Studies On The Regulation Mechanism Of EpCAM By DNMT3A2 And Correlation Analysis With Gastric Cancer Development

Objective:To investigate the regulation mechanism of EpCAM by DNMT3A2 and correlation analysis with Gastric cancer development.Methods:Real-Time Fluorescent Quantitative PCR was performed to analysis the expression pattern of EpCAM in GC cell lines with overexpression-DNMT3A2 or knockdown-DNMT3A2,and BGS was applied to assess the methylation status of EpCAM promoter regions in MKN28-shDNMT3A2 cell lines.IP was constructed to verify the correlation between DNMT3A2 with EZH2,then RT-PCR was used to analyze the effect of EZH2/SUZ12 on EpCAM expression in SiEZH2/SiSUZ12 cell lines.Establishing the stable overexpression and downexpression EpCAM cell lines,CCK-8 cell proliferation assay,Transwell invasion/migration assay and Wound healing assay were used to detect the proliferation,migration and invasion ability of MKN45 cell stable transfected with EpCAM inhibitor and MKN28 cell stably transfected with EpCAM.Finally,RT-qPCR was used to analyzed the expression pattern of EpCAM in GC Tissue samples and statistical analysis the relation of EpCAM with clinicopathologicl features.Results:.1)RT-qPCR detected the expression pattern of EpCAM in MKN28-shDNMT3A2 and MKN45-DNMT3A2 cell lines,indicating that EpCAM was downregulation in MKN45-DNMT3A2 cell line and EpCAM was upregualtion in MKN28-shDNMT3A2 cell line.2)Software predicted the promoter region of EpCAM contained CpG island.Decreased DNMT3A2 could induced the methylation status of EpCAM promoter reduced from 57.8%to 20%.3)IP revealled the combination between EZH2 with DNMT3A2,indicating EZH2 participated in regulation effect of DNMT3A2 on EpCAM.4)RNAi-EZH2 could upregulate the expression level of EpCAM in MKN45-DNMT3A2 cell line,and RNAi-SUZ12 is similar to the effect of RNAi-EZH2.5)EpCAM stable inhibitor remarkedly promoted migration and invasion ability of GC cells,conversely,overexpression of EpCAM significantly suppressed the migration and invasion ability of GC cells.The expression level of EpCAM was non-related with proliferation of GC cells.6)In 46 pairs GC tissues,the expression of EpCAM was significantly decreased.The expression of EpCAM was correlated with the lymph node metastasis of GC patients.Conclusion:1.DNMT3A2 dyregualted the expression of EpCAM,and the possible mechanism is the following,DNMT3A2 could change the methylation status of EpCAM promoter region,maybe directly or combining with EZH2.2.EpCAM could inhibite the migration and invasion ability of GC cells,but be inessential to proliferation ability of GC cells.3.In GC tissues,the experssion of EpCAM was decreased,and was related with the lynph node metastasis of GC patients.