Construction of TFAP2α plasmid and its influence on cell proliferation of clear cell renal cancer

Objective To build the plasmid of pLV-EGFP (2A) puro-TFAP2α (transcription factor AP-2 alpha) and it stable cell line and observe the effect of TFAP2α overexpression on the cell proliferation of clear cell renal cell carcinoma Methods TFAP2α gene coding sequence was amplified from 293T cell's DNA by reverse transcription polymerase chain reaction (RT-PCR). The plasmid pLV-EGFP puro-TFAP2α was reconstructed. The stable transfection cell lines, 786-O and Caki-2 were built by lentivirus packaging system. The expression of TFAP2α was detected by real time quantitative PCR and Western Blot. It effect on cell proliferation was observed through MTS assay, colony formation assay and cell cycle analysis. Results The plasmid pLV-EGFP (2A)-TFAP2α and its stable cell lines were successfully constructed. The expression of TFAP2α in the recombinan plasmid group up-regulated significantly when compared with untransfected group and empty plasmid group (P＜ 0.05). MTS assay showed that the absorbance at 490 nm in recombinant plasmid group reduced significantly (P＜ 0.05) and colony formation assay showed that cell colony numbers also decreased (P＜ 0.05) compared with untransfected group and empty plasmid group. The percentage of G₀/G₁phase cells in recombinant plasmid group was significantly higher than untransfected group and empty plasmid group (P＜ 0.05), and the percentage of S phase cells decreased significantly (P＜ 0.05). Conclusion Overexpression of TFAP2α can significantly reduce cell proliferation of 786-O and Caki-2 cell, through blocking the cell cycle at G₀/G₁phase.