RhoC在胃癌细胞中的表达及其小干扰RNA表达载体的构建与鉴定  

Expression of RhoC in gastric cancer cell lines and construction and identification of RhoC-specific siRNA expression vector

作　　者：刘娜[1] 毕锋[1] 潘阳林[1] 薛妍[1] 韩者艺[1] 刘长江[1] 樊代明[1] 

机构地区：[1]第四军医大学西京医院全军消化病研究所,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2004年第2期148-151,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金部主任基金资助 (No .30 1 40 0 2 6 );国家自然科学基金重点项目资助 (No .30 0 30 1 40 )

摘　　要：目的 :检测RhoC在胃癌细胞系中的表达 ,并构建其小干扰RNA(siRNA)表达载体。方法 :以Westernblot方法检测RhoC在多株胃癌细胞系中的表达。利用计算机辅助设计RhoC特异性siRNA ,体外合成siRNA基因并将其定向克隆入真核表达载体mU6pro。以脂质体法转染RhoC siRNA载体至高转移的人胃癌细胞系AGS中 ,以Westernblot方法鉴定RhoC siRNA对RhoC表达的作用。结果 :RhoC蛋白在具有高转移潜能的胃癌细胞系中表达增强。利用BbsⅠ和XbaⅠ双酶切法鉴定重组载体后 ,DNA测序证实合成的siRNA基因序列正确并已被准确克隆入mU6pro载体。RhoC siRNA载体可特异性抑制AGS细胞中RhoC的表达。结论 :RhoC特异性siRNA表达载体成功构建 。AIM: To study the expression of RhoC in human gastric cancer cell lines and to construct and identify the RhoC-specific siRNA expressing vector. METHODS: 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 expre ssion of RhoC in AGS cells was detected using Western blot. RESULTS: The expression level of RhoC was up-regulated in high metastatic cells lin es. 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. CONCLUSION: The RhoC-specific siRNA expression vector has been successfully constructed, which may provide a novel applicable strategy for gene therapy of gastric cancer.

关 键 词：RHOC 小干扰RNA 胃癌 转移 

分 类 号：Q782[生物学—分子生物学]