微小RNA-128a抑制上皮-间质转化逆转胶质母细胞瘤原发耐药的实验研究

Glioma stem cells (GSCs) and tumor mesenchymal cells are principal cells of primary drug-resistance in glioblastoma multiforme(GBM). Epithelial-Mesenchymal Transition (EMT) can promote cancer epithelial cells transfering to mesenchymal cells and “stemoid” cells. So far, there are few correlative studies evaluate whether EMT inhibition could enhance drug-resistance through decreasing EMT cells and GSCs. Preliminary data indicated that miR-128a could down-regulate multiple protein expressions of different EMT pathways, furthermore, it also inhibits invasion via blocking EMT. Relation between miR-128a and chemotherapy sensitivity is still unknown. Next, we design further experiments to ①detect key gene expressions in EMT pathway through molecular experiments; ②validate the invasion effects of miR-128 on GBM via implanting GBM cells into nude-mice; ③determine synergistic killing effects through treating GBM cells utilizing TMZ combination with miR-128a in vitro and in vivo. Collectively, in this project, we will elucidate mechanism of miR-128a reducing primary drug-resistance on GBM, and verify miR-128a reduce primary drug resistance through down-regulating EMT and change molecular markers in GBM cells surface. Through this project, we want to provide new visions and methods for GBM primary drug-resistance.

胶质瘤干细胞（GSCs）和肿瘤间充质细胞是构成胶质母细胞瘤(GBM)原发耐药的主要细胞。上皮-间质转化（EMT）可诱导肿瘤上皮细胞向间充质细胞和“干细胞样”细胞转化。抑制EMT是否可减少原发耐药细胞生成，增强化疗药物敏感性？目前尚无相关报道。前期实验证实miR-128a可下调多个EMT通路中关键蛋白表达，阻止EMT，减少GBM侵袭。miR-128a是否逆转耐药，仍然未知。本课题进一步通过①分子生物学检测miR-128a对多个EMT通路中关键基因表达影响；②转化GBM细胞裸鼠脑内移植实验检测miR-128a对GBM细胞侵袭能力的抑制效果；③通过细胞学和裸鼠荷瘤实验检测替莫唑胺联合miR-128a治疗GBM的效果，评价其对GBM杀伤的协同效应和分子表型改变。从而证实miR-128a通过抑制EMT，改变GBM细胞形态及分子表型，减少原发耐药，抑制侵袭。最终为提高GBM治疗效果提供新理论和新方法。

胶质母细胞瘤（GBM）是颅内最常见的恶性肿瘤，术后放疗合并替莫唑胺（TMZ）放疗可延长患者生存期，然而由于肿瘤异型性产生原发耐药，敏感亚型向耐药亚型转换导致继发性耐药，从而导致化疗抵失败，肿瘤间充质细胞是构成胶质母细胞瘤(GBM)原发耐药的主要细胞。抑制GBM由前神经（PN）向间充质型（Mes）转化，可显著提高疗效。然而如何抑制上皮-间充质转化及机制是急需解决的问题。miRNAs可调节GBM亚型转化。我们在组织和细胞中发现下调miR-128a增加Mes标志物，促进增殖和侵袭，增加耐药性，证实miR-128a调节PN-Mes转化；还发现miR-128a可能通过下调多个EMT通路中的关键蛋白表达，阻止EMT，减少GBM侵袭。体外研究显示miR-128a 抑制胶质母细胞瘤细胞增殖、侵袭、迁移，其表达上调提示化疗敏感及预后较好。体外研究发现：miR-128a联合TMZ治疗较单独应用TMZ治疗，肿瘤的体积明显缩小，脑内侵袭范围变小。其分子机制与靶向c-met，调节PDGFRα、Notch1 、Slug表达变化抑制EMT相关。因此我们的研究显示：miR-128a通过抑制EMT，减少EMT细胞生成，进而减少原发耐药细胞，逆转胶质瘤耐药。