miR-218 调控FAK-Slit/ Bmi-1-TGF-β 信号通路抑制脑胶质瘤增殖的机制研究

As to glioma therapy, one of the key factors is inhibition of glioma proliferation. Recent studies in our lab showed that, miR-218 inhibits glioma invasion, migration, and proliferation as well as self-renewal of cancer stem-like cells by targeting the polycomb group gene Bmi1. However, transfection with the miR-218-expressing vector in U251MG cells inhibited glioma cell proliferation much better than those observed after knockdown of Bmi1 by siRNA.The data are to be discussed, and it is proposed that the molecular mechanism of miR-218 should be further elucidated, and Bmi1 might only be one of the target genes of miR-218. Further targeting and screening of miR-218 target genes with RIP-chip and iTRAQ indicated that the genes of FAK and TGF-β might also be the candidates for miR-218. The gene of FAK regulates expression of Bmi1 in gliomas, which indicates that FAK is an upstream regulatory molecule of Slit/Bmi1 signaling pathway. Additionally, TGF-β is seen as the downstream molecule of Slit/ Bmi1 pathway. Based on these results, we propose a new hypothesis that miR-218 inhibits glioma cell proliferation through regulating multiple target genes in FAK-Slit/Bmi1-TGF-β signaling pathway. To test such a hypothesis, we will use site-directed mutagenesis，ChIP, and EMSA to study the transcriptional regulatory mechanism of FAK on Bmil. And then we will use gain-of-function and loss-of-function approaches to investigate the influence of miR-218 on glioma cell proliferation through directly regulating multiple target genes in vitro and in vivo. This study will provide a theoretical basis for the application of miR-218 to new targets in gene therapy of gliomas.

胶质瘤治疗的重要策略之一是抑制其增殖。我们证实，miR-218通过下调Bmi1抑制胶质瘤增殖。但是，过表达miR-218抑制胶质瘤增殖的能力远远超过Bmi1 siRNA，提示Bmi1只可能是其靶基因之一，还需进一步阐明miR-218的分子机制。蛋白质谱技术联合RIP-chip对miR-218靶基因的筛选显示，FAK、TGF-β亦可能是其靶基因。我们的预实验显示，FAK调控Bmi1，可能是Slit2/Bmi1通路的上游分子，而TGF-β是Slit2/Bmi1的下游分子。我们提出理论假说，miR-218多环节调控FAK-Slit/Bmi1-TGF-β信号通路抑制胶质瘤细胞增殖。本研究将行定点突变、ChIP、EMSA等实验研究FAK对Bmi1的调控机制，开展干预、拮抗实验研究miR-218多环节调控FAK-Slit/Bmi1-TGF-β通路抑制胶质瘤增殖，将为胶质瘤治疗选择新靶点提供理论指引。

探索脑胶质瘤的发生发展机制、寻找确切的治疗靶点是当前脑胶质瘤研究热点。本项目阐明 miR-218 抑制胶质瘤增殖的分子机制，揭示 miR-218 多环节调控 FAK-Slit/Bmi1- TGFβ 信号通路抑制胶质瘤细胞增殖的机制。我们前期发现，FAK、TGFβ可能是miR-218的靶基因，FAK 调控 Bmi1，可能是 Slit2/Bmi1 通路的上游分子，而TGFβ是 Slit2/Bmi1 的下游分子。我们还发现，过表达 miR-218 能够抑制 FAK、Bmil、TGFβ1、 p-AKT473 等的表达。我们提出假说，miR-218 对FAK- Slit/Bmi1- TGFβ 及 TNC-AKT-AP1-TGFβ 信号通路进行多环节调控、抑制胶质瘤生长及侵袭。多个数据库预测了，miR-218的下游靶基因包括FAK、Bmil、TGFβ1、 p-AKT473等，然而，最终被证实，miR-218 影响IKBKB、WNT2B、腱生蛋白（TNC）等基因的表达。TNC 被确认，参与细胞的增殖、粘附以及侵袭等多种功能，可以通过激活多个重要的信号通路，在胶质瘤的发生发展中发挥重要作用。过表达 miR218 能够显著抑制 TNCmRNA 的表达量、TNC 蛋白表达量；miR-218 可以通过抑制TNC、AKT通路，进而抑制AP-1通路的活性；过表达miR-218可明显抑制pAktS473的表达，不影响pAktT308以及总AKT的表达水平；过表达miR-218不影响AP-1的两个重要成员FOS以及JUN的表达水平；miR-218可抑制TNC- AKT-AP-1通路，下调TGFβ1的表达；TGFβ1可诱导TNC、p-AktS473、 p-JNK，以及TGFβ1自身的表达水平，而过表达miR-218可显著抑制上述TGFβ1的作用。总之，miR-218通过TNC、AKT上调AP-1转录活性，而AP1作为转录因子可以结合TGFβ启动子区并促进其转录。TNC-AKT-AP1-TGFβ就形成了一个正反馈环路，这一环路中任何一个蛋白表达的发生上调都会引起整个环路上其他蛋白的上调，这样的恶性循环会进一步加重肿瘤的进展。而miR-218可以通过靶向TNC来抑制这一环路，进而抑制胶质瘤细胞的增殖、侵袭、迁移、干细胞自我更新等一系列功能。