miR-200c通过MYCN调控MDS细胞恶性生物学特征的分子机制及靶向干预研究

miR-200c becomes a focus in current research due to its inhibition effect on epithelial-mesenchymal transition and cancer stem cells formation. However, the role of miR-200c in pathogenesis of myelodysplastic syndromes (MDS) is still unclear. Our previous work have showed that miR-200c was obviously under-expressed in MDS patients, and up-regulation of miR-200c could inhibit the proliferation of MDS cells and induce increasing apoptosis-sensitive to cytotoxic agents. Further analysis of gene expression profile and real-time PCR array showed that expression of MYCN, a miR-200c-targeted gene, was obviously up-regulated. Therefore, the applicant considered MYCN as a potential oncogene. Lack of miR-200c induced over-expression of MYCN, which contributed to the formation of MDS malignant biological characteristic. In this study, our hypothesis will be verified by accomplishing a series of functional experiments such as ectopic expression/RNAi, FCM, PCR, WB, GST pull-down, ChIP, etc. The purpose of this project was to investigate the mechanism that up-regulation of MYCN induced the formation of MDS malignant biological characteristic, and discuss the possibility of targeted intervention. Finally, this project would provide scientific evidence for novel targeted therapy or combination therapy.

miR-200c因其抑制肿瘤上皮-间质转化及癌症干细胞形成的重要功能而成为研究热点。然而miR-200c在MDS发病机制中的作用尚无报道。申请人前期研究显示骨髓增生异常综合征(MDS)患者miR-200c明显低表达，体外miR-200c的过表达显著抑制MDS细胞增殖且增强其对细胞毒药物的凋亡敏感性，基因表达谱及定量PCR显示miR-200c的靶基因MYCN在MDS中明显过表达且与疾病进展相关。因此，申请人推测MYCN在MDS中是一个原癌基因，miR-200c的缺乏导致MYCN过表达从而有助于MDS细胞恶性生物学特征的形成。本研究在前期工作基础上，采用病毒介导干扰/过表达、FCM、PCR、WB、GST pull-down、ChIP等功能学实验，探讨miR-200c通过MYCN调控MDS细胞恶性生物学特征的分子机制，探讨影响MYCN表达和功能的靶向干预价值。该项目将为拓展新的靶向治疗提供策略。

申请人前期研究显示骨髓增生异常综合征( MDS)患者miR-200c明显低表达，体外miR-200c的过表达显著抑制MDS细胞增殖且增强其对 细胞毒药物的凋亡敏感性，基因表达谱及定量PCR显示miR-200c的靶基因MYCN在MDS中明显 过表达且与疾病进展相关。因此，申请人推测MYCN在MDS中是一个原癌基因， MYCN过表达从而有助于MDS细胞恶性生物学特征的形成。在本项目中，我们研究了MYCN在MDS患者中的表达水平及其致病机制。结果显示MDS患者MYCN表达水平显著高于正常对照，MYCN高表达预示着较差的生存以及较高的白血病转化。在体外试验中，MYCN敲低抑制白血病细胞增殖，诱导细胞凋亡；而MYCN过表达导致细胞过度增殖，同时增强白血病细胞对细胞毒药物的凋亡抵抗以及诱导红系分化阻滞。此外，MYCN敲低也能诱导P21介导的细胞衰老增加。基因表达谱及染色质免疫沉淀联合分析提示EZH2是MYCN的一个靶基因。EZH2过表达明显促进细胞增殖，而EZH2敲低可减弱MYCN过表达产生的促增殖效应。进一步的机制研究显示MYCN通过激活EZH2表达，进而维持抑癌基因P21启动子H3K27me3水平，导致P21失活从而发挥癌基因作用。最后的药物干预试验提示MYCN抑制剂MLN8237诱导通过降低MYCN和EZH2表达，增加P21表达来诱导白血病细胞凋亡增加。这些研究探讨了MYCN-EZH2-P21轴在MDS中的功能特性，探讨MYCN-EZH2介导的H3K27me3在抑癌基因沉默中的关键作用，丰富了MDS表观遗传发病机制，最后也探讨影响MYCN表达和功能的干预靶向价值。