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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 screen small interfering RNA (siRNA) -transfected gastric cancer cell line AGS cells and screen the gene expression profiling by high-throughput microarray to lay a foundation for further study of RhoA signal transduction pathway. Methods: Recombinant plasmid of RhoA / siRNA was constructed by pSilencer3.1 vector. Effectene transfection reagent transfected AGS cells, empty vector pSilencer3.1 transfected control group. The transfected cell lines were screened by G418 to obtain stable transfected cells. The gene expression profile of the gene was detected by Agilent microarray in USA. Results: Gene chips detected the whole genome and found that 1151 genes were up-regulated and 1079 genes were down-regulated, which involved signal transduction, cell cycle and apoptosis related signaling molecules. Bioinformatics analysis showed that apoptosis-related caspase family, as well as cell cycle control genes were significantly different expression. Conclusion: Gene microarray results suggest that RhoA expression may play an important role in the regulation of RhoA-mediated abnormal proliferation signal transduction in RhoA cells and provide important information for further study on its signaling regulatory network Information.