APE1shRNA重组质粒的构建及对人乳腺癌细胞生长的影响

Construction of APE1 gene specific short hairpin RNA expressing plasmid and effect on growth of human breast carcinoma cell

目的设计构建靶向脱嘌呤脱嘧啶核酸内切酶1(APE1/ref-1)特异性短发卡RNA的真核表达载体,并转染人乳腺癌T47D细胞。方法设计、合成针对APE1的特异性的短链寡核苷酸,构建APE1特异性shRNA的重组质粒,稳定转染人乳腺癌T47D细胞;采用聚合酶链反应和免疫印迹法检测转染前后T47D细胞中APE1的表达。采用四甲基偶氮唑盐法观察T47D细胞的生长情况。结果经酶切和测序鉴定,成功构建APE1特异性shRNA的重组质粒,并能够转染T47D细胞。转染后,APE1在mRNA和蛋白水平表达分别下降约53.1%和60.5%,T47D细胞增殖活性受到抑制。结论运用pGenesil-1.1质粒载体构建的靶向APE1特异性shRNA的重组质粒可以成功转染T47D细胞,有效抑制APE1的表达,并抑制肿瘤细胞的生长。

Objective To investigated the effect of short hairpin RNA（shRNA） targeting Apurinic/Apyrimidinic Endonuclease 1（APE1） on the human breast carcinoma T47D cell line.Methods The shRNA targeting APE1 was constructed and transfected into T47D cells through LipofectamineTM 2000.The mRNA and protein levels of APE1 were detected by RT-PCR and Western-blot.Cell proliferation was examined by MTT.Results The APE1-shRNA expression plasmid was successfully constructed and transfected into T47D cells.The expression inhibition rate was about 53.1% at mRNA level and the expression inhibition rate was about 60.5% at protein level.The cell proliferation was also lower because of the APE1-shRNA.Conclusions An APE1-shRNA expression,which we constructed by pGenesil-1.1,can be sucessfully transfected into T47D cells.It can inhibit the expression of APE1,suppress the growth of T47D cells.