FAK-shRNA重组逆转录病毒载体的构建及稳定表达细胞株的筛选

Construction of FAK- shRNA retroviral vector and screening of stable HCC-LM3 cell line with persistent knockdown of FAK

目的:研究利用RNA干扰技术,以黏附斑激酶(FAK)为靶基因,构建FAK-shRNA重组逆转录病毒载体,将其导入包装细胞Phoenix中,筛选出稳定产生FAK-shRNA病毒的细胞克隆,以病毒上清感染并筛选FAK表达沉默的细胞株,观察其对相关蛋白表达的影响。方法:体外合成能转录产生靶向FAK短发夹RNA(shRNA)的寡核苷酸并定向克隆入pSuper.retro逆转录病毒载体,以脂质体法转染Phoenix细胞株,待筛选稳定克隆成功后收获病毒上清,感染人肝癌细胞株HCC-LM3,以嘌呤霉素筛选得到抑制FAK表达的稳定细胞株后用Westernboltting鉴定FAK表达的抑制效果及相关蛋白表达情况。结果:构建了重组逆转录病毒载体pSuper-FAK并抑制了人肝癌HCC-LM3细胞内FAK蛋白的表达。在下调FAK表达的细胞株中p-Akt和p-MAPK1/2表达明显受到抑制。下调FAK的细胞株迁移和侵袭能力下降,细胞周期多被阻止在G_0/G_1期,细胞凋亡增多,增殖率明显下降。结论:重组逆转录病毒载体pSuper-FAK转染包装细胞Phoenix后,其产生的FAK-shRNA病毒可以抑制HCC-LM3细胞内的FAK蛋白表达并抑制Akt及MAPK1/2磷酸化。下调FAK后可以对肿瘤细胞的生物学行为产生明显影响。

AIM：To construct a recombinant retroviral vector of short interfering RNA targeting focal adhesion kinase（FAK）gene and to establish a cell line with stable knockdown of FAK.METHODS：The oligonucleotides that transcribed to short hairpin RNA（shRNA）targeting FAK gene were synthesized in vitro,cloned into retroviral vector pSuper. retro and transfected into Phoenix cell line.The stable clones were screened and high-titer virus was produced.The human hepatocellular carcinoma cell line HCC-LM3 was infected with the virus-rich supernatant.The stable LM3 cell line,which showed significantly to silence FAK and associated proteins,was selected by puromycin.RESULTS：The recombinant retroviral vector was successfully constructed.Persistent knockdown of FAK in the LM3 cell line infected with the supernatant containing the retrovirus was confirmed by Western blotting.Down-regulation of FAK resulted in the inhibition of p-Akt and p-MAPK1/2 expression and led to decreased migration and invasion of the cells.The cell cycle was blocked at G_0/G_1 phase,and apoptosis was increased.The proliferation rate also decreased significantly.CONCLUSION： FAK-shRNA virus generated by recombinant retroviral vector pSuper-FAK can inhibit the protein expression of FAK and phosphorylation of Akt and MAPK1/2 in HCC-LM3 cells.Down-regulation of FAK shows a significant impact on biological behaviors of tumor cells.