Experimental Study Of RUNX3 Gene By Promoter Hypermethylation Is Associated With Cutaneous Malignant Melanoma

Objectives: To investigate the relationship of the methylation of CpG island of RUNX3 gene with its gene expression in human cutaneous malignant melanoma,explore the role and mechanism of methylation of RUNX3 gene in the pathogenesis of human cutaneous malignant melanoma.Methods: reverse-transcription polymerase chain reaction (RT-PCR) analysis the expression of RUNX3 mRNA,methylation-specific PCR (MSP) evaluate for the promoter region methylation status of the RUNX3 gene and western-Blot analysis the RUNX3 protein expression in human cutaneous malignant melanoma cell line A375 . Then the A375 cell line were treated with different concentration of 5-azacytidine (0μmol/l,1μmol/l,5μmol/l,10μmol/l,20μmol/l), using RT-PCR, MSP and western-Blot to determine the difference of mRNA expression, protein expression and the promoter region methylation status of the RUNX3 gene in human cutaneous malignant melanoma cell line A375. The impact of 5-azacytidine on the growth of A375 cell line was observed with MTT assay.Results: No expression of RUNX3 mRNA and protein were found in RUNX3 gene in untreated A375 cell. At the same time RUNX3 gene promoter is hypermethylated in untreated A375 cell. But after the A375 cell treated by methylation inhibitor 5-azacytidine ,the promoter of RUNX3 gene is demethylated partly,expression of RUNX3 mRNA andprotein were found. And the degree of the expression is directlycoorelated with the concentration of methylation inhibitor 5-azacytidine .And 5- azacytidine was demonstrated to inhibit the growth of A375 cellline.Conclusions: The promoter hypermethylation of RUNX3 gene may berelated with the loss expression of RUNX3 gene in cutaneous malignantmelanoma , and it may play an important role in the pathogenesis ofhuman malignant melanoma.