基于斑马鱼活体实时研究Yap1和p53R217S协同驱动肝癌发生和发展机制

Hepatocellular carcinoma (HCC) is a deadly malignancy with limited knowledge on the mechanism and the key driver genes, which bring major obstacles to precise medicine. As major carcinogens detected in Chinese patients, hepatitis B virus (HBV) and aflatoxin B1 (AFB1) can synergistically induce hepatocellular carcinoma. Recent studies revealed that HBV contributes to the activation of an oncogene named Yap1, while AFB1 is responsible for R249S mutation of a tumor suppressed gene p53. Using transgenic and CRISPR/Cas9-based gene editing approaches, we previously generated Yap1 liver overexpressed and p53R217S (the conserved site of R249) mutant zebrafish lines, in which we demonstrated that Yap1 and p53R217S synergistically triggered HCC. These, together with our recent findings that Yap1 and p53R217S amplified the expression to each other, have prompted us to hypothesize that Yap1 and p53R217S form a positive feedback loop to drive hepatocarcinogenesis and tumor progression synergistically. To test this hypothesis, we propose to address the following key points: Establish a zebrafish line (based on casper mutant) that allows us to trace the hepatocarcinogenesis synergistically driven by Yap1 and p53R217S real-time in vivo by screening a Yap1 and p53R217S-specific HCC marker, followed by knocking in a fluorescent protein to the identified marker as fusion gene. Using this model, together with gene editing approaches, we propose to analyze how Yap1 and p53R217S formed a positive feed-back loop, which contributes to our understandings on how Yap1 and p53R217S drive hepatocarcinogenesis and tumor progression synergistically. In long-term, this project promises to make a significant progression to precise medicine for HCC patients, as well as contributes a zebrafish disease model.

驱动肝癌发生和发展的机制及关键驱动基因是制约肝癌精准治疗的重要瓶颈之一。作为我国肝癌患者主要致癌物，乙肝病毒和黄曲霉素可共同诱发肝癌，且可能分别与原癌基因Yap1的激活和抑癌基因p53的R249S突变密切相关。我们前期用转基因和CRISPR/Cas9构建肝过表达Yap1和p53R217S突变(R249保守位点)的斑马鱼，发现Yap1和p53R217S可协同诱发肝癌，且二者之间可能形成正反馈。因此，我们提出假说：Yap1和p53R217S可能以正反馈环方式协同驱动肝癌发生和发展。本课题拟：基于斑马鱼模型，通过构建肝癌标志物-荧光蛋白融合基因以活体实时指示Yap1和p53R217S协同驱动肝癌发生和发展，结合基因编辑技术研究Yap1和p53R217S正反馈的详细机制，以阐明其协同驱动肝癌发生和发展的机制。本研究将为肝癌病人靶向药物开发和精准治疗方案优化提供实验依据及一种可活体实时追踪的模型。

驱动肝癌发生和发展的机制及关键驱动基因是制约肝癌精准治疗的重要瓶颈之一。作为我 国肝癌患者主要致癌物，乙肝病毒和黄曲霉素可共同诱发肝癌，且可能分别与原癌基因Yap1的 激活和抑癌基因p53的R249S突变密切相关。我们前期用转基因和CRISPR/Cas9构建肝过表达Yap 1和p53R217S突变(R249保守位点)的斑马鱼，发现Yap1和p53R217S可协同诱发肝癌，且二者之 间可能形成正反馈。因此，我们提出假说:Yap1和p53R217S可能协同驱动肝癌 发生和发展。本研究采用斑马鱼和人肝细胞，重点研究了Yap1和p53R217S协同促进肝细胞增殖，肝脏过度生长和肝癌发生。机制上，我们发现Yap1和p53R217S在肝细胞中形成相分离，共同结合在METTL16的启动子区，并促进METTL16的转录。METTL16的重要性也通过基因敲除（低）以及恢复拯救进一步被确立。通过m6A和非m6A依赖的方式，METTL16增强了肝细胞的增殖，肝过度生长和肝癌发生。本研究将为肝癌病人靶向药物开发 和精准治疗方案优化提供实验依据及一种可活体实时追踪的模型。