雄激素受体基因慢病毒干扰载体的构建及在肝癌细胞中的表达

Construction of androgen receptor lentiviral interference plasmid and its expression in Hepatoma carcinoma cells

目的:构建敲减人雄激素受体(AR)基因的慢病毒质粒,转染Hep3B细胞使其表达目的蛋白并探索其对PTEN、p-AKT蛋白表达的影响。方法:设计合成3对针对AR基因特异性敲减的寡核苷酸片段,分别连接于重组慢病毒载体pLKO.1,构建3条AR干扰载体。利用验证正确的3个慢病毒载体转染293T细胞得到慢病毒上清液。采用RT-PCR和Western blot在5株肝癌细胞中挑选AR高表达的细胞系进行AR慢病毒上清液感染。RT-PCR和Western blot检测3条AR干扰质粒在Hep3B细胞内的表达及其对PTEN、p-AKT蛋白表达的作用。结果:测序鉴定证实3条AR慢病毒干扰载体构建成功,干扰Hep3B细胞后,RT-PCR和Western blot结果显示其中1条AR干扰后的mRNA和蛋白表达明显降低,细胞内PTEN蛋白表达升高,p-AKT表达降低。结论:成功构建3条AR干扰载体,其中1条干扰效率较高,能在Hep3B细胞中干扰AR的表达,感染细胞后上调抑癌基因PTEN的蛋白表达对p-AKT有抑制作用,提示AR和PTEN/AKT信号通路可能具有相关性。

Objective:Androgen receptor(AR)genes knockdown lentiviral plasmids were constructed,and transfected into Hep3B cells to express target protein and to explore its effect on protein expressions of PTEN and p-AKT.Methods:Three pairs of oligonucleotide fragments specific to AR gene knockdown were designed and synthesized,and respectively ligated to recombinant lentiviral vector pLKO.1 to construct three AR interference vectors.293T cells were transfected with three correct lentivirus vectors to obtain lentivirus supernatant.Cell lines with high AR expression were selected and infected with AR lentivirus supernatant by RT-PCR and Western blot.RT-PCR and Western blot were used to detect expressions of three AR interfering plasmids in Hep3B cells and their effects on expressions of PTEN and p-AKT protein.Results:Sequencing identification confirmed the successful construction of three AR lentiviral interference vectors.After interference of Hep3B cells,RT-PCR and Western blot results showed that mRNA and protein expressions of one AR interference vector decreased significantly,and showed up with high PTEN expression and lower p-AKT expression.Conclusion:Three AR lentivirus interference vectors have been successfully constructed,one of which had high interference efficiency and could interfere with expression of AR in Hep3B cells.After infection,protein expression of tumor suppressor gene PTEN is up-regulated and p-AKT inhibited,suggesting that AR and PTEN/AKT signaling pathway may be correlated.