The Regulation Of TBX15 Methylation On Its Expression And Functional Research In Hepatocellular Carcinoma

Objective Primary hepatocellular carcinoma is one of the most common malignant tumors in our country.It poses a serious threat to people's health and life as a result of the high malignant degree,long incubation period,occult onset,rapid progression,short course of disease and high mortality rate.The occurrence of liver cancer is a complex and gradual process.In this study,promoter methylation of candidate genes significantly different and negatively correlated with its expression level were screened out by online tool MethHC database,and bioinformatics analysis of liver cancer data in TCGA database was used to explore the relationship of the candidate gene in prognosis of liver cancer.This study was conducted to investigate the effect of candidate gene TBX15 promoter methylation on its expression in liver cancer and corresponding para-cancer tissue,and the cell tumor behavior of TBX15 gene in HCC cell lines.Methods 1.Bioinformatics analysis:?1?The significantly different gene promoter methylation during hepatocarcinogenesis was screened out using online tool MethHC Database.?2?Methylation and mRNA data and clinical data from Level 1 were downloaded using the HumanMethylation450 BeadChip?Illumina?platform in the TCGA public database of Cancer Genome Atlas.?3?Using R software to integrate the original data,then the difference of candidate gene TBX15 methylation and mRNA expression was analyzed in included HCC and para-cancerous tissue samples.?4?Spearman rank correlation analysis was used to analyze the effect of TBX15 gene promoter methylation on its mRNA.?5?Log-rank test and COX proportional hazard regression model were used to analyze the association between TBX15 gene promoter methylation and prognosis in hepatocellular carcinoma.2.Gene expression analysis:real-time PCR was used to detect the TBX15promoter methylation on mRNA expression in 30 pairs of hepatocellular carcinoma and its corresponding paracancerous tissue samples.3.Cytomethylation validation and functional experiments:?1?Finding the5'upstream sequence of TBX15 gene by bioinformatics,predicting the promoter region and CPG island location,and designing primers using the related softwares.?2?The methylation level and mRNA and protein expression of TBX15 promoter in three hepatocellular carcinoma cell lines?HepG2,MHCC97H and SNU-449?were detected by BSP,real-time PCR and western blot to screen out the cell line with the highest methylation and the lowest expression.?3?TBX15 overexpressing vector was constructed and transfected into SNU-449 cell,and then the mRNA and protein expression of TBX15 were detected by RT-PCR and WB.?4?Taking empty plasmid and pc3.1 plasmid as control groups,the ability to proliferation,migration and invasion,apoptosis of liver cancer cell lines were detected by CCK-8,transwell and flow cytometry assay.Results 1.Bioinformatics data analysis showed that:?1?Promoter methylation of TBX15 significantly different and negatively correlated with its expression level was screened out by MethHC database.?2?TCGA methylation data analysis showed that the TBX15 promoter was hypermethylated and its expression downregulated in hepatocellular carcinoma.?3?Spearman rank correlation analysis showed that the methylation level of TBX15 gene was negatively correlated with its mRNA expression level.?4?Log-rank test indicated that the survival rate of HCC patients with low level methylated TBX15 promoter was higher than high level methylated TBX15 promoter.?5?COX proportional hazard regression analysis showed that TBX15 promoter methylation of HCC independent prognostic test was statistically significant?P=0.045?.2.The result of gene expression analysis showed that the mRNA expression of TBX15 was downregulated verified by RT-PCR.3.Cytomethylation assay:The methylation rates of three hepatocellular carcinoma cell lines were 57.4%?HepG2?,78.9%?MHCC-97H?,89.5%?SNU-449?.The result indicated that TBX15 methylation was the highest in SNU-449 cell.4.Real-time quantitative PCR:The TBX15 expression was almost not found in MHCC-97H and SNU-449 cells but higher in HepG2 cells.TBX15overexpressing vector was constructed and transfected into SNU-449 cell.Taking the 2^-??Ct value of the wild-type plasmid group as 1,the results showed that the mRNA level of TBX15 in the overexpression group was significantly higher than that in the empty plasmid group and the wild-type plasmid group.5.Western Blot assay:The protein expression of TBX15 in three hepatocellular carcinoma cell lines was detected by WB taking GADPH as an internal reference.The results showed that the expression of TBX15 was downregulated in three hepatocellular carcinoma cell lines.TBX15overexpressing vector was constructed and transfected into SNU-449 cell.Comparing with the wild type plasmid group and the empty plasmid group,the protein expression of the overexpression plasmid group was upregulated.6.The results of CCK-8 assay:OD values of OD450nm in wild-type plasmid group,empty plasmid group and overexpression plasmid group were detected.The results showed that after culturing for 24h and 72h,the differences of proliferation in three hepatoma cells were not significant,but the effect of overexpression plasmid group on proliferate ability of hepatocarcinoma cells was higher than that of wild-type plasmid group?P=0.015?.7.Transwell migration and invasion experiments:The results of stained light microscopy,visual field cytometry and OD values of OD590nm showed that wild-type plasmid group,empty plasmid group and overexpression plasmid group had no effect on the migration and invasion ability of HCC cells.8.Flow cytometry assay:Through the analysis of early and late apoptotic count results,it was found that the effect of overexpression plasmid group on apoptotic ability of hepatocarcinoma cells was higher than that of wild-type plasmid group and empty plasmid group?P=0.036?.Conclusions 1.In hepatocellular carcinoma,TBX15 promoter hypermethylation inhibits the expression of its mRNA.2.TBX15 promoter methylation may be associated with the prognosis of hepatocellular carcinoma.3.TBX15 overexpressing vector was successfully constructed and transfected into SNU-449 cell.Overexpression of TBX15 gene may enhance the apoptosis of hepatocellular carcinoma cells.4.TBX15 gene methylation is associated with the malignant biological behavior of hepatocellular carcinoma,which is a predictive index for the progress of hepatocellular carcinoma and poor prognosis.