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 G0/G1 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.%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Western boltting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G0/G1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.
展开▼
