miR-126通过靶向CXCR4调控RhoA通路抑制结肠癌转移的机制研究

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.

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。

前期发现miR-126在体外能抑制结肠癌细胞增殖及侵袭转移。CXCR4为miR-126直接负调控的靶基因，且miR-126负性调控RhoA通路。然而miR-126在结肠癌中发挥抑癌作用的具体机制尚不明确。本研究中，在细胞水平、动物水平及人结肠癌组织水平三个层面探讨了miR-126对结肠癌的侵袭转移的抑制作用及机制。本课题按照研究计划进行。通过构建miR-126稳定过表达、沉默结肠癌细胞模型及皮下移植瘤模型和尾静脉肺转移模型，发现miR-126能抑制结肠癌生长及侵袭转移（P<0.05）及EMT的发生。通过原位杂交分析发现miR-126在人结肠癌组织中表达下调，且其表达与结肠癌患者TNM分期及转移明显负相关（P<0.05）；生存分析证实低表达miR-126的结肠癌患者生存预后更差（P<0.05）；相关分析提示结肠癌中miR-126表达与CXCR4及RhoA通路重要分子表达明显负相关（P<0.05）。采用Real-time PCR、Western Blot及RhoA G-LISA等技术在稳定miR-126过表达及沉默结肠癌细胞中分析发现miR-126能负性调控CXCR4的表达及RhoA通路的活性，提示其可能为miR-126的抑癌机制。进一步在miR-126稳定过表达或沉默结肠癌细胞中恢复CXCR4的表达后，能改变miR-126对结肠癌生物学行为的抑制作用（p<0.05）及对RhoA信号通路的负调控作用（p<0.05），提示CXCR4为miR-126调控RhoA通路发挥抑癌作用的关键节点。通过蛋白交互作用及RhoA G-LISA实验证实结肠癌中CXCR4可能通过Ga13调控RhoA通路。在miR-126稳定过表达或沉默结肠癌细胞中恢复RhoA的表达后，能逆转miR-126对结肠癌生物学行为的抑制作用、EMT发生及RhoA通路的负调控作用（p < 0.05），提示结肠癌中RhoA通路为 miR-126 发挥生物学功能的关键通路。通过本课题的完成，阐明了miR-126在结肠癌中通过靶向CXCR4调控RhoA通路发挥抑制结肠癌转移的作用。miR-126可以作为结肠癌的预后标志及潜在治疗靶标。有望为临床上结肠癌的治疗提供新的理论依据和生物治疗策略。一系列结果在Oncotarget、Int J Clin Exp Med、Mol Cell Biochem等重要期刊上发表。