Effects of inhibitor of growth 4 (ING4) gene on the proliferation of melanoma cell line M14 / 中华皮肤科杂志

ABSTRACT

Objective To construct the pCDNA3.1-ING4 eukaryotic expression vector and investigate its effect on the proliferation of melanoma cell line M14. Methods The targeted cDNA fragment encoding ING4 was cloned by reverse transcription-PCR with normal gastric mucosa from patients with gastric ulcer, and subcloned into eukaryotie expression vector pcDNA3.1. PCR and DNA sequencing were performed to identify the eukaryotic expression vector pCDNA3.1-ING4, which was then transfected into M14 cells with Lipofectamine 2000 reagent. The expression of ING4 was detected in untransfected, pCDNA3. 1-ING4-transfected and pcDNA3.1-transfected M14 cells by Western blot and immunocyto-chemistry, and cell proliferation by MTT assay. Results A fragment of expected size (750 bp) was amplified by PCR analysis, and DNA sequencing confirmed the correctness of the recombinant plasmid. As shown by immunocytochemistry, the percentage of cells positive for ING4 protein was significantly higher in pCD NA3. 1-ING4-transfected MI4 cells than in non-transfected M14 cells and pcDNA3.1-transfected M14 cells (71.80%±9.88% vs 4.20%±3.35%, P < 0.01). Western blot also revealed an increased expression of ING4 in pCDNA3.1-ING4-transfected cells. Decreased cell viability was observed in ING4-transfected cells compared with nontransfected cells and pCDNA3.1-transfected cells (both P < 0.01). Conclusions The pCDNA3. 1-ING4 eukaryotic expression vector has been constructed successfully, and M14 cells transfected by this recombinant plasmid could effectively express ING4 protein, which may inhibit the cell proliferation of M14 cells.