本课题组研究发现miR-126可抑制结肠癌转移，基因芯片等技术初步揭示miR-126可能靶向CXCR4负调控RhoA通路发挥作用。本课题将采用基因过表达及沉默技术研究miR-126对结肠癌转移指标EMT、血管生成的影响，并用裸鼠移植瘤活体成像技术证实其抑制转移的功能。为明确miR-126的作用机制，本课题将研究miR-126过表达和沉默对结肠癌中CXCR4及RhoA通路活性的调控作用。进一步在miR-126过表达和沉默结肠癌细胞中分别逆转CXCR4及RhoA的表达，采用体内外转移实验及蛋白印迹等技术明确CXCR4及RhoA是否为miR-126抑制转移的关键节点；采用IP探究CXCR4与RhoA、RhoGEF和ARHGAP5的交互作用；并在结肠癌组织中验证上述分子的表达及其与转移、生存率的关系，阐明结肠癌中miR-126→CXCR4→RhoA通路的转移抑制机制，为结肠癌转移诊治提供新靶点。

My group discovered that miR-126 inhibited colon cancer metastasis. Our Preliminary Data from Affymetrix GeneChips, real-time PCR, RhoA activation assay and luciferase report assay revealed a possible mechanism by which miR-126 may inhibit colon cancer metastasis due to inactivity of RhoA signaling pathway mediated by CXCR4. This project proposes a hypothesis-driven study that is directed at uncovering the mechanism of miR-126 inhibiting colon cancer metastasis by suppressing RhoA signaling pathway mediated by CXCR4. Over-expression and knockdown technique will be used to analyze the effect of miR-126 on the EMT and angiogenesis. Nude mouse orthotopic xenograft model will be applied to verify the inhibitory role of miR-126 in colon cancer metastasis in vivo. In order to demonstrate the inhibitory mechanism of miR-126 in colon cancer metastasis, first, we'll examine the effects of miR-126 over-expression and knockdown on the expression of CXCR4 and the activity of RhoA signaling pathway in colon cancer cell lines. Furthermore, the expression of CXCR4 and RhoA will be reversed in miR-126 over-expression and knockdown cell lines respectively, and migration assay, invasion assay, nude mouse orthotopic xenograft model and western blot will be used to examine the role of CXCR4 and RhoA signaling pathway in miR-126 inhibiting colon cancer metastasis. Immunoprecipitation and immunofluorescence will be employed to analyze the biochemical interaction between CXCR4 and RhoA, RhoGEF, ARHGAP5. Immunohistochemistry and statistics will be used to investigate the relationship between expression levels of miR-126, CXCR4, RhoA, RhoGEF, ARHGAP5 and ROCK in patient's colon cancer tissues and analyze the association between the expression levels of these molecular with the clinical stage, metastasis and survival rates of patients with colon cancer. This study will delineate the mechanism of miR-126 in inhibition of colon cancer metastasis by suppressing the RhoA signaling pathway through CXCR4 and facilitate identification of novel potential therapeutic targets for colon cancer metastasis.