c-Myc-miR-199b-5p-β-catenin环路在结直肠癌肝转移中的作用及其机制研究

In our preliminary study, we found that miR-199b-5p was significantly down-regulated in colorectal cancers (CRCs) with liver metastases. However, the roles and underlying mechanisms of miR-199b-5p in CRC and liver metastasis are still unknown. Further experiments showed that ectopic overexpression of miR-199b-5p suppressed CRC cell migration and invasion, and down-regulated the expressions of FZD4 and PIK3CD, which were predicted to be targets of miR-199b-5p and specifically noted as positive regulators of the β-catenin signaling pathway. Moreover, overexpression of miR-199b-5p inhibited β-catenin signaling pathway and the expression of c-Myc, a well-known downstream target of β-catenin signaling pathway. Furthermore, we used the bioinformatics tool and identified that the multiple binding sites for c-Myc were presented within the promoter of miR-199b-5p. In addition, silencing of c-Myc markedly up-regulated miR-199b-5p expression. Thus, we raise the hypothesis that c-Myc-miR-199b-5p-β-catenin loop regulates CRC liver metastasis. In this study, we aim to obtain the reliable evidence that c-Myc-miR-199b-5p-β-catenin loop regulates CRC liver metastais by using luciferase reporter gene, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays, etc. Therefore, this study will help to further elucidate the molecular mechanisms underlying CRC liver metastasis, and provide enough scientific evidence for identification of miR-199b-5p as a novel target for the treatment of CRC liver metastasis.

我们前期发现miR-199b-5p在伴有肝转移的结直肠癌组织中表达显著下调，但其在结直肠癌肝转移中的作用及机制不明。进一步实验显示：过表达miR-199b-5p可抑制结直肠癌细胞迁移侵袭，下调其预测靶基因、β-catenin通路正向调节因子FZD4和PIK3CD的表达，并抑制β-catenin通路及其靶基因c-Myc表达。还预测到miR-199b-5p启动子区含有多个c-Myc结合位点；沉默c-Myc可明显上调miR-199b-5p表达。由此，我们提出c-Myc-miR-199b-5p-β-catenin环路调控结直肠癌肝转移的假设。本项目拟采用报告基因、定点突变、ChIP等方法，旨在获得c-Myc-miR-199b-5p-β-catenin环路调控结直肠癌肝转移的可靠证据，进一步阐明结直肠癌肝转移的分子机制，为确立miR-199b-5p作为结直肠癌及其肝转移治疗的新靶点提供充分科学依据。

结直肠癌是最常见的恶性肿瘤之一，其发生发展的分子机制尚未完全明确。已发现磷脂酰肌醇-3-激酶催化亚基δ(PIK3CD)和微小RNA（miR）-199b-5p参与多种肿瘤的发生发展，但它们在结直肠癌中的作用和分子机制尚不十分清楚。在本项研究中，我们分析了PIK3CD在结肠癌组织和细胞中的表达及其意义，研究了PIK3CD和miR-199b-5p对结直肠癌生物学行为的作用及其分子机制。结果显示PIK3CD在结肠癌组织和细胞中过表达，是结肠癌患者总生存的独立预测因子；过表达PIK3CD能促进结直肠癌的生长、迁移和侵袭，而抑制PIK3CD表达则抑制结直肠癌的生长、迁移和侵袭；并证实PIK3CD通过激活AKT/GSK-3β/β-catenin通路发挥作用。进一步研究发现miR-199b-5p在结直肠癌细胞中低表达，外源性表达miR-199b-5p能抑制结直肠癌细胞的生长、迁移、侵袭和血管生成及上皮-间质转化（EMT），而抑制miR-199b-5p表达则促进结直肠癌细胞的生长、迁移、侵袭和血管生成及EMT；我们还发现C-myc在结肠癌组织过表达，是结肠癌预后的独立预测因子；此外，C-myc能抑制结直肠癌细胞中miR-199b-5p的表达。这些结果提示PIK3CD是结直肠癌患者预后的独立预测因子，PI3KCD通过激活AKT/GSK-3β/β-catenin信号促进结直肠癌的生长和侵袭；miR-199b-5p能抑制结直肠癌细胞的生长、侵袭和血管生成及EMT。因此，本项目不仅进一步阐明了结直肠癌发生发展的新分子机制，而且为确立PIK3CD和miR-199b-5p作为结直肠癌治疗的新的靶点提供充分的科学依据。