核不均一核糖蛋白HNRPDL促白血病细胞生长的研究

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein D-like (HNRPDL) is a member of this family, which is able to bind both RNA and DNA regulating multiple steps of mRNA metabolism. Chronic myeloid leukemia (CML) originates from hematopoietic stem cells acquiring BCR/ABL fusion gene. Previous study has shown that HNRPDL is aberrantly expressed in CD34+ cells from CML patients; however what role it plays in these cells has not been elucidated yet. Herein, we showed that HNRPDL silencing inhibited the colony-forming cell (CFC) capacity of CML CD34+ cells; strikingly, overexpression of HNRPDL in murine BaF3 cells induced lethal leukemia in mice, suggesting HNRPDL is a proto-oncogene. Mechanistically, we showed that HNRPDL impaired the stability of pre-B-cell leukemia homeobox 1 (PBX1) mRNA in K562 cells (BCR/ABL+) and PBX1 silencing inhibited the growth of these cells. In this proposal, we will perform long-term culture initiating cell (LTC-IC) assay to assess the effect of HNRPDL silencing on primitive CML cells, and analyze the effect of HNRPDL silencing on the growth of primitive CML cells in NSG mice. On the contrary, we will study the effect of HNRPDL alone and with BCR/ABL on the growth of murine hematopoietic stem/progenitor cells in vitro and in vivo. Lastly, we will generate microarray data comparing HNRPDL silenced CML CD34+ cells with their control cells, and identify the genes that play a role in the HNRPDL silencing mediated growth inhibition and are regulated by HNRPDL through mRNA decay. The execution of the project will deepen the understanding of the role of hnRNPs in human leukemia and possibly provide new clues for the improvement of the disease management.

核不均一核糖蛋白（hnRNP）家族成员HNRPDL是DNA/RNA结合蛋白，参与RNA代谢调控。慢性髓细胞白血病（CML）是造血干细胞获得BCR/ABL融合基因而诱发的恶性血液病。我们报道HNRPDL在CML患者CD34+细胞中表达增高，但其在CML干/祖细胞中的功能与作用机制尚未知。预研显示HNRPDL沉默抑制患者CD34+细胞生长；过表达HNRPDL恶性转化小鼠造血BaF3细胞；HNRPDL可以影响mRNA（如PBX1）稳定性而行使功能。将检测HNRPDL沉默对CML患者细胞的长期培养起始细胞（LTC-IC）和免疫缺陷小鼠体内生长能力的影响；研究HNRPDL单独或与BCR/ABL共同促进小鼠造血干/祖细胞生长和转化的作用；鉴定mRNA稳定性受HNRPDL调控而参与其促CML细胞生长的功能靶基因。项目实施将深化对hnRNPs促白血病细胞生长功能的认知，并为改善疾病治疗提供新的理论基础。

慢性髓细胞白血病（chronic myeloid leukemia, CML）是源自造血干细胞（HSCs）的恶性血液病，HSCs中获得BCR-ABL融合基因是诱发该疾病重要因素。然而，单独使用靶向BCR-ABL的酪氨酸激酶抑制剂（TKI，如甲磺酸伊马替尼，IM）仅能治愈少数CML患者，这与TKI不能有效清除CML干/祖细胞、诱发其凋亡密切相关。因此，深入研究CML干/祖细胞的生长机制有可能为改 善疾病治疗提供新线索。我们的前期研究发现核不均一核糖核蛋白HNRPDL（heterogenous nuclear ribonucleoprotein D-like）在CML患者CD34+细胞中异常高表达。在本项目中，我们证实HNRPDL能恶性转化NIH-3T3细胞和BaF3细胞，且增强BCR-ABL诱发白血病的能力，具有原癌基因功能。HNRPDL调控CML干/祖细胞的生长与IM反应性。为研究HNRPDL的分子作用机制，建立对照和HNRPDL沉默K562细胞的基因表达谱，验证发现HNRPDL可以调控前B-细胞白血病同源盒基因（pre B cell leukemia homeobox 1, PBX1）的表达。进一步分析显示，HNRPDL作为RNA结合蛋白可以识别PBX1（mRNA）中的特异基序（ACUAGC）而增强其稳定性。PBX1沉默与HNRPDL沉默类似，都能抑制CML干/祖细胞生长并调控CML细胞的IM反应性；PBX1过表达能增强正常造血CD34+细胞的集落生成能力，也能“挽救”HNRPDL沉默导致的生长受抑和IM增敏。综上，本项目的研究表明HNRPDL/PBX1轴促进CML干/祖细胞的生长并使它们对IM耐受。这增进了我们对CML干/祖细胞生长/生存机制的认知，也将为改善该疾病的治疗提供新的理论依据和实验基础。