PCK1通过Wnt信号通路抑制肝细胞癌的作用机制研究

Metabolic reprogramming is recognized as one of the important cancer hallmark. Changes in glucose metabolism are essential for carcinogenesis and cancer development. Liver is the major organ of gluconeogenesis,, recent studies reveal that the expression of PCK1, the key enzyme of gluconeogenesis, was significantly reduced in hepatocellular carcinoma (HCC). Overexpression of PCK1 could inhibit tumor growth, suggesting that PCK1 is a new potential tumor suppressor gene, but its mechanism remains unclear. Our previous studies found that, the expression levels of several genes in the Wnt signaling pathway were downregulated when overexpression of PCK1, speculating that PCK1 suppress HCC through regulating Wnt signaling pathway. This project uses RT-qPCR，Western blot and TOPFlash/FOPFlash Wnt reporter assay to analyze the correlation between expression level of PCK1 and Wnt signaling pathway activation. Chromatography-mass spectrometry (GC-MS) to study the metabolite expression patterns change caused by PCK1 expression. Explore the molecular mechanism of PCK1 regulating Wnt signaling pathway in liver cancer cell lines and tumor xenograft mouse model. This project will not only reveal the molecular mechanisms of Hepatocarcinogenesis, but also expect to provide new target for diagnosis and treatment of HCC.

代谢重编程被视为肿瘤的重要标志之一，其中葡萄糖代谢的改变对恶性肿瘤的发生发展至关重要。肝脏是糖异生的主要器官，肝细胞癌（HCC）中糖异生的关键酶 PCK1表达明显下调，而过表达PCK1基因能够抑制肝癌细胞生长，提示PCK1是一种新的潜在抑癌基因，但其具体机制仍未阐明。我们通过转录组测序发现，过表达PCK1导致肝癌细胞中Wnt信号通路的多个基因表达水平降低，推测PCK1通过调节Wnt信号通路而抑制HCC。本项目利用利用RT-qPCR、Western blot和双荧光素酶报告基因分析PCK1的表达水平与Wnt信号通路激活状态的相关性；利用气相色谱-质谱联用（GC-MS）技术研究PCK1表达差异造成的肝癌细胞代谢变化谱;在细胞和裸鼠皮下移植瘤模型中探索PCK1通过Wnt信号通路抑制HCC的作用机制。本项目不仅能进一步揭示HCC发生的分子机制，而且有望提供新的靶点用于临床肝癌的诊断和治疗。

研究背景：代谢重编程是肿瘤的重要特征之一，葡萄糖代谢的改变对恶性肿瘤的发生发展至关重要。肝脏是糖异生的主要器官，肝细胞癌（HCC）中糖异生的关键酶 PCK1抑制肝癌细胞增殖的机制仍不明确。研究内容: （1）PCK1在肝癌中的表达和肿瘤恶性程度观察；（2）研究PCK1对肝癌细胞增殖能力、细胞周期和代谢物的调控；（3）PCK1对Wnt信号通路和AMPK/p27Kip信号轴的调控；（4）敲除PCK1通过促进ROS产生和Nrf2信号通路的激活。重要结果及数据: （1）PCK1在肝癌组织中低表达且与预后不良相关；（2）过表达PCK1抑制肝癌细胞增殖和克隆形成，PCK1抑制肝癌细胞G1期到S期转换，敲除PCK1影响细胞能量代谢；（3）PCK1下调Wnt信号通多个基因表达，降低β-catenin蛋白水平；PCK1降低ATP水平，激活AMPK/p27信号轴阻滞细胞周期G1/S期转换；体内外实验表明AMPK激动剂二甲双胍能够抑制PCK1缺失的肝癌细胞增殖（4）敲除PCK1促进ROS产生和Nrf2信号通路的激活，上调TXNRD1表达；而TXNRD1抑制剂金诺芬能够显著增加PCK1低表达肝癌细胞对化疗药物索拉菲尼的敏感性。科学意义：本课题揭示了PCK1抑制肝癌进展的新机制，即PCK1影响了代谢物AMP/ATP和ROS水平，过表达PCK1通过抑制Wnt信号通路、AMPK/p27信号轴、Nrf2/TXNRD1信号轴而抑制肝癌细胞增殖。本研究从代谢角度证实了二甲双胍抑制肝癌的作用，并提出了金诺芬联合索拉非尼治疗肝癌的新策略。