基于Wnt/β-catenin信号通路探讨miR-122调控肝癌干细胞的分子机制及鳖甲煎丸的干预作用

In this research, we will use immune magnetic bead separation method to separate liver cancer stem cells, and then take a gene transfection technology to establish an overexpression of miR - 122 in liver cancer stem cell lines. We detect the expression of miR - 122 and its target gene Wnt1, and the beta - catenin and TCF4 which is the key molecular of Wnt/beta -catenin signaling pathway in liver cancer stem cells by real-time fluorescent quantitative PCR method and Western blot method, and observate the proliferation and self-renewal potential changes in liver cancer stem cells. We aim to prove the molecular mechanisms that miR - 122 targeted Wnt1 and Wnt/beta - catenin signaling pathway to regulate liver cancer stem cells. And then we use animal serum that contained biejiajianwan to cultured liver cancer stem cells. We want to Illustrate that the intervention of liver cancer stem cells is involed in the mechanism of biejiajianwan in the liver cancer therapy, and prove a molecular mechanism that miR - 122 targeted Wnt1, thus affected the Wnt/beta - catenin signaling pathway in liver cancer stem cells. In all, we aim to explore the molecular mechanism of traditional Chinese medicine in liver cancer therapy in molecular biology level and reflect the ethnic characteristics of folk medicine. We expected to provide a new way of targeting therapy to liver cancer stem cells, and a new, more specific targets for liver cancer prevention. This research will have a great significance in improving the current status of liver cancer therapy.

采用免疫磁珠法分选肝癌干细胞，利用基因转染技术建立过表达miR-122的肝癌干细胞株。通过实时荧光定量PCR法和Western-blot法检测miR-122及其靶基因Wnt1，以及Wnt/β-catenin 信号通路的关键分β-catenin和TCF4在肝癌干细胞的表达，观察肝癌干细胞的增殖和自我更新潜能的变化。阐明miR-122靶向Wnt1，影响Wnt/β-catenin信号通路，从而调控肝癌干细胞的分子机制。再通过鳖甲煎丸含药动物血清培养肝癌干细胞，证明鳖甲煎丸对肝癌干细胞有干预作用，阐明其分子机制为调节miR-122表达，从而影响肝癌干细胞的Wnt/β-catenin信号通路。本课题从分子生物学层面探讨中医药治疗肝癌的分子机制，体现民族医药特色，为针对肝癌干细胞的靶向治疗提供新思路，为肝癌的防治提供新的、更有针对性的靶点，对改善肝癌治疗现状具有十分重要的意义。

肝细胞肝癌(HCC)是世界范围内发病率第5位的恶性肿瘤，其病死率、复发率高，而且治疗效果不理想。肿瘤干细胞理论认为，肝癌干细胞（HCSCs）是HCC发生、发展、侵袭转移和治疗后复发的根源。在肝脏特异性高表达的miR-122具有抑制肝癌形成和发展的抑癌基因样功能。miR-122可能成为针对肝癌干细胞靶向治疗的靶点。前期临床和实验研究证明，鳖甲煎丸治疗HCC效果显着。但鳖甲煎丸对肝癌干细胞是否也有作用，其分子机制是否涉及调控miR-122靶向Wnt1，并靶向调控Wnt/β-catenin信号转导通路，目前尚未明确。.本研究通过分选CD133+SMMC-7721肝癌干细胞作为研究对象。利用基因转染技术，验证miR-122通过作用Wnt/β-catenin途径调控肝癌干细胞的增殖、侵袭特性。然后制备鳖甲煎丸含药血清，验证miR-122和Wnt/β-catenin信号通路在鳖甲煎丸干预肿瘤干细胞中的作用。.研究结果表明，CD133+SMMC7721细胞具有更强的增殖和侵袭能力；miR-122可通过下调Wnt1、β-catenin和TCF4蛋白来抑制CD133+SMMC7721细胞的增殖；鳖甲煎丸可以抑制CD133+SMMC7721细胞的增殖，其机制是通过抑制Wnt1、β-catenin和TCF4蛋白的表达来完成，但这一过程暂未观察到与miR-122有关。最后，通过转录组数据分析发现，鳖甲煎丸可通过141个miRNA分子来调控Wnt/βcatenin信号通路的表达，其中，hsa-miR-483-3p、hsa-miR-3195和hsa-miR-4508可直接调控Wnt1的表达，hsa-miR-3196则可作用于TCF4基因。因此，进一步验证这4个miRNA与Wnt/βcatenin信号通路的互作关系，对于明确其在肿瘤干细胞中的作用以及挖掘新的HCC治疗靶点具有重要意义。.本研究证明了miR-122可通过作用于其靶基因Wnt1，从而影响Wnt/β-catenin 信号通路，调控肝癌干细胞；鳖甲煎丸对肝癌的治疗机制涉及对肝癌干细胞的干预，其机制为调控hsa-miR-483-3p、hsa-miR-3195、hsa-miR-4508和hsa-miR-3196靶向Wnt/β-catenin 信号通路抑制肝癌干细胞的增殖。这些研究结果对改善肝癌不理想的治疗状况具有重要意义。