VIPR1启动子甲基化在肝癌发生中的作用及机制研究

The morbidity and mortality of liver cancer in Guangxi province are significantly higher than the national level. Its occurrence and development are related to the abnormal expression of various genes, but its exact pathogenesis still remains unclear. Our previous research revealed that the DEC1 function as transcription factors by non-specific binding to E-box region in the proximal promoter of target genes allowed it to regulate a number of potential target genes and play important roles in pathological processes such as cancer. Bioinformatics method predicted the interactions between VIPR1 and DEC1. These results indicated that VIPR1 might play a role in tumorigenesis. Gene-chip data and quantitative PCR analysis displayed a low expression level of VIPR1 in hepatocellular carcinoma (HCC). DNA methyltransferase inhibitor and histone deacetylase inhibitor could upregulate significantly the expression level of VIPR1 in human HCC cell line. These results suggested that epigenetic regulation，such as DNA methylation and histone acetylation, was the main way of VIPR1 silencing. GO analysis revealed that VIPR1 might play a role in the occurrence of HCC by regulating the MAPK signal pathway. This project aims to investigate the correlation between HCC and the date of DNA methylation and histone acetylation and the regulation of DNA methylation and histone acetylation on VIPR1 expression level by using HCC tissues, cell lines and nude mice. Furthermore, we aim to study the effect of promoter methylation on cell biological behavior and the role of VIPR1 in the regulation of MAPK signaling pathway. This study helps to elucidate the role of VIPR1 in the pathogenesis of liver cancer and provides new clues for the prevention of HCC.

广西肝癌的发病率和死亡率明显高于全国水平，其发生涉及多种基因的异常表达，但分子机制尚不明确。我们前期发现转录因子DEC1与E盒的非专一性结合使其可调控多种靶基因参与肿瘤发生，生物信息学预测VIPR1与DEC1有互作关系，提示VIPR1可能在肿瘤发生中起作用。基因芯片数据和定量PCR分析揭示VIPR1在肝癌中低表达，且此现象可被DNA甲基化转移酶抑制剂和组蛋白去乙酰化酶抑制剂逆转，提示DNA甲基化和组蛋白乙酰化可能是调控VIPR1表达的主要方式。GO分析表明VIPR1可能通过调控MAPK信号通路影响肝癌发生。本项目拟在组织、细胞系和裸鼠中研究DNA甲基化和组蛋白乙酰化与肝癌的相关性以及对VIPR1表达的调控作用，观察启动子甲基化对细胞生物学行为的影响，分析验证VIPR1对MAPK信号通路的调控模式。本研究不仅有助于阐明VIPR1影响肝癌发生的分子机理，而且能为肝癌的防治提供新线索。

肝癌是世界上最常见的癌症之一，每年约有84万新发病例，至少有78万人死于肝癌。肝细胞癌（HCC）是最常见的原发性肝癌。尽管目前已经广泛研究了HCC中一些分子的变化，但HCC的发生、发展及转移的分子机制尚未得到很好的探索。有研究表明血管活性肠肽受体1（VIPR1）在不同的肿瘤中有不同的功能，然而其在HCC患者中的表达水平，临床意义、转录调控机制和生物学功能尚不清楚。我们研究发现VIPR1在HCC组织中的表达水平显著降低。VIPR1的表达与患者性别、年龄、肿瘤大小、有无肝硬化、TNM分期等没有明显相关性，而与组织病理分级显著相关。VIPR1低表达与患者低生存率显著相关。VIPR1的启动子区域存在甲基化修饰，DNA甲基化抑制了VIPR1转录。VIPR1启动子中H3K27的去乙酰化抑制了VIPR1转录。我们进一步发现VIPR1启动子区域的甲基化促进了转录因子AP-2α的结合，AP-2α作为转录抑制因子起作用，抑制了VIPR1的表达。VIPR1的过表达导致细胞周期阻滞在G2/M期，同时促进细胞凋亡并抑制细胞增殖，体内实验表明VIPR1过表达可抑制肿瘤生长。Vimentin的表达与VIPR1负相关，且VIPR1与vimentin蛋白相互作用，提示VIPR1可能通过与vimentin蛋白相互作用而抑制HCC细胞的增殖。我们的研究结果表明，VIPR1可能在HCC中作为一种抑癌基因，并为HCC的诊断和治疗提供潜在的新靶点。