FAMLF新基因及其内含子miR-181a/b在急性髓细胞白血病发病机制中作用的研究

In-depth study of familial leukemia will help to clarify the mechanism underlying leukemogenesis in sporadic cases. A novel gene, FAMLF,was previously identified from patient in a unique AML-predisposed family.And the corresponding antibody was prepared to prove the existance of FAMLF as a protein-ecoding gene. We found that both FAMLF gene and it's intronic miR181a/b were overexpressed in some AML samples, related to cell differentiation, and can serve as a prognostic predictor. However, their roles in leukemogenesis remain obscure. Our study will aim at fully determining the expression signature of FAMLF in various subtypes of AML and further defining its clinical relevance; comfirming the molecular interaction between FAMLF gene and miR181a/b; constructing the cell models of FAMLF gene and miR181a/b up-regulated or down-regulated either independently or cooperatively to observe their influence on cell proliferation, differentiation and apoptosis; establishing the bone marrow transplantation mouse model of FAMLF gene overexpressed or knock-out to observe their influence on overal survival and bone marrow cell phenotypes of the mice as well as the tumorigenic effect on nude mice. The multi-level functional research of FAMLF gene and its intronic miRNA will reveal their roles in leukemogenesis ,and meanwhile provides the necessary theoretical basis for targed drugs development.

对家族性白血病深入研究有助于揭示散发白血病发病机制。我们利用AML高发家系基因资源，克隆出FAMLF新基因，制备FAMLF抗体，验证其为编码蛋白基因，发现FAMLF基因及其内含子miR181a/b高表达于AML细胞，可能与细胞分化有关，是AML预后判断指标。但FAMLF基因及其miR181a/b在白血病发病机制中的作用未明。本项目将明确FAMLF基因在AML亚型表达谱及临床意义；明确FAMLF基因与miR181a/b分子水平上的相互作用关系；分别构建FAMLF基因及miR181a/b过表达与敲除的细胞模型，观测其对细胞增殖、细胞周期、分化、凋亡等的影响。从分子、细胞水平研究FAMLF基因的功能，以揭示FAMLF及其miR181a/b在白血病发病机制中的作用，为开发靶向药物奠定基础。

家族性白血病是一种与遗传因素高度相关的血液系统恶性肿瘤，它的基因资源具有独特的研究价值。前期从福建省一个急性髓系白血病（AML）高发家系患者中克隆出FAMLF基因后，为深入研究FAMLF在白血病发生发展中的作用机制，本项目应用流行病学调查方法、生物信息学分析以及系列分子生物学技术等，通过基因表达谱、突变谱分析FAMLF基因表达、突变在AML亚型中的临床意义，在细胞水平初步探讨FAMLF功能以及参与AML发生发展的分子机制。①经Northern blot证实FAMLF的全长为2.3kb，该基因在AML患者与细胞株中高表达；②QPCR检测显示，FAMLF在AML患者中表达水平比正常人高，进一步统计分析显示其在急性粒细胞白血病部分分化型（M2）患者中的表达水平比非M2（non-M2）及M5患者高；③AML患者FAMLF表达量与AML外周血幼稚细胞比例、血红蛋白浓度和血清HBDH浓度有关，并显示在M2患者中的FAMLF表达水平与外周血幼稚细胞比例、白细胞计数、血红蛋白浓度和血清HBDH浓度有关，而与non-M2及M5亚型AML患者的临床血液学特征无明显关系；④FAMLF高表达的AML患者 CR率较高，而RFS、远期OS较低；FAMLF高表达M2患者的CR率较高，FAMLF的表达水平与non-M2的CR率无关；⑤统计学分析显示FAMLF与其内含子miR-181a1之间的表达水平正相关；⑥基因分型、单倍型分析及关联分析显示，FAMLF基因家族的GTAGG单倍型是M2患病危险性增高的因素；⑦通过慢病毒介导，构建出FAMLF下调的Kasumi-1细胞模型，通过绘制生长曲线、克隆形成实验、流式细胞术、QPCR、Western blot等技术检测FAMLF基因沉默后对Kasumi-1细胞生物学特性的影响，发现FAMLF基因沉默可抑制Kasumi-1细胞增殖，使细胞周期阻滞于G0/G1期，促进Kasumi-1细胞分化成熟，可能与Akt通路有关。⑧FAMLF基因沉默后，其内含子miR-181a1的表达水平未降低，显示FAMLF不依赖于MiR-181a1下调而独立发挥作用，这些结果表明FAMLF基因与miR-181可能在急性粒细胞白血病（M2）发生发展中起着重要作用。本项目研究对于阐明急性白血病的发病机制，发现与开发新的分子标志，具有重要的研究价值与临床意义