miR-218多靶点调控Sp1-Slit/Robo-srGAP2信号通路抑制胃癌转移的实验研究

Our previous studies confirmed that miR-218 inhibits invasion and metastasis of gastric cancer through suppressing the Robo1 receptor. However, knockdown of Robo1 by siRNA in MKN28-M cells inhibited cell invasion, which is far less than those observed after transfection with the miR-218-expressing vector. These observations suggest that Robo1 is only one of miR-218 target genes. The mechanism of action of miR-218 has not been fully clarified. Further screening of miR-218 target genes with RIP-chip and iTRAQ suggested that Robo2, srGAP2 and Sp1 also may be targets for miR-218. Transcription factor Sp1 regulates expression of the Robo1 and Robo2 receptors in gastric cancer, indicated that Sp1 is an upstream regulatory molecule of Slit/Robo signaling pathway. In addition, srGAP2 is a downstream molecule of Slit/Robo pathway, thus creating a Sp1-Slit/Robo-srGAP2 signaling pathway. Based on these results, we propose a new hypothesis that miR-218 inhibits metastasis of gastric cancer through directly regulating multiple target genes in Sp1-Slit/Robo-srGAP2 signaling pathway. To test this hypothesis, we will use ChIP, EMSA and truncated-gene reporter system to study the transcriptional regulatory mechanism of Robo1 and Robo2 by Sp1. And then we will use gain-of-function and loss-of-function approaches to investigate the influence of miR-218 on gastric cancer metastasis through directly regulating multiple target genes in vitro and in vivo. This study will provide a theoretical basis for the miR-218 as a target for gene therapy in gastric cancer.

我们前期研究证实miR-218通过下调Robo1抑制胃癌转移。然而Robo1 siRNA抑制胃癌侵袭的能力远低于miR-218，提示Robo1只是其靶基因之一，miR-218的分子机制仍未阐明。RIP-chip联合iTRAQ对miR-218靶基因进一步筛选的结果表明Robo2，srGAP2，Sp1也可能是其靶基因，其中Sp1调控Robo1、Robo2，是Slit/Robo通路的上游分子，srGAP2是Slit/Robo的下游分子。基于此我们提出了miR-218多靶点调控Sp1-Slit/Robo-srGAP2信号通路抑制胃癌转移的理论假设。本项目将通过ChIP、EMSA、截短体报告基因实验研究Sp1对Robo1、Robo2的调控机制，通过干预、拮抗实验研究miR-218多靶点调控Sp1-Slit/Robo-srGAP2通路对胃癌转移的影响，为以miR-218为靶点的基因治疗提供理论基础。

前期研究中，我们发现miR-218通过调控Slit2/Robo1信号通路抑制胃癌转移。然而用RNAi 降低Robo1的表达时，胃癌细胞的迁移、侵袭能力降低，但其降低的程度远远低于miR-218过表达时的效果。提示Robo1只是介导miR-218参与胃癌转移的靶基因之一。我们用RIP-chip联合iTRAQ高通量的方法对miR-218调控胃癌转移的靶基因进行了进一步的挖掘。结合生物信息学分析，提出了： miR-218多靶点调控POU2F2-Slit/Robo-srGAP2信号通路抑制胃癌转移的理论假设。然后从以下四个方面进行了研究：1. POU2F2对Robo1直接表达调控的研究。结果发现POU2F2在高转移潜能的胃癌细胞中表达升高，降低其表达抑制胃癌转移。POU2F2通过结合ROBO1启动子区 -880～ -873正性调控ROBO1的转录表达。2. NF-ƙB直接激活POU2F2在胃癌转移中的转录。在胃癌细胞中POU2F2的表达水平与NF-ƙB的活性显著正相关，NF-ƙB通过结合POU2F2启动子区-531 ～ -522区域调控POU2F2。3. POU2F2是NF-ƙB 和 slit2/Robo1信号通路的联接点。NF-Kb活化诱导POU2F2过表达，表达升高的POU2F2上调膜受体ROBO1，活化Slit2/ROBO1信号通路，促使胃癌细胞转移。4. miR-218同时抑制IKK-β、POU2F2、ROBO1基因，抑制NF-Kb和Slit/ROBO1信号通路活性抑制胃癌转移。.本研究不仅发现了2个新的miR-218的靶基因，并且揭示了一个以218为中心的调控网络：Slit2-miR-218-(NF-kB/ POU2F2/Robo1)。在这一相互作用网络中，miR-218的表达缺失，失去对IKK-β的翻译抑制，使NF-Kb活化，激活POU2F2的转录，使其过表达，POU2F2的表达导致ROBO1表达上调，与其配体Slit2相互作用后，活化Slit2/ROBO1信号通路，促进胃癌的侵袭和转移。而miR-218可同时直接调节Slit2-(NF-kB/ POU2F2/Robo1) axis上多个基因的表达，分别从转录因子、膜受体不同的层面同时抑制这一信号通路的活性，抑制胃癌的侵袭和转移。