Skp2靶向RNA干扰质粒的构建及其对人舌鳞癌Tca8113细胞的影响  

Construction of targeting-Skp2 shRNA plasmids and observation of their inhibitory effect on Tca8113 cells

作　　者：方亮[1] 胡勤刚[1] 华子春[2] 李淑锋[2] 

机构地区：[1]南京市口腔医院 南京大学医学院附属口腔医院口腔颌面外科,210008 [2]南京大学医药生物技术国家重点实验室

出　　处：《中华口腔医学杂志》2007年第10期624-628,共5页Chinese Journal of Stomatology

摘　　要：目的运用 RNA 干扰技术阻断人舌鳞状细胞癌 Tca8113细胞中 Skp2基因的表达,观察Skp2基因沉默后对 Tca8113细胞的影响。方法采用真核转录质粒 pRNAT-U6.1/Neo 构建针对 Skp2基因的重组转染质粒。经聚乙烯亚胺法将其转染 Tca8113细胞,通过反转录聚合酶链反应(reversetranscrip-tase polymerase chain reaction,RT-PCR)、Western blot 检测 Skp2、p27表达的变化;流式细胞仪、甲基噻唑基四唑(methyl thiazolyl terrazolium,MTT)法检测转染后 Tea8113细胞的细胞周期、生长速度的变化。结果转染重组质粒 Skp2shRNA-2、Skp2shRNA-3后,Tca8113细胞内 Skp2基因在 mRNA 和蛋白水平上均下调表达(P<0.01),p27基因蛋白水平上调表达(P<0.01);而各重组质粒转染后 p27基因的 mRNA 水平无明显变化。重组质粒 Skp2shRNA-2、Skp2shRNA-3转染 Tca8113细胞后,与对照组相比,G_1～G_0期细胞增加了约22%(P<0.01),G_2～M期和 S 期细胞减少了约10%和12%(P<0.01),细胞的生长速度明显变慢(P<0.01)。结论初步证明了 Skp2、p27基因在口腔鳞状细胞癌细胞分化增殖中所扮演的重要角色;筛选出了高效 RNA 干扰重组质粒 Skp2shRNA-2、Skp2shRNA-3,为进一步研究以 Skp2基因为靶点的人舌鳞状细胞癌基因治疗奠定了基础。Objective To construct the recombinant plasmids expressing Skp2 short hairpin RNA （shRNA） by pRNAT-U6. 1/Neo plasmid vector and observe the effects of RNAi-mediated Skp2 gene silencing on Tca8113 cells. Methods Five recombinant eukaryotic expression vectors were successfully constructed using pRNAT-U6. 1/Neo plasmid vector separately. After they were transfected into Tca8113 ceils with PEI, the interference effects no Skp2 and p27 were detected by RT-PCR and Western blot. The ceil cycle of Tca8113 cells were tested by flow cytometry. The proliferation of Tca8113 cells were examined by MTT. Results In Skp2shRNA-2 and Skp2shRNA-3 vectors, the expression of Skp2 protein of Tca8113 cells was down-regulated and p27 protein up-regulated（P 〈 0. 01 ）. The cell number during G1/G0 phases increased 22% （ P 〈 0. 01 ） and during G2/M and S phases the number decreased 10% and 12% （ P 〈 0.01）. The proliferation of Tca8113 ceils slowed down and the cells number decreased （P 〈 0.01 ）. Conclusions Skp2shRNA-2 and Skp2shRNA-3 vectors of shRNA for Skp2 were successfully constructed. They could influence expression of Skp2 and p27 gene. Skp2 may be a promising target of gene therapy on human tongue squamous cell carcinoma.

关 键 词：癌 鳞状细胞 舌肿瘤 基因疗法 

分 类 号：R739.8[医药卫生—肿瘤]