RNA干扰Skp2基因抑制T47D乳腺癌细胞生长

RNA interference of Skp2 inhibits the growth of breast tumor cell T47D

目的构建S期激酶相关蛋白2（skp2）RNA干扰真核细胞表达载体，研究skp2基因沉默对乳腺癌T47D细胞生长的抑制作用。方法应用Ambion公司在因特网上的设计程序设计小干扰RNA。构建3个不同的重组体转染T47D细胞，RT-PCR和Western Blotting检测skp2基因表达，细胞计数分析Skp2RNA干扰对肿瘤细胞增殖的影响。结果重组体经PCR和测序证实准确连接。转染重组体的细胞skp2的表达在mRNA和蛋白水平均明显下降。重组体转染的细胞增殖明显降低。结论用RNA干扰下调skp2表达抑制乳腺癌细胞生长，skp2可能是乳腺癌治疗的潜在靶点。

Objective To construct plasmids for eukaryotic expressions of shRNA （short hairpin RNA） of S - phase kinase-associated protein-2 and investigate if knockdown of Skp2 by RNAi would inhibit T47D cell growth. Methods siRNAs targeting human Skp2 mRNA were designed with a web program provided by Ambion. Three Skp2 siRNAS were recombinated and transient transfected into human breast cancer T47D cells in vitro. Expressim of Skp2 was examined by RT - PCR and Western blotting. The proliferation activities of transfected cells were assayed by counting cells. Results The structures of recombinant plasmids were comfinaed by PCR and sequencing. Skp2 mRNA and protein expression were decreased by Skp2 siRNA transfection in T47D cells. Skp2 siRNA inhibited the T47D cell proliferation in vitro. Conclusion Downregulation of Skp2 expression by RNAi inhibits breast cancer cell growth. Skp2 might be a potential therapeutic target for human breast cancer.