Liu Na, Bi Feng, Pan Yang-lin, Xue Yan, Han Zhe-yi, Liu Chang-jiang, Fan Dai-ming
Institute of Digestive Disease, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Mar;20(2):148-51.
To study the expression of RhoC in human gastric cancer cell lines and to construct and identify the RhoC-specific siRNA expressing vector.
The expression of RhoC in human gastric cancer cell lines was detected by Western blot. According to the computer aided design(CAD),RhoC-specific siRNA gene was synthesized and cloned into the expression vector mU6pro. The constructed RhoC-siRNA was transiently transfected into high-metastatic gastric cancer cell line AGS and the inhibition effect of RhoC-siRNA on expression of RhoC in AGS cells was detected using Western blot.
The expression level of RhoC was up-regulated in high metastatic cells lines. Double enzyme digestion analysis and DNA sequencing confirmed that the RhoC-specific siRNA expression vector was constructed successfully. RhoC expression in AGS cells was specifically suppressed after transfection of RhoC-siRNA.
The RhoC-specific siRNA expression vector has been successfully constructed, which may provide a novel applicable strategy for gene therapy of gastric cancer.
研究RhoC在人胃癌细胞系中的表达,并构建和鉴定RhoC特异性小干扰RNA(siRNA)表达载体。
采用蛋白质免疫印迹法检测RhoC在人胃癌细胞系中的表达。根据计算机辅助设计(CAD),合成RhoC特异性siRNA基因并克隆至表达载体mU6pro。将构建成功的RhoC-siRNA瞬时转染至高转移人胃癌细胞系AGS,采用蛋白质免疫印迹法检测RhoC-siRNA对AGS细胞中RhoC表达的抑制作用。
RhoC在高转移细胞系中的表达水平上调。双酶切分析及DNA测序证实RhoC特异性siRNA表达载体构建成功。转染RhoC-siRNA后,AGS细胞中RhoC的表达受到特异性抑制。
成功构建了RhoC特异性siRNA表达载体,为胃癌的基因治疗提供了一种新的可行策略。