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目的探讨干扰RNA沉默生存素(survivin)基因表达对人胃癌BGC-823细胞增殖和凋亡的影响。方法设计并合成3条靶向survivin的小分子干扰RNA(siRNA),构建表达性干扰RNA质粒(shRNA)——shRNA-survivin-1、shRNA-survivin-2和shRNA-survivin-3,分别转染胃癌BGC-823细胞,实时定量PCR检测干扰RNA沉默survivin mRNA表达效果,Westernblot观察对胃癌BGC-823细胞survivin蛋白质表达的抑制,MTT(四甲基偶氮唑盐)比色法分析检测细胞生长抑制率,流式细胞计数检测各组细胞周期和凋亡率,探讨干扰RNA对胃癌BGC-823细胞生长的影响。结果在体外,shRNA-survivin-1有效沉默人胃癌BGC-823细胞survivin mRNA的表达,使sur-vivin mRNA相对水平明显降低(P<0.05),survivin蛋白质表达抑制,72h细胞生长抑制率达74.92%(P<0.05),shRNA-survivin-1使G2/M期细胞百分比明显增加,凋亡率显著增加(P<0.05)。结论 shRNA-survivin-1可以沉默survivin基因的表达,可以显著抑制胃癌BGC-823细胞的增殖,在一定程度上诱导其自发凋亡。本研究为靶向sur-vivin的RNA干扰在胃癌的基因治疗提供了有力的理论依据和技术储备。
Objective To investigate the effects of survivin gene silencing on the proliferation and apoptosis of human gastric cancer cell line BGC-823. Methods Three small interfering RNAs (siRNAs) targeting survivin were designed and synthesized. ShRNA-survivin-1, shRNA-survivin-2 and shRNA-survivin-3 were constructed and transfected separately The expression of survivin mRNA was detected by real-time quantitative PCR in gastric cancer cell line BGC-823. The protein expression of survivin in gastric cancer cell line BGC-823 was detected by Western blot. The cell growth inhibition was detected by MTT assay The cell cycle and apoptosis rate of each group were detected by flow cytometry to explore the effect of interfering RNA on the growth of BGC-823 gastric cancer cells. Results In vitro, shRNA-survivin-1 effectively silence survivin mRNA expression in human gastric cancer BGC-823 cells, the relative level of sur-vivin mRNA was significantly decreased (P <0.05), the expression of survivin protein was inhibited, and the cell growth inhibition rate was 74.92% (P <0.05). The percentage of cells in G2 / M phase was significantly increased by shRNA-survivin-1, and the apoptosis rate was significantly increased (P <0.05). Conclusion shRNA-survivin-1 can silence the expression of survivin gene and can significantly inhibit the proliferation of gastric cancer BGC-823 cells and induce its spontaneous apoptosis to a certain extent. This study provides a strong theoretical basis and technical reserve for gene therapy of gastric cancer targeting sur-vivin RNA interference.