全基因组CRISPR文库筛选鉴定NAE1是HCC缺氧存活的关键基因

目前，肝细胞癌（HCC）尚缺乏有效治疗方法。缺氧是肝癌的重要特征。我们通过全基因组CRISPR/Cas9敲除文库筛选，鉴定并验证了对HCC缺氧存活起到关键作用的基因，发现调控类泛素化（Neddylation）修饰的蛋白NAE1在筛选结果中位于前列。NAE1与HCC的无病生存期及总生存期负相关。我们将探索HCC临床样本中NAE1的表达水平及临床意义。有趣的是，我们发现NAE1通过调控HIF-1α的Neddylation修饰，稳定HIF-1α表达。我们将通过质谱和免疫沉淀鉴定并验证NAE1调控的HIF-1α的Neddylation修饰以及具体修饰位点，进一步探索NAE1通过稳定HIF-1α影响HIF-1调控的血管生成和代谢编程。更有意义的是，我们将证明NAE抑制剂能够在体外及体内影响HCC的血管生成和代谢重编程，特异性阻断缺氧HCC细胞的生长。NAE1有望成为HCC的一个新的诊疗靶点。

Hepatocellular carcinoma (HCC) is a primary liver malignancy which has no effective therapeutic strategy. Hypoxia is an important characteristic of HCC. Genome-wide CRISPR/Cas9 knockout library screening was a powerful tool used to identify and validate genes that are critical for HCC hypoxia survival. We found that NAE1, a critical regulator of protein Neddylation, was listed as top gene of our screening results. NAE1 was negatively correlated with disease-free survival and overall survival of HCC. We will explore the over-expression and clinical significance of NAE1 in HCC clinical samples. Interestingly, we found that NAE1 stabilizes HIF-1α by facilitating its Neddylation. We will use mass spectrometry and immunoprecipitation to confirm the Neddylation of HIF-1α mediated by NAE1 and validate specific Neddylation sites of HIF-1α. We will further explore the role of NAE1 in regulating HIF-1-mediated angiogenesis and metabolic reprogramming in normoxic and hypoxic conditions. More importantly, we will demonstrate that NAE inhibitor, MLN4924, specifically targets hypoxic HCC cells and blocks HCC growth in vitro and in vivo through inhibition of HIF-1-mediated angiogenesis and metabolic reprogramming. NAE1 is expected to be a new diagnostic target for HCC.