ZNF191在肝癌发病中的作用及其分子机制研究

Hepatocellular carcinoma (HCC) is a primary cancer of the liver and one of the most common diagnosed cancers in Chinese population. Various risk factors have been associated with HCC. The molecular mechanisms leading to the development of HCC are extremely complicated, consisting of prominent genetic and epigenetic alterations, and are far from being clear presently. Our previous research showed that ZNF191 plays dual functions in HCC development and progression. In the early stage of HCC, ZNF191 plays its pro-proliferation role by directly binding to CTNNB1 promoter, and positively regulate transcription of β-Catenin. In the late stage of HCC, ZNF191 inhibits HCC metastasis via DLG1-mediated YAP1 inactivation by directly binding to DLG1 promoter. Combining the data of Chromatin immunoprecipitation-sequencing (ChIP-seq) with ZNF191 antibody, the transcription data with ZNF191 knockdown, and the co-expression score of ZNF191 mRNA in TCGA(The Cancer Genome Atlas) data, we identified DNMT1 gene, encoding an essential DNA methyltransferase enzyme, is a potential target gene of ZNF191. Some studies have reported that the mRNA and proteins expression of DNMT1 were significantly higher in liver cancerous tissue than those in the corresponding non-cancerous liver tissues. The hepatocarcinogesis effect of DNMT1 is mediated through silencing the expression of tumor suppressor genes and activating oncogenes via abnormal methylation of 5-CpGs of target gene promoters. In this research we will investigate: (1) the relationship between ZNF191 and DNMT1 mRNA and protein expression in human HCCs; (2) the molecular mechanism of ZNF191 in regulation of DNMT1 expression; (3) the regulation of ZNF191on methylation of DNMT1 target genes and expression of the corresponding downstream genes. We hope the research can elucidate a new molecular mechanism of transcription regulation of the DNMT1 gene in HCC, unearth the role of ZNF191 in regulating epigenetic alterations in the development and progression of HCC, and provide the rationale for the use of ZNF191 as a possible prognostic marker for HCCs.

肝癌是我国人群中高发的恶性肿瘤之一。作为多基因病其发病分子机制极其复杂，包括基因改变和表观遗传改变。前期研究发现转录因子ZNF191在肝癌发病中扮演双重角色：在肝癌早期靶向激活β-Catenin促进肝癌细胞增殖；而在晚期靶向激活DLG1发挥抑制转移作用。本研究通过综合分析ChIP-Seq数据、TGCA公共数据库及表达谱数据，发现重要甲基化酶基因DNMT1很可能受ZNF191靶向调控。已有研究表明DNMT1在肝癌中表达水平增高，使DNA异常甲基化导致肿瘤发生。本项目通过体内外实验明确：(1)肝癌中ZNF191与候选基因DNMT1相关性；(2)ZNF191对DNMT1启动子的调控；(3)ZNF191对受DNMT1甲基化调节的基因及信号通路的调控作用。本项目将有助于阐明肝癌内一个新的DNMT1转录调控机制，揭示ZNF191调控肝癌发生的表观遗传改变，为肝癌发生的危险评估及预后提供依据。

本项目致力于转录因子ZNF191在肝癌发病中的作用及机制，主要两方面开展工作：1）完善ZNF191靶向调控Wnt8B促进肝癌细胞增殖的机制研究；2）ZNF191靶向调控甲基转移酶DNMT1在肝癌发生中的作用及机制。研究结果：i) Wnt8B在HCC中表达上调，与患者不良预后相关。ZNF191对Wnt8B mRNA和蛋白表达具有正向调控作用。ZNF191蛋白能够直接与WNT8B启动子结合，结合位点分别位于-1491 (ATTAATT) 和-1178(ATTCATT)。此外，Wnt8B参与了ZNF191对肝癌细胞增殖的影响，并且肝癌临床样本中Wnt8B与ZNF191表达呈正相关。由ZNF191直接转录调控的Wnt8B通过经典的Wnt信号在肝癌细胞增殖中起关键作用，有望成为HCC患者新的预后标志物和潜在的治疗靶点。ii) DNMT1在肝癌中上调表达，与患者不良预后相关。在肝癌标本和肝癌细胞系内ZNF191对DNMT1的mRNA和蛋白水平有调控作用，可能通过DNMT1/PTEN/AKT通路促进肿瘤发生。荧光报告素酶实验、凝胶迁移实验(EMSA)实验等证实ZNF191蛋白能直接结合并激活DNMT1基因的启动子，ZNF191结合DNMT1基因的启动子的调控元件为-113位的ATTCATTCATTCATTC序列。综上，ZNF191是促进肝癌形成发展演变的重要转录因子，调控不同的靶基因如Wnt8B、DNMT1等发挥其生物学功能。我们的研究结果为ZNF191可能作为潜在的肝癌治疗靶点提供理论依据。