胆碱激酶α调控上皮间质转化促进肝癌侵袭转移的实验研究

The prognosis of patients with HCC remains pool largely due to the high frequency of tumor recurrence or metastasis. Therefore, it is particularly important to elucidate the molecular mechanism underlying HCC progression and metastasis in order to improve patient outcomes. Recently, choline kinase α (CHKA) has been implicated in several human cancers including breast, lung and colorectal carcinomas, however, its potential role in HCC progression and metastasis has not been investigated. Our preliminary work revealed that chka gene copy number was elavated in HCC and CHKA was frequently overexpressed in human HCC tissues and cell lines, which may play a positive role in promoting the migratory and invasive properties of HCC cells. We also found that CHKA could facilitate EMT process, a critical early event for the invasion and metastasis of many carcinomas. Based on these preliminary data, the aim of this project is:(1) to intensively explore the role of CHKA in HCC progression and metastasis using gene overexpression and silencing technology both in vitro and in vivo; (2) to dissect the molecular events by which CHKA induces EMT using the classical signal transduction research methods; (3) to evaluate the clinical and prognostic significance of CHKA overexpression in HCC by analyzing the correlation of intratumoral CHKA expression with clinicopathologic features and survival of more than 180 HCC patients. It is expected to clarify the contribution of CHKA in HCC progression and metastasis and reveal its underlying mechanism, which may have important theoretical significance and potential clinical value.

肝癌侵袭转移是导致肝癌患者死亡的主要原因，深入研究肝癌侵袭转移的分子机制对于指导治疗以提高肝癌患者生存率具有重要意义。近年研究发现胆碱激酶α(CHKA)参与影响乳腺癌、肺癌等多种肿瘤的恶性表型，但其在肝癌侵袭转移中的作用尚未见报道。我们前期研究发现chka基因拷贝数在肝癌基因组中扩增，且CHKA在肝癌组织和细胞系中呈普遍高表达、参与正向调控肝癌细胞的侵袭转移并可诱导细胞发生上皮间质转化。基于以上基础，该项目拟运用基因过表达及沉默技术、可视化肝癌转移研究体系和经典信号转导研究手段，全面深入分析CHKA对肝癌侵袭转移的影响及其调控上皮间质转化的分子机制；同时结合180例以上的肝癌临床样本，分析CHKA表达与肝癌患者临床病理特征及生存预后的关系。预期从信号转导角度阐明CHKA调节肝癌侵袭转移的分子机理，明确关键节点分子；为寻找新的肝癌干预靶点和预后指标奠定基础，具有重要理论意义和潜在临床应用价值。

一、项目背景.肝癌侵袭转移是导致肝癌患者死亡的主要原因，然而肝癌细胞侵袭转移的分子机制仍不十分清楚。近年研究发现胆碱激酶α(CHKA)在多种恶性肿瘤中呈现高表达并参与多种肿瘤的恶性表型，然而, CHKA是否参与调控肝癌侵袭转移过程及其发挥作用的机制尚未见报道。.二、主要研究内容.1.明确CHKA在肝癌组织和细胞系中表达情况。.2.研究肝癌组织标本中CHKA的预后价值。.3.明确CHKA在肝癌侵袭转移过程中的生物学功能。.4.研究CHKA参与调控肝癌的侵袭转移过程的分子通路机制。.5.分析CHKA在肝癌组织中异常高表达的原因。.三、重要结果.1.CHKA在肝癌组织和细胞系中呈普遍高表达。.2.肝癌组织中CHKA的表达水平与肿瘤的微血管侵犯、疾病分期、肿瘤复发、患者死亡存在正向关联，是肝癌患者肿瘤复发和死亡的独立危险因素。.3.过表达CHKA在体外能明显促进肝癌细胞的迁移和侵袭能力，在体内能明显促进肝癌细胞的肺转移能力；反之，沉默CHKA表达则明显抑制肝癌细胞在体外体内的迁移和侵袭潜能。.4.CHKA可诱导肝癌细胞发生EMT表型改变，并诱导EMT相关基因表达。.5.CHKA促进肝癌细胞侵袭转移是通过激活AKT通路所介导的。.6.机制分析发现，CHKA促进AKT活化及其促侵袭转移效应主要是通过增强与膜EGFR相互作用，不仅促进膜EGFR的二聚化和磷酸化激活，同时抑制膜EGFR内化介导的信号衰减过程，从而激活EGFR实现的。.7.联合CHKA和磷酸化AKT的表达水平这两指标具有更好的预后价值。.8.chka基因在肝癌基因组中存在拷贝数扩增，其拷贝数扩增水平与其表达水平存在正相关性。.四、科学意义.本项目发现CHKA在肝癌中高表达，与chka基因在肝癌基因组中拷贝数扩增有关。肝癌中CHKA高表达对患者肿瘤复发和死亡具有独立预后价值。肝癌细胞中CHKA高表达通过增强与膜EGFR相互作用，促进膜EGFR二聚化和磷酸化激活，并抑制膜EGFR内化介导的信号衰减，从而激活EGFR，进而活化下游AKT，诱导肝癌细胞发生EMT，发挥促肝癌侵袭转移的生物学功能。研究结果提示EGFR-AKT通路在CHKA促侵袭转移的过程中发挥重要作用，联合CHKA和p-AKT两指标具有更好预后价值。研究结果为寻找肝癌侵袭转移干预新靶点和预后分子标志物奠定基础，具有理论意义和潜在应用价值。