miR-10a通过RYBP调控MDS克隆细胞凋亡抵抗的分子机制

In recent years, miR-10a is gaining particular attention in solid tumors due to its positive role in malignant proliferation and migration. However, the role of miR-10a in pathogenesis of myelodysplastic syndromes is still unclear. Our previous work have showed that miR-10a is overexpressed in MDS patients, and downregulation of miR-10a could induce increasing apoptosis-sensitive of MDS clonal cells to cytotoxic agents and activation of P53-associated apoptosis pathway. Further analysis of miR-10a-targeted gene expression profile showed that expression of RYBP, a miR-10a-targeted gene, was obviously downregulated. Previous studies have revealed that RYBP may be considered as a potential tumor suppressor. Taken together, we speculated that overexpression of miR10-a may contributes to apoptosis resistance of MDS clonal cells through inhibiting RYBP function. Based on the previous data, we will confirm our hypothesis by performing a series of experiments such as RNAi, ectopic expression, ChIP, PCR, WB, FCM, etc. The purpose of this project was to investigate the mechanism of action that miR-10a induced apoptosis resistance of MDS clonal cells through inhibiting RYBP function, and explore the relationship of RYBP with activation of P53-associated apoptosis pathway. Finally, this project would contribute to better understanding to biological mechanism of apoptosis resistance of MDS clonal cells, and provide scientific evidence for novel targeted t biological intervention.

近年来，miR-10a因其参与实体肿瘤及白血病的恶性增殖及迁移而引发关注，然而miR-10a在MDS发病机制中的作用罕见研究。申请人前期工作显示MDS患者存在miR-10a异常高表达，miR-10a的下调增强肿瘤细胞对细胞毒药物的凋亡敏感性以及诱导P53凋亡通路的激活，进一步的基因表达谱及定量PCR分析显示miR-10a的靶基因RYBP在MDS患者中明显下调。既往研究结合我们的前期研究提示RYBP可能是一个潜在的抑癌基因。因此，我们推测miR-10a可能通过抑制RYBP来调控MDS克隆细胞的凋亡抵抗。本研究在已有工作基础上，采用慢病毒介导的干扰/过表达技术、PCR、ChIP、WB、FCM以及细胞功能学实验，探讨miR-10a通过RYBP调控MDS细胞凋亡抵抗的作用机制，探索RYBP与P53凋亡通路的关系。该项目有助于我们理解MDS克隆细胞凋亡抵抗的生物学机制，为拓展相应的靶向干预提供依据。

近年来，miR-10a因其参与实体肿瘤及白血病的恶性增殖及迁移而引发关注，然而miR-10a在骨髓增生异常综合征(MDS)发病机制中的作用罕见研究。申请人前期工作显示MDS患者存在miR-10a异常高表达，miR-10a的下调增强肿瘤细胞对细胞毒药物的凋亡敏感性以及诱导P53凋亡通路的激活，进一步的基因表达谱及定量PCR分析显示miR-10a的靶基因RYBP在MDS患者中明显下调。既往研究结合我们的前期研究提示RYBP可能是一个潜在的抑癌基因。本项目在国自然资助下，围绕“miR-10a通过RYBP调控MDS克隆细胞凋亡抵抗”这一假说，按照任务书计划已完成以下三个方面的内容：第一，研究了原癌miR-10a与RYBP靶向关系，预测软件分析显示RYBP是miR-10a的一个靶基因，MDS患者miR-10a表达与RYBP表达呈反相关，进一步的体外荧光报告基因检测显示miR-10a作用于RYBP基因的3’-UTR区域，这些研究证实了RYBP是miR-10a的靶基因；第二，研究了RYBP在MDS患者中的表达水平及其基因功能特征，结果显示RYBP在MDS患者中表达水平降低，其低表达预示着较高的白血病转化风险。体外实验显示RYBP过表达抑制细胞增殖，诱导TP53介导的细胞凋亡。然而，RYBP敲低促进细胞增殖，诱导对化疗药物如依托泊甙和地西他滨的凋亡抵抗；第三，我们也研究了RYBP调控MDS克隆细胞凋亡抵抗的机制。体外实验显示RYBP敲低导致减少的H2AK119ub1和H3K27me3水平，H2AK119ub1和H3K27me3与基因转录沉默相关，随后的体内体外基因表达谱联合ChIP-qPCR分析显示BCL2是RYBP的一个靶基因。RYBP通过移除BCL2基因启动子区域的H2AK119ub1和H3K27me3，导致BCL2转录抑制解除而诱发持续性激活，BCL2是经典的抗凋亡蛋白，通过抑制TP53介导的细胞凋亡通路从而授予MDS克隆细胞对化疗药物的凋亡抵抗。这些结果提供了MDS凋亡研究的新视点，国内外未曾有所报道，该项目有助于我们理解MDS克隆细胞凋亡抵抗的生物学机制，为拓展相应的靶向干预提供依据。