ISL1在肝癌干细胞中的作用及其分子机制

Hepatocellular carcinoma (HCC) is associated with a high morbidity and mortality due to its high rate of recurrence and metastasis. Tumor stem cells are considered to be the root of recurrence and metastasis. However, little is known about the molecular mechanism of tumor stem cells. A single patient's primary(Hep11)and recurrent(Hep12)HCC-derived cell lines, Hep12 cells are enriched for tumor stem cells, whereas Hep11 cells were non-tumorigenic. And we generated a monoclonal antibody against recurrent HCC, 1B501, which bound the calcium channel α2δ1 subunit and identified a subset of tumor initiating cells with stem cell-like properties. These two cell lines and Mab1B501 provide a unique cellular model for researching the liver tumor stem cells. Our previous work found that the high expression of ISL1 in recurrent HCC-derived cell lines Hep12 and Mab1B501 positive tumor stem cells, and knock down ISL1 declined the the cell self-renewal capacity and tumorigenesis. These results suggested that ISL1 may promote the malignant properties of liver tumor stem cells.The high expression of ISL1 in liver tumor tissue is negative correlation with patient survival period. In this study, we will want to further explore the the ISL1 high expression of clinical significance and the role in the regulation of malignant characteristics of tumor stem cells and molecular mechanisms, and provide clues to the prognosis and therapy of liver tumor.

肝癌术后复发和转移是导致患者死亡的最主要因素，肿瘤干细胞被认为是复发和转移的根源，但有关肝癌干细胞调控的分子机制却知之甚少。Hep11和Hep12是我们实验室从同一肝癌病人原发和复发灶建立的一对细胞，其中Hep12多数具备肿瘤干细胞特性，而Hep11则不致瘤。我们从这对细胞中制备了能有效识别肝癌干细胞的Mab1B501单克隆抗体，Hep-11和Hep-12细胞对以及Mab1B501阳性和阴性细胞亚群是肝癌干细胞研究的良好模型。我们前期工作发现ISL1在Hep12细胞以及Mab1B501阳性肝癌干细胞中高表达，干扰ISL1表达后细胞自我更新能力和成瘤能力明显下降，提示该基因对肝癌干细胞恶性生物学特性有促进作用。在肝癌组织中ISL1高表达与患者生存期负相关。本研究拟此基础上，深入探讨ISL1表达的临床意义及对肝癌干细胞恶性生物学特性调控作用和分子机制，为揭示肝癌预后和治疗提供依据。

肝癌术后复发和转移是导致患者死亡的最主要因素，肿瘤干细胞被认为是复发和转移的根源，但有关肝癌干细胞调控的分子机制却知之甚少。Hep11和Hep12是我们实验室从同一肝癌病人原发和复发灶建立的一对细胞，其中Hep12多数具备肿瘤干细胞特性，而Hep11则不致瘤。我们从这对细胞中制备了能有效识别肝癌干细胞的Mab1B501单克隆抗体，Hep-11和Hep-12细胞对以及Mab1B501阳性和阴性细胞亚群是肝癌干细胞研究的良好模型。我们通过Super Array RT-PCR及Western blot，免疫荧光确认ISL1在同一病人复发（Hep-12）中的表达高于原发（Hep11）。并且ISL1高表达的在肝癌的病人预后比较差。我们利用1B501抗体筛选出具有肿瘤干细胞特性1B501+的Hep11，Huh7, HepG2及三例肝癌病人的PDTX。通过RT-PCR、western-blot的检测方法发现ISL1在1B501+的的细胞群中的表达明显高于其在1B501-的细胞群。并且转染ISL1慢病毒过表达载体Hep11，HuH7，HepG2及1B501-的两例PDTX后，肝癌肿瘤干细胞球体形成能力和小鼠体内成瘤能力及体内二次成瘤能力增强。反之，敲低后成球能力和成瘤能力均减弱。我们构建了HuH7-ISL1稳定细胞株后进行ChIP-sequence和RNA-sequence，寻找到ISL1的下游潜在靶基因ZEB1/ZEB2，并且经过western-blot，免疫荧光，荧光素酶分析实验，ChIP等验证了ISL1与NPM1形成复合物协同调控ZEB1/ZEB2的来发挥增强肝癌肿瘤干细胞干性的功能。本研究拟此基础上，深入探讨了ISL1表达的临床意义及对肝癌干细胞恶性生物学特性调控作用和分子机制，为揭示肝癌预后和治疗提供依据。