目的:应用RhoA小干扰RNA(small interfering RNA,siRNA)转染胃癌细胞系AGS细胞,采用高通量基因芯片技术,筛选基因表达谱的变化,为进一步研究RhoA信号转导通路奠定基础。方法:用pSilencer3.1载体构建RhoA/siRNA重组质粒。Effectene转染试剂转染AGS细胞,空载体pSilencer3.1转染对照组。应用G418筛选转染细胞系,获得稳定转染细胞;美国Agilent基因芯片检测细胞基因表达谱变化。结果:基因芯片检测全基因组,发现表达上调的有1151个基因,表达下调的有1079个基因,涉及信号转导、细胞周期、细胞凋亡等相关信号分子。生物信息学分析显示,与细胞凋亡有关的caspase家族,以及细胞周期调控基因存在明显的差异表达。结论:基因芯片结果提示干扰RhoA表达后,与凋亡和增殖有关的分子在RhoA介导的肿瘤细胞异常增殖信号转导中起着重要的调控作用,为下一步研究其信号调控网络机制提供重要的信息。
Objective :To analyse the alterations of gene expression in gastric cancer cell line AGS transfected with RhoA small interfering RNA (siRNA) by DNA gene microarray technology. Methods: The recombinant RhoA/siRNA plasmid pSilencer 3.1 was constructed and transfected into gastric cancer cell line AGS with effectene reagent. pSilencer 3.1 was transfected with the same cell line as the control . The two groups were subjected to G418 selection. The gene expression profile was analyzed by Agilent Gene Chip. Results : The gene microarray chip demonstrated that 1151 genes were up -regulated and 1079 genes down -regulated, which involved in signal transduction, cell cycle , apoptosis et al. Bioinformation analysis showed that the caspase family which related to cell apoptosis, the cell cycle related molecules,were important in RhoA mediated signal transduction. Conclusion: The gene microarray is a good method with advantages of high flux and high efficiency , which brought some new clues to study the molecular mechanism of RhoA signaling in cancer .