SPTBN1缺失介导肝干细胞增殖与恶性转化的作用及其分子机制研究

Hepatocellular carcinoma (HCC) arises from the abnormal differentiation and proliferation in the liver stem cells. Wnt/β-catenin pathway plays an important role in this process. SPTBN1 is the most common nonerythrocytic member of the β-spectrin gene family. Down regulation of SPTBN1 confers susceptibility to liver cancer but the mechanism is unknown. We previously found loss of SPTBN1 in HCC cells can promote metastasis through Wnt activation. ASCL1 can promote glioblastoma cancer stem cell properties and Wnt pathway activation, and we found that the number of liver stem cell and the expression of ASCL1 increased in SPTBN1 +/- mice liver. So we hypothesize loss of SPTBN1 can promote proliferation and malignant transformation of liver stem cells by activating ASCL1-induced Wnt pathway. In this project, we firstly establish DDC -induced liver stem cell activation model with SPTBN1+/- and WT mice. Using liver tissue or sorting liver stem cells, we will study the role of SPTBN1 on proliferation and malignant transformation of liver stem cells and on activation of ASCL1/Wnt pathway. Then using liver stem cells or MEF cells, the mechanisms of SPTBN1 on Wnt pathway activation by inducing ASCL1 expression will be explored. Finally, correlation between expression of SPTBN1 and clinic pathological features, liver stem cell markers and Wnt pathway will be demonstrated by immunohistochemistry stain on HCC tissue. Our project will provide a theoretical basis for the search for new markers and therapeutic targets for liver cancer.

肝癌起源于肝干细胞的异常分化和增生，Wnt通路在此过程起重要作用。SPTBN1是收缩蛋白（spectrin）的βII亚基，其缺失导致小鼠发生肝癌而机制未明。我们前期发现下调肝癌细胞SPTBN1能活化Wnt促进肝癌转移。ASCL1可激活Wnt通路，促进肿瘤干细胞特征和致瘤性，而SPTBN1+/-小鼠肝脏ASCL1表达上调、肝干细胞增多。因此设想SPTBN1缺失通过诱导ASCL1表达、活化Wnt通路而调控肝干细胞的增殖及恶性转化。本课题先用SPTBN1+/-及野生小鼠建立DDC诱导的肝干细胞活化模型，体内外研究SPTBN1对肝干细胞增殖及恶性转化、ASCL1/Wnt通路的影响，并利用MEFs、肝干细胞研究其调节ASCL1/Wnt通路的机制，最后用人肝癌组织研究SPTBN1与肝癌干细胞标志物表达、临床病理特征及ASCL1/Wnt通路的相关性。本研究将为寻找新的肝癌标志物和治疗靶点提供理论依据。

肝细胞癌（Hepatocellular carcinoma，HCC）是严重威胁人类健康的恶性疾病之一，其致死率在世界范围内占肿瘤相关疾病的第三位。深入研究肝癌发病的分子机制对于提高肝癌的防治效果具有十分重要的意义。肝干细胞增殖、分化的异常可能是肝脏肿瘤发生中的重要事件，肝干细胞与原发性肝癌关系非常密切。另有报道，自噬缺陷可诱导肿瘤发生。在小鼠的肝脏中特异性敲除自噬相关重要基因Atg5和Atg7能导致肝脏肿大并伴有肝癌发生，而敲除p62能抑制自噬缺陷诱发的肝癌。SPTBN1是TGF-β信号通路的调节蛋白，40-70%SPTBN1+/-小鼠会在15月龄自发肝癌，说明SPTBN1缺失与肝癌的发生密切相关。因此，本课题首先利用SPTBN1+/-及WT小鼠建立DDC（Dicarbethoxydihydrocollidine，1,4-二氢-2,4,6-三甲基-3,5-吡啶二甲酸二乙酯）诱导的肝干细胞活化模型，并腹腔注射自噬诱导剂，1月后提取肝组织并分选肝干细胞，体内外研究SPTBN1对肝干细胞增殖、自噬及恶性转化的作用及可能机制。其次，利用肝癌细胞系，研究SPTBN1缺失与肝癌细胞干化、自噬的关系及机制。此外，利用人肝癌组织样本，研究SPTBN1与自噬的关系。结果发现，在DDC诱导的小鼠模型中，SPTBN1缺失可抑制肝干细胞自噬、促进肝干细胞增殖及恶性转化。其机制与SPTBN1抑制Hippo信号通路有关。而自噬诱导剂可逆转SPTBN1缺失导致的肝干细胞恶性转化和癌变。其次，利用Huh-7和PLC/RPF/5肝癌细胞系发现SPTBN1敲低后通过抑制Hippo信号通路,抑制细胞自噬，增加肝癌细胞干性；过表达SPTBN1可以激活Hippo信号通路，提高肝癌细胞自噬水平。最后我们利用组织芯片，发现与癌旁组织相比，肝癌组织中SPTBN1表达明显下降且与P62成正相关。综上， 我们的研究结果说明SPTBN1缺失可通过抑制Hippo信号通路，降低肝干细胞自噬水平，促进其增殖、恶性转化，从而促进肝癌发生。本研究将为肝癌的防治提供理论基础和实验依据以及新的靶点。