SCUBE1调控Shh/SMO/Gli1信号通路在肝癌细胞干性转化与恶性进程中的作用及其预后预测研究

Many studies have found that liver cancer stem cells(LCSCs) is an important factor affecting the recurrence and metastasis of liver cancer, and the tumor microenvironment plays an important role in the process of liver cancer cells get stem cell phenotype. In our previous study, we found the liver cancer cells would get stem cell phenotype when they were co-cultured with activated hepatic stellate cell(LX2). Moreover, using gene chip, cancer associated fibroblasts(CAFs) primary culture and qPCR method, we screened a key gene, SCUBE1, which was produced by CAFs and could promote liver cancer cells transform into LCSCs. In this project, by constructing Lentivirus loaded SCUBE1 shRNA plasmid, co-culture experiment, orthotopic co-transplantation model of HCC in nude mice, we would verify the effect of SCUBE1 on the process of the liver cancer cells get stem cell phenotype in vivo and vitro. By using luciferase reporter system and co-immunoprecipitation technique, we will investigate the mechanism of how SCUBE1 control the process of liver cancer cells get stem cell phenotype. Through constructing prospective cohorts, we will establish the liver cancer recurrence and metastasis prediction model which combined the SCUBE1 with the clinical information by using parametric and nonparametric methods. The findings of this study will help to clarify the possible mechanism involved in the generation of LCSCs and the mechanism of SCUBE1 play a key role in the process of generation of LCSCs. Furthermore, the prediction model can be used to screen high risk patients who would have worse prognosis and then to achieve the early prevention final goal.

许多研究发现肝癌干细胞是影响肝癌复发转移及预后的重要因素，而肿瘤微环境对肝癌细胞的干性转化及维持起重要促进作用。申请人近期研究发现与活化的肝星状细胞共培养后肝癌细胞可获得干性表型，并筛选出了原代肿瘤相关成纤维细胞（CAFs）促肝癌细胞干性转化的关键基因SCUBE1。在本项目中，我们拟进一步从分子、细胞及动物水平验证SCUBE1基因是否对肝癌细胞干性转化及侵袭转移有促进作用；探讨SCUBE1/Shh/Gril1信号通路调控肝癌细胞干性转化的具体分子机制；并通过前瞻性队列利用参数及非参数方法构建联合SCUBE1水平与临床数据的肝癌术后转移复发预测模型。本研究的结果，将有助于阐明肝癌干细胞产生的可能机制及SCUBE1基因在其中发挥的关键作用，并且以SCUBE1表达水平联合临床数据构建的预测模型可应用于肝癌转移复发高危人群的早期筛检与预后评估，从而达到早发现、早诊断、早治疗的预防目标。

肝癌干细胞是影响肝癌转移及预后的重要因素，在许多研究中也被发现是复发转移及化疗耐药的关键因素之一，而肿瘤微环境对肝癌细胞的干性转化及维持起重要促进作用。本研究发现与活化的肝星状细胞共培养后肝癌细胞可获得干性表型，且极大促进了肝癌细胞的增殖转移能力，据此进行基因测序筛选出原代肿瘤成纤维细胞（CAFs）中可能促肝癌细胞干性转化的关键基因SCUBE1. 从肝癌病人组织中分离提取的波形蛋白及α-SMA阳性、CD31阴性的原代肿瘤相关成纤维细胞（CAFs）中SCUBE1均具有较高表达水平。在本项目中，我们进一步明确了SCUBE1在肝癌患者进展中发挥的作用，并从分子、细胞及动物水平验证CAFs中高表达的SCUBE1基因能对肝癌细胞干性转化及侵袭转移产生促进作用。利用调控基因表达技术，改变提取得到的肝癌成纤维细胞中SCUBE1的表达水平，检测其表达水平变化对肝癌恶性进程及干性转化的影响，并从分子、细胞水平及裸鼠皮下移植瘤研究后，确定SCUBE1可通过促进Shh/SMO/ Gli1信号通路来促进肝癌细胞恶性进程和干性转化。在肝癌组织及血清中，分别通过WB和Elisa实验手段检测后可发现SCUBE1也呈现出高水平，这也说明SCUBE1在肝癌病人的发生发展过程中起重要作用。有助于阐明肝癌干细胞产生的可能机制及SCUBE1基因在其中发挥的关键作用，从而为肝癌患者的治疗提供新的方案，并且有望以该靶点为肝癌患者提供新的治疗手段