RhoCsiRNA表达载体构建及其对肝癌细胞体外侵袭能力影响.docVIP

RhoCsiRNA表达载体构建及其对肝癌细胞体外侵袭能力影响 　 作者：郑文建 梁平 赵弘智 韩克强 【摘要】 目的 构建RhoCsiRNA表达载体，研究其对肝癌细胞体外侵袭潜能的影响。方法 以脂质体转染法稳定转染人SKHep1肝癌细胞株，Western印迹鉴定转染细胞中RhoC蛋白表达的抑制情况，观察转染前后细胞黏附、运动及体外侵袭能力的改变。结果 酶切及测序证实RhoCsiRNA真核表达载体构建成功。通过Western印迹检测证实，转染RhoCsiRNA表达载体的肝癌细胞中RhoC蛋白表达抑制达60%，其黏附、移动及体外侵袭能力均降低。结论 RhoCsiRNA表达载体能有效抑制RhoC在肝癌细胞株SKHep1中的表达，同时抑制细胞的侵袭转移潜能，为肝癌的生物学治疗提供新的方法。 【关键词】 肝肿瘤； 侵袭； RhoCsiRNA 【Abstract】 Objective To construct a RhoCsiRNA expression vector and study its role in invasion and metastasis ability of hepatoma carcinoma cells (HCC). Methods RhoCsiRNA gene was synthesized and cloned into the expression vector pSilencer 2.1. The constructed RhoCsiRNA expression vector was stably transfected into SKHep1 of HCC. By using Western blotting, the inhibitive effect of RhoCsiRNA on expression of RhoC in transfected HCC was detected, the ability of cell adhesion, cell migration, and cell invasion before and after transfection were observed too. Results Enzyme digestion and DNA sequencing confirmed that the RhoC specific siRNA expression vector was constructed successfully. After transfection, the RhoC expression was inhibited by 60%, and the ability of cell adhesion, cell migration, and cell invasion was decreased markedly, which was proved by Western blotting. Conclusions RhoCsiRNA expression vector can effectively suppress RhoC expression in SKHep1 of HCC and decrease potential of cell invasion and cell metastasis, which may provide a novel applicable strategy for gene therapy of HCC. 【Key words】 Liver tumors; Invasion; RhoCsiRNA Rho(ras homologous)基因亚家族成员(RhoA、B、C)是一类与ras同源的小GTP结合蛋白，Rho亚家族蛋白的异常表达可能与恶性肿瘤的侵袭、转移有关。有资料表明，RhoC基因产物的过量表达与肝癌发展及侵袭、转移有密切关系［1］。本研究通过构建RhoC特异性siRNA真核表达载体，并稳定转染到肝癌细胞株SKHep1中，以探讨抑制RhoC蛋白表达对肝癌细胞侵袭转移能力的影响。 1 材料与方法 1.1 材料 1.1.1 细胞株、菌株及质粒：人肝癌细胞株SKHep1购自中科院上海细胞所，大肠杆菌DH5α感受态细胞由新桥医院中心实验室保存，质粒pSilencer 2.1为Ambion公司产品。 1.1.2 酶和试剂：新生牛血清购自PAA，RPMI 1640购自Hyclone，G418购自BPI，DOTAP购自Roche，羊抗人RhoC多克隆抗体购自Santa Cruz，HRP标记的兔抗羊二抗购自北京中杉公司，质粒小量抽提试剂盒、琼脂糖凝胶回收试剂盒购自中鼎公司，