hsa-miR-200c调控大肠癌干性相关基因及其信号通路新机制

In our previous studies, we found that the expression of hsa-miR-200c is negatively correlated with metastatic potentials of colorectal cancer, and that knock-down of hsa-miR-200c in SW480 cell line led to resultant increase of cells growth, clonogenicity, cells migration, invasion, and the acquisition of stemness, which was consistent with increased stem cell sphere-forming capacity and increased expression of cancer stem cell markers such as Sox2, CD166 and β-catenin. By using the luciferase reporter assay, we found that Sox2 can be directly targeted and negatively regulated by miR-200c. Therefore, we had proved that Sox2 may be new targeted gene for miR-200c, and we put fordward that "hsa-miR-200c-Sox2-FRAT2-GSK3β-c-myb-β-catenin(CD166)-to form metastatic stem- like cells" as a novel signal pathway may drive cancer metastasis. We speculated that there may be feedback loop mechanisms between hsa-miR-200c and Sox2 in regulation of colorectal cancer stemness and metastasis. Interestingly, the downstream genes of Sox2 include a series of stemness associated genes, e.g. c-Myb, c-Myc, KLF4 and NANOG. For this reason, we hypothesized that hsa-miR-200c affects colorectal cancer metastasis probablely through stemness associated genes and the signaling pathway of stemness in which Sox2 involved. To make this mechanisms explicit, we will transfect different cancer cells with lentivirus particles of hsa-miR-200c overexpression or suppression, and co-transfect with miR-200c and Sox2 vectors uncontained of it's 3'UTR, and then observe the change of stemness, cell proliferation, migration and invasion of these cell lines in vitro, as well as oncogenesis, invasion and metastatic effects in vivo by using the nude mice models of whole-body visualization. Dual Luciferase reporter assay and Chromatin Immunoprecipitation (CHIP) analysis will be carried out to validate direct association of Sox2 with miR-200c promoters. By using the established CD133(+) single cell-derived progenies of colorectal cancer cell line with different invasive and metastatic potential, and CD133-binding peptides(LS-7,TR-7) targeting cancer stem cells, We will detect the association between cancer stemness and metastasis. By comparing the colorectal cancer cell lines with hsa-miR-200c overexpression and low expression by gene transfection or siRNA, we will investigate the candidate downstream genes of Sox2, and to explore the signaling pathway that we hypothesized. Our research will provide novel insights into cancer stemness and metastasis, and new way for targeting hsa-miR-200c-related signaling to transformed colorectal cancer stemness and to suppress metastasis.

我们前期工作发现hsa-miR-200c表达与大肠癌侵袭转移潜能呈负相关，并初步证明Sox2为其新靶基因，稳定干扰miR200c能上调干性相关基因Sox2、β-catenin及CD166的表达，使大肠癌增殖、迁移及干细胞球形成能力显著增强。我们提出了"miR200c-Sox2-FRAT2-GSK3β-c-myb-β-catenin(CD166)-转移性干样细胞形成"的新假说。本项目将应用双荧光素酶报告基因、染色质免疫共沉淀等技术探索miR200c-Sox2的调控反馈机制,将利用我们已建立的大肠癌可视化裸鼠转移模型、CD133阳性大肠癌干样细胞系与CD133阴性细胞系模型，深入探讨 miR200c对大肠癌干性相关基因调控的信号分子通路。本研究将癌细胞干性与侵袭转移的关系结合起来，旨在揭示microRNA调控大肠癌干性相关新信号通路及侵袭转移的分子机制，为抗转移治疗提供新的思路和靶点。

肿瘤细胞具有无限增殖和自我更新能力，肿瘤组织中存在一小部分细胞表达干细胞标记物并具有干细胞样特性(stem-cell-like ability)，可以维持肿瘤的生长、转移和复发。最新研究发现，通过产生新的基因突变或环境因子作用可产生新的肿瘤干细胞( cancer stem cells, CSCs)，亦即肿瘤细胞可能获得新的突变而成为新的肿瘤干样细胞, 并可能使肿瘤细胞获得转移表型。干性相关基因（stemness associated genes），如Sox2， KLF4， Bmi1 和 Oct4，通常被认为可以维持胚胎干细胞的干性，干性相关基因及其信号通路异常对肿瘤发生和转移具有极其重要的作用。.我们对MiR-200c调控结直肠癌干性相关基因中的作用及机制进行了较为系统的研究，研究表明，miR-200c可以抑制上皮间质转化（epithelial-mesenchymal transition，EMT）发生，其在结直肠癌细胞中表达下调可以促进肿瘤的侵袭和转移。我们证实了miR-200c在结直肠癌组织及高转移结直肠癌细胞中表达下降；我们发现了干细胞相关基因Sox2是miR-200c的靶基因；上调miR-200c的表达能抑制结直肠癌细胞体内外的生长和转移能力，而恢复Sox2的表达，结直肠癌细胞体内外的生长和转移能力亦即恢复；下调miR-200c的表达能使结直肠癌细胞获得干性特征，即肿瘤球形成能力增强，干细胞标记物β-catenin、CD133和CD166表达升高；我们发现了miR-200c与Sox2通过负反馈机制相互调节；miR-200c通过靶向抑制Sox2而抑制PI3K-AKT信号通路。.我们还筛选CD133+的造血干细胞，并与结直肠癌细胞共培养，CD133+的造血干细胞可以促进结直肠癌细胞体内外的生长和转移，并诱导转移前微环境（pre-metastatic niche）的形成而促进结直肠癌细胞的转移。.总之，我们发现了miR-200c通过抑制P13K-AKT信号通路，并通过miR-200c-Sox2负反馈机制调节结直肠癌的干性、生长和转移。这一新的信号通路机制对于揭示结直肠癌的转移机制具有重要意义，为miR-200c作为新的抑癌靶标提供了重要实验依据。