LncRNA-NA2332/CUEDC2在急性髓系白血病发生发展中的作用机制研究

Leukemia stem cells (LSC) are the origins of relapse occuring in leukemia patients after remission, and effective removal of LSC is difficult in leukemia therapy. Most studies supported that the LSC might be orginated from the dysregulation of the key signaling pathway in the differentiation of hemopoietic stem cell/hemopoietic progenitor cell. The hyperactivation of JAK1-STAT3 signaling pathway is the key factor responsible for the transformation of hemopoietic stem cell/hemopoietic progenitor cell to LSC. Our previous studies suggested that the hyperactivation of JAK1-STAT3 mediated by the downregulation of CUEDC2 played critical roles in the maintenance of stem cell self-renewal and the pathogenesis of acute myeloid leukemia (AML). However, the exact mechanism of CUEDC2 downregulation in AML has not been reported previously. Through gene chip analysis, we found the long noncoding RNA-NA2332 was closely related to the regulation of JAK1-STAT3 signaling pathway. Moreover, the Lnc-RNA NA2332 was a potential negative regulator of CUEDC2. Further studies indicated that the higher expression of NA2332 was observed in AML cells and competitive inhibition of its’ expression enhanced the expression of CUEDC2 and affected the proliferation of AML cell lines. In the project, we propose to elucidate the regulation mechanism of NA2332 on the expression of CUEDC2 and the activation of JAK1-STAT3 signaling pathway in the epigenetic, transcription and post-transcription level using in vivo and in vitro model, and then explicit the role of NA2332 in the maintenance of stem cell self-renewal and the pathogenesis of AML. If finished on schedule, this study may enhance our understanding on the pathogenesis of leukemia and characteristics of LSC.

白血病干细胞（LSC）是白血病患者缓解后复发的根源，有效清除LSC是白血病治疗的难点。大部分研究支持LSC起源于造血干/祖细胞关键通路失调。JAK1-STAT3通路过激活是干/祖细胞向LSC转化的关键因素之一。我们前期研究提示CUEDC2下调介导的JAK1-STAT3通路过激活在LSC的干性维持和AML的发生发展中起重要作用，但是其下调机制尚未见报道。通过基因芯片，我们发现长链非编码RNA-NA2332参与JAK1-STAT3通路调控并且是CUEDC2潜在的负调控子。进一步研究显示NA2332在AML细胞中高表达，竞争抑制其表达则CUEDC2表达上调，抑制白血病细胞的增殖。本课题拟采用离体和在体模型，从表观遗传学、转录和转录后水平探索NA2332调控CUEDC2以及JAK1-STAT3通路的机制，明确其在LSC干性维持和AML发生发展中的作用。如期完成，有望加深对白血病发病和LSC的认识。

白血病干细胞（LSC）是患者缓解后复发的根源。CUEDC2表达下调导致JAK1-STAT3通路过激活是LSC干性维持和AML发生的关键因素，但机制尚不清楚。本研究结合LncRNA研究的新技术探索了AML中CUEDC2下调的机制。结果如下：①在AML细胞株和原代细胞中NA2332高表达，CUEDC2低表达，过表达NA2332下调CUEDC2促进JAK1-STAT3通路过激活，提示NA2332通过调控CUEDC2调节JAK1-STAT3通路的活性。②过表达NA2332促进细胞的增殖、抑制其凋亡，使其发生G1-S期的转变，提高对化疗药物的耐药性和克隆形成率促进LSC的干性维持；而竞争抑制其表达，则结果相反。③过表达NA2332促进AML原代细胞增殖、抑制对化疗药物的敏感性、细胞凋亡和分化、使细胞从G1向S期转化；而竞争抑制其表达，则结果相反，提示其通过调控CUEDC2影响AML细胞的生物学行为。④Chip分析提示NA2332在表观遗传学水平调控CUEDC2的甲基化和乙酰化；启动子区域敲除实验提示其招募结合蛋白调控CUEDC2的转录。⑤序贯移植结果提示：过表达NA2332提高LSC的含量、抑制其分化、使其处于静息状态；竞争实验结果提示：过表达NA2332提高LSC的数目、比例和干性维持能力；而竞争抑制其表达，则结果相反；提示其可以调控LSC的干性维持。⑥序贯移植提示：过表达NA2332缩短小鼠生存期、降低生存率、促进AML细胞的骨髓浸润；而竞争抑制其表达延长小鼠生存期、提高其生存率、减少AML细胞的骨髓浸润；CUEDC2敲除后，过表达NA2332不影响小鼠的生存率、生存期和AML细胞的骨髓浸润，提示NA2332需要通过调控CUEDC2的表达参与AML的发生发展。本研究基本阐明了NA2332调控CUEDC2的表达、SOCS1的泛素化降解和JAK1-STAT3通路在LSC干性维持和AML发生发展中的作用机制，为AML的治疗提供了新的潜在靶点。意义：基本阐明了NA2332通过调控CUEDC2的表达、SOCS1泛素化降解进而调控JAK1-STAT3的活性在LSC干性维持和AML发生发展中的作用，为其治疗提供了新的潜在靶点。