PCNA相关因子（Paf）在人白血病细胞中的功能及其机制

The competition between leukemic and normal hematopoietic cells determines the outcome of leukemia patients. Leukemia cells and hematopoietic stem cells （HSCs）share the ability to self-renew; thus, the genes and pathways associated with maligancies often also regulate HSC function. Much efforts has gone into identifying genes that are differentially expressed in leukemia relative to HSCs. In previous study, we have defined the different expression pattern of proliferating cell nuclear factor (PCNA) associated factor (Paf), also known as KIAA0101, between leukemia stem cells and normal HSCs. Paf gene encodes 15-KD protein (p15PAF), which originally identified in a yeast two-hybrid screen to identify novel PCNA-interacting proteins. The biological function of p15PAF has been linked to the functions of PCNA, which has essential roles in DNA replication, repair and cell cycle control. With further investigation, the expression of Paf gene was suppressed in HSC isolated from patients with de novo and relaped leukemia, whereas, up-regulated in HSC of patients achieved complete remission. Moreover, its expression pattern is strongly associated with the prognosis of leukemia patients. In the present project,we will further identify the association between expression pattern of Paf gene and prognosis in a expanded case-control study. The specific function of Paf in leukemic cells will be investigated through gene, protein, cell and on the whole. And the aberrant up-stream signal pathway regulated Paf in de novo and relapsed leukemia will be investigated through the animal model. The results obtained from cell lines will be verified in humanized leukemia model and clinical samples. The project will specify the function and mechanism of aberrant Paf expression in human leukemia and provide evidences for the novel target of Paf and its pathway in leukemia therapy.

在前期工作中，我们比对了白血病患者体内白血病干细胞（LSC）和正常造血干细胞（HSC）的基因表达谱，发现36条有关细胞周期调控的基因发生改变，其中，最为显著的是，KIAA0101，又称Paf基因在LSC和HSC呈差异性表达。进一步研究发现，Paf基因在白血病患者初发、复发时均高表达，其表达水平与白血病的发生、治疗后缓解和复发具有很强的相关性。本项目拟进一步扩大病例研究，从mRNA表达水平、蛋白表达水平和细胞水平分析Paf基因与白血病患者预后的关系；利用基因敲减及相应的功能实验，研究Paf在白血病细胞中的确切功能；通过高通量检测方法和生物信息学，明确调控Paf基因的信号通路；通过构建白血病复发模型，研究白血病复发前后Paf及其相关信号通路的改变，为明确白血病复发机制，以及Paf及其作用通路成为白血病治疗靶点奠定实验基础。

本课题组前期比对了白血病患者体内白血病干细胞（LSC）和正常造血干细胞（HSC）的基因表达谱，发现KIAA0101基因（Paf），在LSC和HSC呈差异性表达。本项目通过病例对照研究，研究Paf基因表达水平与白血病的发生、治疗后缓解和复发的相关性并筛选可干预的相关分子靶点。研究通过对急性白血病患者临床特征分析，明确了Paf在急性白血病（急性髓细胞白血病、急性T淋巴细胞白血病、急性B淋巴细胞白血病）作为微小残留病监测标志的临床预警意义。在此基础上，以白血病干细胞的临床应用为入口，在CBF-AML患者用Flow-FISH方法比RT-PCR及流式更早筛选出微小残留病灶阳性高复发风险群体，可用于指导预后分层及个体化治疗。针对Paf基因在白血病细胞中的生物学功能，进行了人源化动物模型和体外实验，证实Paf可以通过调控白血病细胞细胞周期促进白血病细胞进入细胞周期，促进白血病增殖。磷酸化Paf蛋白是Paf起作用的有效形式。下调Paf基因可以延长动物实验中白血病荷瘤小鼠的生存时间。