A Study In Vitro In The Biological Behavior Of Melanoma Cell Line M14 Regulated By Tumor Suppressor ING4

Malignant melanoma is a severe and life-threatening skin malignant tumor,and its incidence is the third in skin malignancies with poor prognosis.The mechanism of melanomagenesis is incompletely understood.Deregulation of tumor suppressor genes is probably one of the key factors contributing to the occurrence and progression of melanoma. ING4 is a novel tumor suppressor gene,and the exact functions of ING4 have been recognized limitedly.Previous studies demonstrated that ING4 presented with significant abnormality in several human malignancies and played an inhibitory role on several malignant tumor cell lines.In this research,in order to confirm the role of ING4 on melanomagenesis,we investigated in vitro the level of ING4 in melanoma tissues and its effect on proliferation,apoptosis and invasion of melanoma by a cDNA fragment encoding human ING4 transduction into human melanoma cell line M14.(1) The expression of tumor suppressor ING4 in cutaneous melanomasImmunohistochemical examination revealed the level of ING4 was markedly reduced in melanoma tissues and mostly no staining was observed in nuclei of most tumor cells. However,normal skin and benign nevi tissues presented with positive staining of nuclei. There was a statistically significant difference in the expression of ING4 between melanoma and benign melanocytic nevi tissues(p＜0.01 )(2) Construction of pcDNA3.1-ING4 eukaryotie expression plasmid and cell transfectionDNA fragment encoding ING4 was obtained by RT-PCR method from normal human gastric mucosa and was subcloned into empty vector pcDNA3.1.PCR and DNA sequencing showed that the recombinant plasmid pCDNA3.1-ING4 was constructed correctly. pCDNA3.1-ING4 was transfected into cell line M14 with Lipofectamine2000 reagent,and the expression of ING4 was detected by western blot analysis and immunocytochemistry. Compared with non-transected M 14 cells and M14 cells with pcDNA3.1,the level of ING4 was significantly higher in M14 cells with exogenous 1NG4 gene.(3) The regulatory effect of ING4 on the growth of melanoma M14Cell proliferation and apoptosis of M14 cells with or without exogenous ING4 gene were detected by MTT,FCM and TUNEL method.M l4 cells with exogenous ING4 gene presented with growth suppression and enhanced apoptosis,with the G2/M arrest.Moreover, western blot analysis revealed that M14 cells with exogenous ING4 gene presented with the upregulation or downregulation of cell cycle regulating proteins and the activation of apoptosis pathway.(4) The effect of ING4 on the invasion of melanoma M14The effect of ING4 on the invasion,adhesion and movement activities of M14 cells were assayed by Transwell zeta method and MTT method.ING4 could inhibit the adhesion and invasion abilities of M14 cells with no effect on its moving ability.Moreover,gelatin Zymography was applied to assay the effect of ING4 on the production and activity of MMP2 and MMP9.ING4 could inhibit the expression of MMP2 and MMP9,which led to decreased activation on degradation of extracellular matrix and basement membrane.ConclusionsThe low-expression of ING4 in melanoma tissues indicates that ING4 may be involved in the tumor occurrence and progression of melanoma.ING4 can effectively inhibit the growth of M14 and enhance its apoptosis by the regulation on cell cycle and the activation of apoptosis signal pathway.Moreover,ING4 can effectively inhibit the invasion of M I 4 by decreasing the adhesion activity of tumor cells and the secretion of MMP2 and MMP9.