Effect Of Methylation Of PTEN Promoter Region On The Sensitivity Of Molecular Targeting Drugs In Hepatocellular Carcinoma

Objective: This study aims to detect the methylation rate of the tumor suppressor PTEN promoter region in hepatocellular carcinoma(HCC)tissues,and to explore the regulatory mechanism of the promoter region methylation of the PTEN promoter region and its influence on the sensitivity of molecular targeted drugs to the treatment of HCC.Methods: Fifty-two pairs of tumor tissue and para-cancerous tissue specimens from 52 HCC patients treated with Sorafenib,a molecular targeted drug,were collected and saved,the level of PTEN m RNA and the methylation rate of the PTEN promoter region in these samples were detected using q PCR technology and next generation sequencing(NGS)technology,respectively.Patients were divided into 2groups(high-level group and low-level group)according to the median of the PTEN expression level or the methylation rate of the PTEN promoter region,and the clinical significance of PTEN promoter region methylation in HCC was evaluated by comparing and analyzing the time to progression(TTP)and overall survival(OS)of the two groups of patients.The MHCC97-H cell and Hep G2 cell,two representative HCC cells,were cultured in vitro,and these cells were treated with Decitabine,a DNA-methyltransferase 1(DNMT1)inhibitor,to detect the methylation rate of the PTEN promoter region of MHCC97-H and Hep G2 cells and the expression level of PTEN,and the recruitment of DNMT-1 in the PTEN promoter region was detected by chromatin immunoprecipitation(Ch IP).Then,the MHCC97-H and Hep G2 cells were combined treatment with Decitabine and molecular targeted drugs,the IC50 value of cells was determined by the MTT method,and the cell metastasis or invasion in vitro was detected by the transwell experiment.Results: The expression level of PTEN m RNA in HCC tissues was significantly lower than that in para-cancerous tissues(p<0.05),and the methylation rate of PTEN promoter region in HCC tissues was significantly higher than that in para-cancerous tissues(p<0.05).The prognosis of patients in the PTEN high expression group receiving the molecularly targeted drug Sorafenib was better than that of the PTEN low expression group,and there were statistically significant differences in TTP and OS between the two groups(p<0.05).The prognosis of patients in the PTEN promoter hypomethylation group receiving Sorafenib treatment was better than that of the PTEN promoter hypermethylation group;There were statistically significant differences in disease progression TTP and OS between the two groups(p<0.05).The methylation of the PTEN promoter region is regulated by DNMT-1.Decitabine treatment can down-regulate the methylation level of the PTEN promoter region of MHCC97-H and Hep G2 cells,up-regulate the expression level of PTEN,and inhibit the recruitment of DNMT-1 in the PTEN promoter region.Treatment with Decitabine or overexpression of PTEN in MHCC97-H and Hep G2 cells can significantly reduce the IC50 value of molecularly targeted drugs on cells.Decitabine(5μmol/L)combined with Sorafenib(1μmol/L)can significantly enhance the inhibitory effect of Sorafenib on the migration and migration of MHCC97-H and Hep G2 cells in vitro.Conclusion: This study successfully established a new method for detecting the methylation level of the PTEN promoter region based on next-generation sequencing,which can be used to detect the methylation rate of the PTEN promoter region in HCC tissues.It was found that the methylation rate of the PTEN promoter region was negatively correlated with the prognosis of HCC patients.The methylation rate of the PTEN promoter region is regulated by DNMT1.Inhibition of the methylation of the PTEN promoter region enhance the sensitivity of HCC cells to molecular targeted drugs.The results of this study suggest that PTEN promoter region methylation may be used as a diagnostic indicator or therapeutic target for HCC.