miR-125b抑制剂增敏脱甲氧基姜黄素和替莫唑胺对胶质瘤干细胞的作用及其机制研究

Malignant glioma is one of the highest degree of human intracranial malignancy tumors, which is easy to recur, but difficult to cure. Nowadays, the presence of glioma stem cells is considered to be one of its main reasons. We found that miR-125b inhibitors can effectively inhibit the primary glioma stem cells growth and invasion, and its mechanisms are mediated by PI3K/AKT signaling pathway inhibition, Bcl-2/Bax-Caspase 3 pathway activation and MMPs/TIMP pathway inhibition.We also observed that it can sensitize glioma stem cells to demethoxycurcumin (DMC) and temozolomide (TMZ)；DMC can also enhance the chemosensitivity of TMZ by inhibiting ABCG2 gene expression while the ABCG2 gene is regulated by miR-125b expression. Accordingly, we hypothesize: the process of miR-125b inhibitor → DMC → TMZ exists progressively enlarged sensitizing effects, and miR-125b takes part in the signaling pathway involving proliferation, invasion and apoptosis process of glioma stem cells. This project intends to establish the primary glioma stem cells in vitro and intracranial planted model, and finish the anti-tumor experiments of proliferation, invasion and apoptosis, and so on. And further explore the molecular mechanisms of the above enhanced chemosensitivity by the regulation of miR-125b in order to provide a theoretical basis for the new mechanisms and prevention strategies on gliomas.

胶质瘤干细胞是恶性胶质瘤易复发、难治愈的重要原因之一。我们研究发现miR-125b抑制剂能有效抑制胶质瘤干细胞的生长和侵袭, 并参与调控PI3K/AKT、Bcl-2/Bax-Caspase 3和MMPs/TIMP等多条癌信号通路。深入研究发现它可增强脱甲氧基姜黄素(DMC)、替莫唑胺(TMZ)对胶质瘤干细胞的敏感性；DMC亦可通过抑制ABCG2基因增敏TMZ，而ABCG2基因又受miR-125b调控。据此，我们提出假说：miR-125b抑制剂→DMC→TMZ存在逐级放大的增敏效应，并通过miR-125b参与的上述癌信号通路调控胶质瘤干细胞增殖、侵袭和凋亡进程。本项目拟通过建立原代胶质瘤干细胞体外培养及颅内种植的模型，进行增殖、侵袭、凋亡等多方面抗肿瘤实验，深入探讨miR-125b抑制剂增敏DMC和TMZ的分子机制，为明确胶质瘤发病的新机制和防治策略提供理论依据。

胶质瘤干细胞（GSCs）是恶性胶质瘤易复发、难治愈的重要原因之一。多项研究表明miR-125b是重要的肿瘤调控miRNA，可调控多种化疗药物对肿瘤的敏感性，而我们前期研究提示miR-125b与GSCs的发生发展关系紧密。在本项国家自然科学基金的资助下，我们通过体内外论证实验明确抑制miR-125b可有效增强替莫唑胺（TMZ）对GSCs的敏感性，而通常情况下作为临床一线药物TMZ对GSCs是不敏感的，并且实验明确其机制是通过调节PI3K/AKT信号通路和靶项调控BAK1蛋白表达实现的。同时，我们实验论证作为天然提取化合物去甲氧基姜黄素（DMC）其体外抗GSCs作用要优于TMZ，且与TMZ联合使用有增强效果，其体外抗GSCs机制与ROS依赖的caspase-3活化的线粒体细胞凋亡途径有关；我们进一步开展了GSCs裸鼠皮下移植瘤实验，证明DMC体内效果要优于TMZ，并且指出两者联合治疗GSCs移植瘤并不优于单独使用DMC，并且申请者证明DMC在体内抗GSCs依赖JAK/STAT3信号通路。并且，体内外实验也表明DMC抗胶质瘤效果与TMZ作用相比也呈现相似趋势。据此，我们实验认为DMC是一种较TMZ具有前景的抗胶质瘤天然药物。但是，在颅内原位动物实验中我们发现DMC抗胶质瘤的作用远远低于TMZ，其可能的因素是DMC不能很好的透过血脑屏障。为此，我们在DMC的基础上进行结构修饰改良，发现并证明DMC衍生物DMC-BH420具有良好的抗颅内原位胶质瘤效果，表明其能很好透过血脑屏障发挥抗胶质瘤作用，并且我们发现其发挥作用的机制可能与GSCs中TCTP表达有关。相关衍生物已申报发明专利，并且其中一项已获得授权。