miR-106b调控结直肠癌细胞干性及增强放化疗敏感性的作用及分子机制

In our previous study, it is found that the expression of miR-106b is negatively correlated with metastatic potentials of colorectal cancer, and that knock-down of miR-106b in SW480 cell line led to increased 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 CD133. We found that Tiam1 may be direct targeted and negatively regulated by miR-106b. Therefore, we hypothesize that Tiam1 may be new targeted gene for miR-106b. We speculate that there may be feedback loop mechanisms between miR-106b and Tiam1 in regulation of colorectal cancer stemness and metastasis. We hypothesize that miR-106b affects colorectal cancer metastasis and radiochemosensitivity probablely through stemness associated genes and the signaling pathway of stemness in which Tiam1 involved. To make this mechanisms explicit, we will transfect different cancer cells with lentivirus particles of miR-106b overexpression or suppression, and co-transfect with miR-106b and Tiam1 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 Tiam1 with miR-106b promoters. By using the established CD133(+) single cell-derived progenies of colorectal cancer cell line with different invasive and metastatic potential, We will detect the association between cancer stemness and radiochemosensitivity. By comparing the colorectal cancer cell lines with miR-106b overexpression and low expression by gene transfection or siRNA, we investigate the candidate downstream genes of Tiam1, and to explore the signaling pathway that we hypothesize. Our project will provide novel insights into cancer stemness and radiochemosensitivity, and new way for targeting miR-106b-related signaling to transformed colorectal cancer stemness and to suppress metastasis and radiochemosensitivity.

我们前期工作发现miR-106b表达与大肠癌侵袭转移潜能呈负相关，并初步筛选出Tiam1可能为其新靶基因，稳定干扰miR-106b能上调CD133等干性相关基因的表达，miR-106b过表达则能降低大肠癌细胞的侵袭和运动能力，初步发现miR-106b和大肠癌EMT及干性（stemness）相关，且miR-106b过表达能增强放化疗敏感性。本项目将探索miR-106b-Tiam1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、双荧光素酶报告基因、染色质免疫共沉淀等技术，深入探讨miR-106b对大肠癌干性相关基因调控的信号通路机制及其与放化疗敏感性的关系。从临床病理、细胞水平和动物模型3个层面，探讨miR-106b抑制干性信号通路及增强放化疗敏感性的分子机制，将为大肠癌治疗和药物筛选提供新的思路和新靶点。

结直肠癌(colorectal cancer，CRC)的转移是患者死亡的主要原因，临床上有一半以上的结直肠癌患者在行根治性手术前已出现了微转移，50%术后患者发生肝转移，为提高局部控制率和远期生存率，这部分患者应该同时进行标准的直肠癌辅助治疗。放疗抵抗是肿瘤患者放疗治疗失败的主要原因，不同个体显示对放疗治疗反应的明显差异。因此寻找有效治疗靶点，增强肿瘤放疗敏感性及降低放疗抵抗，是放疗治疗的关键。miR-106b在许多常见肿瘤异常表达，并在肿瘤的发生、发展转移中发挥重要的作用，但miR-106b和肿瘤细胞“干细胞特性”以及肿瘤放疗抵抗之间的关系尚未见文献报道。本项目对miR-106b在结直肠癌的“干细胞特性”以及肿瘤放疗抵抗所扮演的作用及机制进行了较为系统的研究。.采用MTT、平板克隆、激光共聚焦以及WESTERN BLOT等技术，我们发现过表达miR-106b后CRC细胞的生存分数明显升高，在放疗DNA损伤后的修复能力显著增强；相反在敲除miR-106b的表达后，其DNA损伤后的修复能力明显减弱，对放疗的敏感性明显增强。过表达miR-106b 后细胞所形成的干细胞球数目明显增多，单个克隆球体积明显增大；相反敲除miR-106b 后所形成的干细胞球数量减少，体积变小。同时miR-106b 能够显著增强CRC细胞干细胞相关标志物CD133、SOX2、OCT4、Bmi1 mRNA表达；相反敲除miR-106b，干细胞的相关标志物表达水平明显降低。在放疗处理条件下，我们发现过表达miR-106b同样能够显著增强CRC细胞的肿瘤球形成能力和“干细胞特性”。我们通过生物信息学的方法预测、QRT-PCR、Western blot、荧光素酶报告系统等技术证实PTEN和p21是miR-106b直接作用的靶基因。恢复CRC细胞中PTEN、p21的表达可逆转miR-106b导致的放疗抵抗增强。通过以上系列研究，我们首次证实了miR-106b能够通过靶向调控PTEN、p21增强CRC细胞的“干细胞特性”并进而增强肿瘤细胞的放疗抵抗作用。