白血病干细胞相关miR-99a诱发髓系白血病耐药的机制研究

Leukemia stem cells (LSCs) can resist available treatments and result in disease progression and/or relapse. It is important to explore the potential role of LSC-related miRNAs in maintaining LSC properties, such as substantially higher self-renewal and therapy resistance. Based on our preliminary data about LSC-related miRNA microarray and functional studies, we reported that miR-99a was significantly upregulated in the LSCs and miR-99a may be used as a biomarker for the prognosis and progression of AML. miR-99a promoted cell growth in myeloid leukemia cells, led to increased colony forming ability and provoked drug resistance in vitro and in vivo. However, the molecular mechanisms of miR-99a involving in resistance and self-renewal remain unclearly, deserve a thorough investigation. Our previous data show that miR-99a upregulated mitochondrial protein, such as ATPase-β, increased membrane potential. We will further investigate the mechanisms. 1. Investigate the mechanisms of miR-99a involving in the regulation of resistance by mitochondrial quantitative proteomics analysis, flow cytometry, and western blot. 2. Identify the therapeutic targets of resistance in AML mouse model and patient samples. The intensive study of this project will discover novel molecular targets and open a new perspective to improve the current therapies for resistant and relapsed leukemia.

白血病干细胞（LSCs）是白血病难治和复发的根源。我们基于前期复发病人LSCs相关miRNA表达谱分析和功能研究，报道了miR-99a在LSCs中异常高表达，可作为AML进展和预后的分子标记。miR-99a过表达不仅加速白血病细胞周期，增强LSC的干性维持，而且促进白血病细胞化疗药处理后的存活，但分子机制尚未明确。我们前期结果显示miR-99a上调线粒体蛋白表达，增强药物处理后线粒体膜电位和ATPase-β表达。本课题将在此基础上对分子机制和潜在临床应用展开系统研究：1、利用流式分析、蛋白质谱、免疫共沉淀等方法研究miR-99a通过调节线粒体特性和蛋白表达，促进白血病细胞耐药的机制；2、利用MLL-AF9白血病小鼠模型寻找调控网络中治疗靶点。此项研究从“miRNA-线粒体-耐药”的角度研究miR-99a参与化疗耐药的机制，为白血病复发难治的机制研究提供新视角，为临床治疗提供新靶点。

白血病干细胞（LSCs）是白血病难治和复发的根源。我们基于前期复发病人LSCs相关miRNA表达谱分析和功能研究，报道了miR-99a在LSCs中异常高表达，可作为AML进展和预后的分子标记。miR-99a过表达不仅加速白血病细胞周期，增强LSC的干性维持，而且促进白血病细胞化疗药处理后的存活，但分子机制尚未明确。本课题将在此基础上对分子机制和潜在临床应用展开系统研究：1、利用流式分析、蛋白质谱、免疫共沉淀等方法miR-99a通过调节线粒体特性和蛋白表达，促进白血病细胞耐药的机制；2、利用小分子库筛选技术，寻找增强白血病细胞对化疗药敏感的线粒体小分子；3、借助MLL-AF9白血病小鼠模型验证联合用药体内的疗效。我们的研究结果显示miR-99a过表达通过上调RAS信号通路，一方面增强PGC1a表达，促进线粒体生成，维持线粒体膜电位；另一方面抑制caspase蛋白。除此之外，miR-99a过表达导致脂代谢的重编程。总之，miR-99a过表达引起的以上变化均有利于白血病细胞的存活，抵抗化疗药处理后的凋亡。利用157个线粒体小分子处理过表达miR-99a的白血病细胞，筛选到30多个显著抑制白血病细胞的小分子，进一步实验发现FKB和SBI分别与阿糖胞苷具有最显著的联合效应，急性髓系白血病小鼠模型体内联合效应得到验证。此项研究从“miRNA-线粒体-耐药”的角度研究miR-99a参与化疗耐药的机制，为白血病复发难治的机制研究提供新视角，为临床治疗提供新靶点。