Rac1-siRNA表达质粒的构建及Rac1对SW480细胞单克隆形成的影响  

Effect of small interfering RNA targeting Rac1 gene on colony formation of SW480 cells in vitro

作　　者：南清振[1] 高蕾 肖冰[1] 张振书[1] 姜泊[1] 

机构地区：[1]南方医科大学南方医院消化系疾病研究所,广东广州510515 [2]广州中医药大学第二附属医院放疗科,广东广州510120

出　　处：《南方医科大学学报》2010年第6期1339-1342,共4页Journal of Southern Medical University

摘　　要：目的构建针对Rac1的siRNA表达载体,并观察其对大肠癌SW480细胞中Rac1蛋白表达的抑制作用,同时研究Rac1蛋白表达抑制后细胞在体外软琼脂糖中单克隆形成情况。方法化学合成用于产生针对Rac1的发卡状RNA的寡核苷酸,各64个碱基,退火后插入线性化的pSUPER载体的H1启动子下游。重组载体经限制性酶切和测序。把构建的载体转染大肠癌细胞系SW480,观察其对Rac1蛋白表达的干扰作用,软琼脂糖体外克隆形成实验研究SW480抑制Rac1蛋白表达后的效果。结果限制性酶切和序列测定表明成功构建了可以表达针对Rac1的发卡状RNA表达载体,该载体表达的发卡状RNA在SW480细胞中能够有效的下调Rac1蛋白的表达。体外软琼脂单克隆形成实验显示,抑制Rac1蛋白表达后,形成的单克隆数目明显减少,直径也明显缩小。结论成功构建了针对Rac1的发卡状RNA表达载体;体外软琼脂单克隆实验表明Rac1蛋白对SW480细胞克隆形成数目及大小有明显影响,说明Rac1蛋白在大肠癌的体外生长过程中具有重要作用。Objective To construct a vector expressing small interfering RNA（siRNA） against Rac1 gene and observe its effect on soft agar colony formation of SW480 cells in vitro.Methods Oligos of 64 base pairs for hairpin RNA targeting Rac1 were chemically synthesized and annealed.The siRNA constructs for Rac1,produced by inserting the annealed oligos into the downstream of H1 promoter of linearized pSUPER,were confirmed by restriction digestion and DNA sequencing.The constructed Rac1-siRNA was transfected into SW480 cells and Western blotting was performed to assess the expression and interference efficiency of siRNAs against Rac1.The soft agar colony formation assay was used to study the effect of Rac1 gene silencing on SW480 cells.Results Restriction digestion and DNA sequencing showed that the siRNA targeting Rac1 gene was successfully constructed.The siRNA could effectively down-regulate the expression of Rac1 in SW480 cells.Soft agar colony formation assay showed that the colony number and diameter of SW480 cells was reduced after siRNA transfection.Conclusion A vector expressing hairpin RNA against Rac1 gene are successfully produced,which significantly reduces the colony numbers and size of SW480 cells in vitro,suggesting that Rac1 plays an important role in the growth of colorectal cancer in vitro.

关 键 词：RAC1 RNA干扰 软琼脂单克隆形成 SW480细胞 

分 类 号：R735.3[医药卫生—肿瘤]