miR-200c/GATA4/Nanog反馈环促结肠癌干细胞侵袭转移的分子机制研究

As a transcription factor, Nanog functions to maintain the stemness of cancer stem cells and keep their self-renewal. The present evidence indicates that Nanog is involved in the carcinogenesis and further development. We have found that Nanog was highly expressed and miR-200c was significantly reduced in colon cancer stem cells and their derived neoplastic tissues from those patients who had the metastasis. The miR-200c mimics treatment in colon cancer stem cells led to increased Nanog expression and reduced GATA4 expression. Howerver, how do miR-200c, GATA4 and Nanog integrate and balance, and further promote the invasion and metastasis of colon cancer stem cells is largely unknown. The study is aimed to confirm whether Nanog transcriptionally regulates miR-200c through binding to the proximal promoter of miR-200c, miR-200c posttranscriptionally regulates GATA4 through its binding to the 3’-UTR of GATA4 mRNA, and GATA4 inhibits Nanog expression through its binding to the enhancer of Nanog. The data will provide the evidence of 200c/GATA4/Nanog negative feed-back loop in colon cancer stem cells. Then we investigate the biological significance of 200c/GATA4/Nanog negative feed-back loop in colon cancer stem cells via observation of the change of colon cancer stem cells after modulating of the feed-back loop. The negative feed-back loop of 200c/GATA4/Nanog in colon cancer stem cells will be helpful to understand the occurrence of the neoplastic invasion and metastasis and provides the possible therapeutic targets for colon cancer.

转录因子Nanog有维持干细胞多能性及自更新的功能，参与肿瘤的发生发展。我们发现结肠癌干细胞及有转移结肠癌患者肿瘤组织内Nanog高表达及miR-200c表达下调；miR-200c刺激结肠癌干细胞Nanog表达上调及GATA4表达抑制，但miR-200c、GATA4和Nanog之间是如何调控促进结肠癌干细胞侵袭转移的机制不清。本课题拟用报告基因、EMSA、ChIP、RNA pulldown等实验，明确Nanog 结合miR-200c启动子而转录抑制其表达，miR-200c靶向GATA4 3’-UTR区转录后调控GATA4，GATA4调控Nanog增强子抑制其表达，从而证明miR-200c/GATA4/Nanog反馈环的存在；通过上述基因表达调控后结肠癌干细胞行为改变明确该反馈环在肿瘤干细胞的生物学意义。该反馈环调控结肠癌干细胞的分子机制有助于认识结肠癌侵袭转移的发生，为其防治提供新的靶标。

结肠癌是世界上发病率高、预后较差、病死率第三的恶性肿瘤，早期诊治意义重大。干性基因Nanog作为维持未分化胚胎细胞的多能性和自我更新的关键转录因子之一，在多种恶性肿瘤中异常高表达并可促进其侵袭转移。但干性转录因子Nanog是如何调控结肠癌细胞EMT-MET的转录调控机制不清，且Nanog调控miR-200s的机制尚未见任何文献报道。. 因此，我们在临床样本、动物、细胞、分子水平开展一系列研究，发现：1.在结肠癌组织样本及结肠癌细胞株中，Nanog在mRNA水平分别与miR-200c、miR-200b表达呈极显著负相关。2. miR-200c、miR-200b分别在Nanog过表达、Nanog干扰的结肠癌细胞中下调和上调，再次验证Nanog与miR-200c、miR-200b表达负相关。3. Nanog过表达可致结肠癌细胞增殖、迁移、侵袭、致瘤能力显著增强。4. Nanog过表达的结肠癌细胞上皮向间质化转变（EMT）能力增强，Nanog干扰的结肠癌细胞上皮向间质化转变能力降低并呈间质向上皮转化（MET）。5.应用8条含Nanog顺式作用元件的miR-200c、miR-200b启动子的双荧光素酶报告基因质粒发现，Nanog可直接靶向抑制miR-200c、miR-200b启动子转录活性。6.在染色质免疫共沉淀实验中，进一步发现Nanog可分别直接靶向结合到miR-200c启动子区-761处、miR-200b启动子区-630处关键顺式作用元件。7.点突变Nanog关键顺式作用元件后，miR-200c、miR-200b启动子转录活性可恢复。8.miR-200s mimics可部分逆转Nanog过表达的结肠癌细胞增殖、侵袭、转移能力及EMT进程。. 结论：我们在转录调控水平阐明了结肠癌细胞EMT-MET可塑性的上游调控机制中干性转录因子Nanog调控miR-200c及其它家族成员的作用。强有力地提出Nanog可通过直接转录抑制miR-200s表达而调节结肠癌细胞EMT-MET的可塑性。Nanog/miR-200s/EMT轴将实现对肿瘤发展的调控，可能作为结肠癌耐药性及生物治疗的潜在新靶点，为结肠癌的早期诊断及治疗控制拓展一种新思路。