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

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.