Effects Of Aberrant Methylation Of ZNF750 Promoter In The Development Of Esophageal Squamous Cell Carcinoma

Objective:To study the change of aberrant methylation of zinc finger protein 750(ZNF750)promoter on its expression level and effects of methylation inhibitor on biological behavior of esophageal squamous carcinoma cells.Moreover,to explore its regulatory mechanism preliminarily.Methods:Analysed the correlation between the expression of ZNF750 and the degree of methylation by using the cancer genome atlas(TCGA)database of esophageal cancer;Genome database of the University of California at Santa Cruz(UCSC)and CpG Island Prediction were used to predict the methylated site of ZNF750 promoter;Bisulfite sequencing PCR was used to detect the methylation level of ZNF750 promoter in different esophageal squamous carcinoma cell(ESCC)lines,and detected the expression of ZNF750 in different ESCC lines by Quantitative Real-time PCR(RT-qPCR);Screened hypermethylation of ESCC lines KYSE140 and KYSE150 and detected the expression of ZNF750 after treated by methylation inhibitor 5-Aza-dC,then the changes of cell biological behavior before and after 5-Aza-dC treated were observed by MTT assay,transwell assay and flow cytometer.Western blot was performed to detect the expression of poly ADP-ribose polymerase(PARP),Cysteinyl aspartate specific proteinase-3(Caspase-3),CyclinB-1(CCNB-1),and CyclinE-1(CCNE-1).To examine the effect of DNA methylation on ZNF750 promoter activity,we transfected pGL3-Basic-P600,pGL3-Basic-P600-Hpa II and pGL3-Basic-P600-HhaI fluorescent carriers in the KYSE150 cell line.Results:1.Bioinformatics predicted that the ZNF750 promoter region(-456 to-110)exists the methylation site of the CpG Island and the expression of ZNF750 has a significant correlation with the degree of methylation;2.The ZNF750 expression level correlates inversely to the methylation status of the CpG island in its proximal promoter region in ESCC lines.3.The methylation inhibitor 5-Aza-dC could induce the expression of ZNF750 and inhibited the proliferation,migration and invasion in KYSE140 and KYSE 150 cell lines.In addition,the 5-Aza-dC promoted the apoptosis of cells and blocked cell cycle at G2/M stage.4.The PARP and Caspase-3 were sheared evidently and CCNB-1 protein was up-regulated while the expression of CCNE-1 was down-regulated after being exposed to5-Aza-dC in KYSE140 and KYSE 150 cell lines.5.DNA methylation inhibited the transcriptional activity of ZNF750 promoter.Conclusion:The ZNF750 expression level correlates inversely to the methylation status of the CpG island in its proximal promoter region in ESCC lins.After treated KYSE140 and KYSE150 cell lines with methylation inhibitor 5-Aza-dC,the expression of ZNF750 was recovered.Besides,the proliferation,migration and invasion abilities were inhibited and promoted the apoptosis of cells and blocked cell cycle at G2/M stage.Dual-luciferase reporter assay indicated that DNA methylation inhibited the transcriptional activity of ZNF750 promoter.