肝脏条件性敲除极性蛋白PAR6γ导致小鼠自发性肝癌的机制研究

Cell polarity is a common feature of many different types of cells，and it is essential to normal differentiation and function of cells. Partitioning defective 6（PAR6）gene encodes PAR6 protein，which is crucial to asymmetric cell division and polarized growth. PAR6γ protein as a member of the PAR6 protein polarity complex，affects the synthetic of centrosome and protein recruitment to the centrosome. The abnormal number of centrosome and the loss of cell polarity may eventually lead to the occurrence of tumor. Here we reported that PAR6γ expression is reduced in clinical HCC tissues and chemical-induced hepatocellular carcinoma (HCC) model. Loss of PAR6γ expression in HCC tissues is associated with poor overall survival and high recurrence. Moreover, liver-specific deletion of PAR6γ results in spontaneous liver fibrosis at 6th month and spontaneous HCC at 9th month. We further conducted co-immunoprecipitation combined with mass spectrum assay, and found that PAR6γ could interact with YAP and regulate YAP expression at protein level. Indeed, the expression of YAP is remarkably increased after hepatocyte-specific deletion of PAR6γ. These results proposed a previously unrecognized role for PAR6γ in carcinogenesis, which may regulate hepatocyte polarity via Hippo-Yap pathway. In the present study, we aim to study the role of PAR6γ in different stages of HCC carcinogenesis. Our study will provide new clues to understand the role of PAR6γ-associated polarity signaling in HCC carcinogenesis.

细胞极性的破坏是肝癌发生、发展的先决条件，但其上游调控机制尚不清楚。我们前期发现，调控细胞极性的关键蛋白PAR6γ，在人原发性肝癌组织和化学诱导肝癌动物模型中，表达均显著降低，癌组织中PAR6γ的降低与肝癌的预后不良相关；进一步构建了肝脏条件性敲除PAR6γ基因小鼠，发现该小鼠6个月发生自发性肝纤维化，9个月发生自发性肝癌；利用免疫沉淀联合质谱筛选，找到可与PAR6γ相互作用的候选蛋白——Yes相关蛋白（Yes-associated protein, YAP），且YAP在肝脏条件性敲除PAR6γ基因小鼠中蛋白水平显著升高。提示肝脏表达的PAR6γ可能通过调控YAP水平，调控肝癌极性。本课题拟利用条件性敲除基因小鼠、原代肝细胞和临床肝癌资料，在动物水平、细胞水平和人体水平，探讨肝癌极性异常的机制，课题的完成将为肝癌靶向治疗提供新策略(纠正异常极性)和动物工具(筛选干预药物)。

细胞极性的破坏是肝细胞癌的主要特征之一，本课题旨在研究极性蛋白PAR6γ在肝细胞癌中的作用。通过qPCR、Western blot、免疫染色检测和肝功能分析，检测从肝细胞癌患者和条件性敲除小鼠（DEN诱导的HCC，PAR6γf/f; Alb-cre和PAR6γ f/f; Yapf/f; Alb-Cre小鼠）获得的肝组织的特征变化。我们研究了PAR6γ与肝细胞癌的临床特征和预后之间的关系，以及PAR6γ条件性敲除小鼠的动物表型。我们采用了分子和生物化学策略，深入研究PAR6γ对于肝细胞癌的作用。结果显示：PAR6γ在肝细胞癌组织中下调，并与肝细胞癌患者的不良预后和侵略性肿瘤表型有关。PAR6γ缺失的小鼠，其血清酶活性（AST、ALT、ALP、γ-GT）和胆红素（TBA、DBIL、TBIL）水平升高。在11个月大时，PAR6γ缺失小鼠的肝细胞癌发病率很高。早在3个月时，PAR6γ缺失小鼠的促炎症细胞因子（TGF-β，IL-6）、肝纤维化标志物因子（Colla1，α-SMA）和趋化因子（Timp1，CXCL1，CXCL2，MCP1）的表达增加。此外，PAR6γ缺失小鼠表现出肝脏增大，紧密连接的完整性被破坏，极性指数和细胞分化下降，肝细胞的多倍体状态和增殖增加，并出现自发的肝脏肿瘤发生。分子机制研究表明：这种表型与Yap信号异常有关。PAR6γ分别直接与Yap和泛素连接酶β-Trcp结合，以增强Yap和泛素连接酶β-Trcp之间的结合，这一作用增加了Yap泛素化及蛋白体降解，随后，Yap的入核减少。而Yap敲除以及维替泊芬（VP，Yap的抑制剂）治疗可以抑制PAR6γ缺失小鼠的促肿瘤作用。我们的研究结果发现了一种新型的自发性肝癌动物模型，该模型很好的模拟了肝癌极性缺失的特性，可以用来进行肝癌治疗药物的动物实验，为肝癌研究提供了一个独特的动物模型。