SIX1在HBx诱导肝癌细胞干性获得中的功能和机制研究

Liver cancer is a common malignant tumor in our country. How to prevent recurrence and metastasis is an urgent problem in the treatment of liver cancer. The previous research revealed that cancer stem cells are the main cause of tumor recurrence and metastasis. HBx plays an important role in the formation of liver cancer stem cells. However, the mechanism remains not clear. Our previous study found that SIX1, which is a novel core molecule in acquisition of cancer stem cell-like properies, could interact with and regulated by HBx. This study investigates the function and regulation mechanism of SIX1 in acquisition of cancer stem cell-like properies induced by HBx in liver cancer. Based on previous researches, explored the molecular mechanism of SIX1 regulated by HBx in protain level and transcriptional activation. SIX1 knock-out cells and HBx knock-in mice analysis to determine the biological function and core function of SIX1 in acquisition of cancer stem cell-like properies induced by HBx in liver cancer. Clinical samples will be used to reveal the clinical significance and relationship between the protein level of SIX1 and HBV infection. The results help to clarify the mechanism of SIX1 in liver cancer stem cell-like properies, and provide a novel target for prevention and clinical treatment in liver cancer recurrence and metastasis.

肝癌是我国常见的恶性肿瘤，抑制复发和转移是肝癌治疗中亟需解决的问题。根据以往研究发现，肿瘤干细胞是复发和转移的主要原因，HBx在肝癌干细胞形成过程中发挥重要作用，但其机制还不明确。我们前期研究发现了新的能与HBx相互作用，且受HBx调控的肿瘤细胞干性获得相关分子，即SIX1。本研究拟对SIX1在HBx诱导肝癌细胞干性获得中的功能和调控机制进行研究，以前期研究为基础，在蛋白水平、转录激活等方面探讨HBx调控SIX1的分子机制；通过SIX1基因敲除细胞和HBx敲入小鼠，分析SIX1在HBx诱导肝癌细胞干性获得中的生物学功能和关键作用。利用临床标本确定SIX1蛋白表达与HBV感染的相关性和临床意义，为阐明SIX1在肝癌细胞干性获得中的功能和分子机制提供理论依据，为肝癌复发和转移的预测和临床治疗提供新的靶点。

HBx在肝癌干细胞形成过程中发挥重要功能，肿瘤干细胞又与癌症的复发转移密切相关，但是HBx调控肝癌干细胞特征的具体机制仍不明确。我们前期研究发现了新的能与HBx相互作用的肿瘤细胞干性相关分子SIX1。我们通过蛋白质质谱分析，免疫共沉淀，GST pull-down等方法检测HBx与SIX1的相互作用及功能结构域。检测HBx对SIX1蛋白表达和转录活性的影响。通过建立SIX1敲除细胞模型检测SIX1在HBx调控肝癌细胞干性获得中的功能。收集肝癌患者的组织标本检测SIX1与HBx的相关性及临床意义。我们发现：HBx和SIX1蛋白可以直接相互作用，这种相互作用发生在HBx蛋白的1-110aa区域及SIX1的HD结构域。HBx可以通过降低类泛素化修饰进而增加SIX1蛋白的水平。HBx还通过促进SIX1与EYA2转录复合物的形成增加下游干细胞标志物ALDH1、CD44和CD133的表达。细胞学实验发现HBx可以明显的促进肝癌细胞的干性特征，SIX1敲除可以明显抑制肝癌细胞的干性特征同时抑制HBx的功能。动物实验发现SIX1敲除后可以明显抑制肝癌的转移发生。通过病例分析发现SIX1在HBV感染的患者中表达明显升高，并且高表达预示预后较差。该研究阐明了SIX1在HBV相关肝癌中调控肿瘤干细胞获得的功能和新机制，为肝癌复发和转移提供新的预测和治疗靶点