Mechanism Of Promoter Methylation And H3K27 Deacetylation Regulating VIPR1 And Its Effect On The Prognosis Of Hepatocellular Carcinoma

Chapter 1 Effect of VIPR1 gene expression on clinicopathological features and prognosis of hepatocellular carcinomaObjective: To detect the expression of VIPR1 in hepatocellular carcinoma and analyze the relationship between VIPR1 expression and clinicopathological features and prognosis.Methods: A tissue chip was obtained from Guilin Medical College,which contains 95 cases of hepatocellular carcinoma tissue and 80 matched pairs of adjacent tissues.The expression of VIPR1 in hepatocellular carcinoma was analyzed by Oncomine database and tissue-microarray-immunohistochemistry.Pearson chi-square test was used to analyze the relationship between VIPR1 expression and clinicopathological parameters of hepatocellular carcinoma patients.Kaplan-Meier method was used for survival analysis.Univariate and multivariate analysis were performed using Cox regression model.Results: The expression level of VIPR1 decreased significantly in hepatocellular carcinoma.VIPR1 expression was not correlated with gender,age,tumor size,cirrhosis,TNM stage,but significantly correlated with histopathological grade.Low expression of VIPR1 was significantly associated with low survival.Multivariate analysis revealed that VIPR1 expression was an independent prognostic factor in hepatocellular carcinoma patients(P=0.006).The risk of death in patients with low expression of VIPR1 was significantly higher than that in patients with high expression of VIPR1.Conclusions: The expression of VIPR1 is down-regulated in hepatocellular carcinoma,which is closely related to the low survival rate of patients.VIPR1 may serve as a clinical prognostic marker and a potential therapeutic target of hepatocellular carcinoma.Chapter 2 Promoter methylation inhibits VIPR1 expressionObjective: The decreased expression of VIPR1 in hepatocellular carcinoma may be related to the abnormal DNA methylation.This study will measure the methylation of VIPR1 in hepatocellular carcinoma and explore the relationship between the methylation modification and the expression of VIPR1.Methods:Ten pairs of paraffin-embedded hepatocellular carcinoma tissues and adjacent tissues were obtained from the Fourth Affiliated Hospital of Guangxi Medical University(Liuzhou Workers' Hospital).MHCC97-L and MHCC97-H cell lines were purchased from Shanghai Fudan IBS Cell Resource Center(FDCC).SK-Hep-1 and Hep3 B cell lines were purchased from Kunming Cell Bank,Kunming Institute of Zoology,Chinese Academy of Sciences.Gs-Hep G2 and Huh7 cell lines were preserved and subcultured by the Surgical Laboratory of the Fourth Affiliated Hospital of Guangxi Medical University.c Bio Portal was used to analyze genetic alteration of the VIPR1 gene in TCGA hepatocellular carcinoma dataset.Cp G island of VIPR1 gene was searched using UCSC.Meth HC database was used to analyze the methylation of VIPR1 in hepatocellular carcinoma on-line and the correlation between DNA methylation and VIPR1 m RNA.The methylation level of VIPR1 in hepatocellular carcinoma was detected by pyrosequencing.Pyrosequencing,q RT-PCR and Western blot were used to detect the methylation and expression of VIPR1 in hepatocellular carcinoma cells treated with DAC(a DNA methyltransferase inhibitor).Results: The genetic alterations of VIPR1 in hepatocellular carcinoma were only 1.6%,suggesting that the genetic alterations may not be the main cause of low expression of VIPR1 in hepatocellular carcinoma patients.There is a Cp G island with a length of 1704 bp and 127 Cp G sites at the 5' end of VIPR1 gene,which crosses the transcription starting site.7 Cp G sites(located in the-423 to-388 bp region upstream of the transcription initiation site)in the Cp G island are hypermethylation sites.The methylation level of VIPR1 in hepatocellular carcinoma was significantly higher than that in paracancerous tissues,and the m RNA level was significantly lower than that in paracancerous tissues.The methylation level of VIPR1 was negatively correlated with m RNA expression level.DNA methyltransferase inhibitor DAC treatment could inhibit the methylation of VIPR1 promoter region and enhance the expression level of VIPR1 mRNA and protein.Conclusions: The genetic alteration of VIPR1 gene is not the main reason for the decrease of vipr1 expression in hepatocellular carcinoma.The increased methylation of the promoter region of VIPR1 gene accounts for the down-regulation of VIPR1 expression in hepatocellular carcinoma.Chapter 3 H3K27 deacetylation inhibits VIPR1 expressionObjective: Histone H3K27 deacetylation is closely related to gene transcription silencing.Histone deacetylation and DNA methylation can co-regulate gene expression.The decreased expression of VIPR1 in hepatocellular carcinoma may be related to the abnormal deacetylation of H3K27.This study will detect the enrichment of H3K27 Ac around VIPR1,and explore the relationship between H3K27 deacetylation and VIPR1 expression.Methods: SK-Hep-1 and Hep3 B cell lines were purchased from Kunming Cell Bank,Kunming Institute of Zoology,Chinese Academy of Sciences.Gs-Hep G2 and Huh7 cell lines were preserved and subcultured by the Surgical Laboratory of the Fourth Affiliated Hospital of Guangxi Medical University.H3K27 Ac enrichment on VIPR1 promoter was analyzed using Cistrome Data Browser.Ch IP-q PCR was used to detect the concentration of H3K27 Ac bound to the promoter region of VIPR1 in hepatocellular carcinoma cells treated with PBA(a histone deacetylase inhibitor).The level of VIPR1 m RNA was detected by q RT-PCR in hepatocellular carcinoma cells co-treated with DAC and PBA.Results: The enrichment of H3K27 Ac in the promoter region of VIPR1 in Hep G2 and Huh7 cells was not enriched,suggesting that the down-regulation of VIPR1 may be related to the abnormal deacetylation of H3K27.HDACs inhibitor PBA significantly increased the expression of VIPR1 m RNA in hepatocellular carcinoma cells.The combination of PBA and DAC could increase the expression of vipr1 m RNA in hepatocellular carcinoma cells.PBA treatment enhanced the binding of H3K27 Ac to the promoter region of VIPR1 gene promoter,and this trend was enhanced with the increase of PBA concentration.Conclusions: The expression of VIPR1 can be regulated by H3K27 deacetylation.Deacetylation of H3K27 in the promoter of VIPR1 inhibits the transcription of VIPR1 in hepatocellular carcinoma.