Construction of eukaryotic expression plasmid pcDNA3.1(+)-A1CF and its expression in lung cancer

Objective To construct an eukaryotic expression plasmid Apobec-1 complementary factor (A1CF) and its expression in lung cancer A549 and H1299 cells after transfection. Methods Total RNA was extracted from renal cancer 786-o cells. Then, human A1CF gene was amplified by RT-PCR and inserted into pcDNA3.1(+) vector by the enzyme digestion method. The reconstructed plasmid was digested with BamHI and Hind Ⅲ and confirmed by sequencing analysis, before transfection into lung cancer cells by Lipofectamine 2000. The expression of A1CF was detected by Western blot. The effect of A1CF on the migration and invasion of lung cancer was observed with the wound healing assay and Transwell assay. Results The results of enzyme digestion and DNA sequencing showed that the sequence of fragments inserted in pcDNA3.1(+)-A1CF was consistent with human A1CF sequence. Western blot results showed that the protein expression of A1CF in A549 and H1299 cells significantly increased and the expression of E-cadherin and P-ERK increased (P＜0.05). Conclusions The cDNA of human A1CF gene is successfully cloned and the eukaryotic expression plasmid pcDNA3.1(+)-A1CF was constructed. A1CF promotes the migration of lung cancer cells, which may be associated with phosphorylation of ERK.