ZNF23 Suppresses Cutaneous Melanoma Cell Malignancy Via Mitochondria-dependent Pathway

Background and Objective:Cutaneous melanoma is one of the leading causes of cancer deaths with an increasing incidence worldwide.A KRAB-containing zinc finger protein member,zinc finger 23(ZNF23),was reduced in some types of tumors and inhibited cell growth by inducing cell cycle arrest.However,the role of ZNF23 expression is still poorly understood in cutaneous melanoma.The aim of this study is to investigate the epigenetic regulation and mechanism of ZNF23 in melanoma.Methods:The level of ZNF23 expression was detected in cutaneous melanoma,adjacent normal skin tissues and cutaneous melanoma cell lines(SK-MEL-1,SK-MEL-2,SK-MEL-5,SK-MEL-28 and human immortal keratinocyte cell line HaCat)using immunohistochemistry and western blotting,respectively.The correlations between ZNF23 expression and other clinicopathologic parameters were analyzed in melanoma patients.Ectopic expression of ZNF23 plasmid was transfected into melanoma cells,SK-MEL-2 and SK-MEL-28 with no expressing of ZNF23.MTT,flow cytometry and transwell assay were used to measure cell proliferation,apoptosis,invasion and migration abilities,respectively.In order to reveal the mechanism of decreased ZNF23 in melanoma,we have further found that there is a clear CpG island in the promoter region of ZNF23 gene through the web site http://www.urogene.org,which may be related to the methylation of ZNF23 promoter region.The treatment of melanoma cells by 5-Aza-dc can increase the expression level of ZNF23 mRNA.Further we used MSP and bisulfite genomic sequencing to methylation analysis of the promoter region of this gene to detect the varaitions of ZNF23 methylation site in the melanoma cells and paired tissues,as well as the role or rlationships of ZNF23 methylation status on the prognosis of melanoma patients and other clinicopathological features.Mitochondrial functions and structures were detected by mitochondrial membrane potential assay and Transmission electron microscopy(TEM)methodin melanoma cells transfected with overexpressing ZNF23 plasmid or empty vector.Western blotting was performed to detect the levels of ZNF23,p53,p27,Bcl-2 and cleaved caspase-3 after overexpressing of ZNF23 in melanoma cells.Moreover,semi-quantitive RT-PCR and methylation specific PCR(MSP)are employed to detect the expression and promoter region methylation of ZNF23 in melanoma cells and tissues.And 72 cases of melanoma were selected in this study.Results:ZNF23 was elevated in adjacent normal skin tissues compared with melanoma tissues.Patients with low level of ZNF23 expression exhibited higher incidence of lymphoid metastasis,thicker size of tumors and worse outcome.By using Cox’s regression analysis,ZNF23 expression,tumor thickness and lymph node metastasis were the independent prognostic factors for overall survival(p<0.05).Results from cellular experiments indicated that ectopic expression of ZNF23 induced cell apoptosis by activation of caspase-3,p27,p53 expression and down-regulation of Bcl-2 through mitochondria-dependent pathway.And frequent methylation of ZNF23 gene was found in melanoma,and its expression was regulated by promoter region methylation.Loss of ZNF23 expression and complete methylation or partial methylation of promoter region was detected in melanoma cell lines.Restoration of ZNF23 expression or increased expression of ZNF23 was achieved by treating with 5-Aza-2’-deoxy-cytidine(5-Aza)in unexpressed and reduced melanoma cells.These results demonstrated that the expression of ZNF23 was regulated by DNA methylation in promoter region in melanoma cells.DNA hypermethylation was detected in 48.6%(35/72)in melanoma tissuses.There is intense correlation between DNA methylation of ZNF23 and lymph node metastasis,tumor thickness.Conclusion:Decreased ZNF23 was contributed to melanoma progression and poor survival with mitochondria-dependent pathway.The expression of ZNF23 gene is regulated by DNA methylation in melanoma cells and it is frequently methylated in primary cutaneous melanoma patients.It indicated that ZNF23 could be a promising therapeutic biomarker candidate for cutaneous melanoma.And its biological function and clinical application should be further studied in future.