effect of human breast cancer line MCF-7 cell cycle and apoptosis using RNAi hTERT mRNA
目的:观察载体介导的RNA干扰技术能否有效抑制人乳腺癌细胞株MCF-7的hTERT的表达及其对细胞周期及凋亡的影响。方法:构建2个针对hTERT基因表达短发夹状siRNA的真核表达载体(pshRNA-hTERT)并转染人乳腺癌细胞株MCF-7,通过逆转录-聚合酶链反应(RT-PCR)检测该基因mRNA,western blot蛋白质检测,端粒酶活性检测,流式细胞仪检测细胞周期及凋亡等方法检测RNAi效果,从中筛选出基因沉默效果好的靶位点。结果 酶切与测序证实真核表达载体pshRNA –hTERT1,2,3构建成功。转染乳腺癌细胞后,pshRNA - hTERT1,2质粒均能抑制hTERT基因表达,mRNA表达分别下调了54.6%,55.2%;蛋白表达分别下调46.6%,47.8%。端粒酶活性均明显下降。对乳腺癌细胞周期中S期细胞明显减少,G1/G0期细胞显著增加。结论:RNAi在体外明显抑制乳腺癌细胞中hTERT基因的表达和瘤细胞增殖。
Objective:To investigate whether RNA interference(RNAi) induced by plasmid vector could suppress hTERT expression in human breast carcinoma cells MCF-7 and the effect on cell cycle and apoptosis. Methods:The small hairpin RNA(shRNA) eukaryotic expression vector targeting hTERT gene,named pshRNA-hTERT-1/pshRNA-hTERT-2,were constructed and transfected into cultured human breast carcinoma cell of line MCF-7 via GenEscort™Ⅱ;The inhibited effects on hTERT gene were determined by semi-quantitative reverse transcription PCR detecting hTERT-mRNA, western blot detecting its protein expression, TRAP-Hyb Kit detecing telomerase activation and flow cytometry analysis Cell cycle and apoptosis .Then screening gene silencing effective site. Results:The successful construction of pshRNA-hTERT1,2,3 plasmid was identified with sequencing.After the shRNA expression vector was successfully transfected into MCF-7 cells via GenEscort™Ⅱ, pshRNA-hTERT1,2 reduced hTERT gene expression by compared with the control group, hTERT–mRNA expression decrease 54.6%,55.2%,protein expression decrease 46.6%,47.8%.Telomerase activation decrease significantly. Cell cycle analysis showed accumulation of cells in S phase significant decrease , G0-G1 phase significant increase. Conclusions:RNAi obviously inhabit hTERT gene expression of the breast cancer cell line MCF-7 and cell proliferation.
王淑英、许景伟、樊丽
肿瘤学基础医学分子生物学
乳腺癌细胞MCF-7端粒逆转录酶RNA干扰
breast cancer cell MCF-7hTERTRNAi
王淑英,许景伟,樊丽.RNA干扰hTERT基因对人乳腺癌细胞株MCF-7细胞周期及凋亡影响的研究[EB/OL].(2009-04-06)[2025-08-18].http://www.paper.edu.cn/releasepaper/content/200904-152.点此复制
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