GLS2基因在原发性肝癌转移与侵袭中的作用及机制研究

As one of the most common malignancies in the world, hepatocellular carcinoma (HCC) has a very high morbidity and mortality. It is a major global health challenge that affects an estimated 500,000 people worldwide each year. The overall survival of HCC patients remains unsatisfactory because of a high incidence of recurrence and metastasis after hepatic resection. Understanding the mechanisms of HCC recurrence and metastasis should improve HCC treatment and outcomes. In our study, the alteration in expression of genes between normal HCC samples and HCC sample which were proved to have a metastasis result were screened using microarrays. The results revealed a GLS2 low expression that was 18-fold less than HCC samples without metastasis and 21-fold less compared with normal liver tissue. This result was further conformed by analyzing the expression GLS2 in 203 HCC tissue samples obtained with informed consent from patients who underwent hepatectomy in the First Affiliated Hospital of Harbin Medical University from 2009 to 2012, suggesting that, GLS2 low expression might play an important role in HCC metastasis. GLS2 gene is well conserved at the genomic level during evolution. A growing body of literature has emerged in the last decade on the involvement of energy metabolism in the pathogenesis of cancer. Importantly, some group reported that aberrant expression of some gene regulate metabolism might regulate Epithelial-mesenchymal transition (EMT), an important physiological process contributing to HCC metastasis. Based upon these evidence and our previous findings, we hypothesize that GLS2 can inhibit HCC metastasis by regulating EMT and the mechanism of this regulation may be through targeting ROS. In conclusion, in the present study we aimed to determine the involvement of GLS2 in HCC metastasis and our study will provide new insight into the mechanism of HCC metastasis.

原发性肝细胞癌是全球最常见的恶性肿瘤之一, 占世界恶性肿瘤死亡率的第三位。肝癌的主要死因是转移与复发，因此阐明肝癌复发转移机理,是提高肝癌总体疗效的根本。因此，我们通过基因芯片筛选出与肝癌转移相关的基因GLS2，并应用Real time PCR在203例临床肝癌病例和不同转移潜能的肝癌细胞系中证实GLS2与肝癌的转移呈负相关性。并且，通过预实验在肝癌细胞系与裸鼠肝癌转移模型中初步证实GLS2基因的表达可以抑制肝癌细胞转移。但确切的分子生物学作用机制仍需进一步研究。因此，本项目在前期研究的基础上，进一步扩大临床标本量（300例），并通过结合慢病毒介导的RNA干扰技术沉默/过表达差异GLS2，在多种肝癌细胞系以及裸鼠肝癌转移模型中检验与论证GLS2基因对于肝癌转移的影响，并从肿瘤上皮细胞间质转化（EMT）及能量代谢入手，分析GLS2基因调控转移的作用机制。

肝癌导致死亡的原因主要是其高度转移的特性，研究表明肝癌根治性切除术后5年转移率高达60-70%，因此找到肝癌转移和侵袭的关键因素并发现其影响肝癌转移的分子机制，对于提升肝癌的治疗的效果具有重要意义。基于上述问题及研究现状，本研究小组利用最新型的基因芯片对正常肝脏组织、无转移的单纯肝癌组织、肝切除术后发生肝内转移的肝癌组织以及肝切除术后发生肺脏转移的肝癌组织进行筛选，通过生物信息学分析，发现谷氨酰胺酶-2（Glutaminase 2，GLS2）与肝癌转移（无论是同时性还是异时性）具有显著负相关性，并且，GLS2的表达水平与肝癌患者的预后显著相关。提示GLS2具有潜在抑制肝癌转移的作用，为了进一步探索GLS2抑制肝癌转移的潜在机制，我们开展了相应的体外及体内实验。本项目的研究结果表明：①在不同转移潜能的肝癌细胞系、肝癌细胞系构建的裸鼠原位肝癌模型中，GLS2可以明显的抑制肝癌的转移；②GLS2抑制肝癌转移的作用机制之一在于抑制肝癌的EMT；③GLS2对于EMT的抑制作用在于GLS2可以抑制肝癌的糖酵解及ROS。④证实肝癌的转移潜能与肝癌的能量代谢水平密切相关，为肝癌转移的治疗提供了新的思路。本研究的部分研究结果发表在知名杂志Cancer Letters，影响因子（5.992）。