去泛素化酶USP7在骨髓增生异常综合征患者中的作用及机制研究

Myelodysplastic syndromes (MDS) are clonal and heterogeneous diseases of the hematopoietic stem / progenitor cell. Dyserythropoiesis in the bone marrow is found in about 80% of early MDS. However, the mechanism of ineffective erythropoiesis in MDS is poorly understood. USP7 plays an important role in variety of life processes by regulating the stability of different proteins. Our preliminary data found that USP7 can influence normal erythroid differentiation by regulating the GATA1 protein level.Further,we found USP7 mRNA was increased, and GATA1 mRNA was also increased in MDS. Subsequently,we found that specific inhibitors of USP7 resulted in inhibition of cell proliferation,and apoptosis induction in MDS-L and SKM-1 cell lines;In addition, we found the level of GATA1 and RUNX1 proteins declined.According to the preliminary results we propose the hypothesis that the abnormal increase of USP7 in patients with MDS results in the up-regulation of GATA1,repression of erythroid differentiation and disordered erythropoiesis;And USP7 may be involved in the pathogenesis of MDS by GATA1 interaction with RUNX1.In this study, we will enlarge the samples of MDS patients and health donors;and clarify the role of USP7 in ineffective erythropoiesis and pathogenesis in MDS in vitro;and carry out the study of therapeutic action by specific inhibitor of USP7 in MDS.The demonstration of the above hypothesis would provide a new therapeutic strategy in MDS.

骨髓增生异常综合征（MDS）是一组克隆性异质性造血干/祖细胞疾病，约80%的早期MDS患者可出现红系发育异常。但对MDS红系无效造血的机制所知甚少。去泛素化酶USP7通过调控蛋白稳定性参与细胞多种重要生命过程。既往研究发现USP7通过调控GATA1影响人正常终末红系分化;本研究发现MDS中USP7异常高表达，GATA1表达亦增高。用USP7抑制剂处理MDS-L、SKM-1细胞系，发现其抑制细胞增殖，诱导凋亡；进一步研究发现抑制剂处理前后GATA1和RUNX1蛋白表达下调。我们据此推测：MDS中USP7表达异常增高，导致GATA1表达上调，从而抑制红系分化，引起红系无效造血；USP7可能通过GATA1影响RUNX1参与MDS发生发展。本项目将扩大MDS患者和正常样本，通过体外功能实验阐明USP7致MDS红系无效造血及发病的机制，研究USP7抑制剂对MDS的治疗作用，为临床治疗提供新的策略。

骨髓增生异常综合征（MDS）是一组克隆性异质性造血干/祖细胞疾病，约80%的早期MDS患者可出现红系发育异常。但对MDS红系无效造血的机制所知甚少。去泛素化酶USP7通过调控蛋白稳定性参与细胞多种重要生命过程。本研究发现MDS患者中USP7异常高表达，用USP7抑制剂处理MDS-L、SKM-1细胞系，发现其抑制细胞增殖，诱导凋亡；MDS细胞系中巨核系标志物CD10,CD41,CD44,CD61等表达升高；RNA‐seq分析和western blot结果显示USP7抑制剂显著上调了GSN表达。为探究GSN的功能，我们利用2个不同的siRNAs序列在MDS-L和SKM-1细胞系敲低GSN, 发现GSN敲低显著抑制了P5091诱导的细胞凋亡效应。流式分析显示，GSN 敲低显著抑制了P5091诱导的CD41阳性细胞增加。以上结果提示USP7抑制剂通过上调GSN表达抑制MDS细胞增殖、诱导MDS细胞向巨核系分化。为明确USP7抑制剂对MDS细胞的体内作用，我们应用USP7抑制剂P5091治疗MDS-L荷瘤小鼠，发现该抑制剂显著抑制了小鼠肿瘤生长，TUNNEL分析显示，P5091处理组的肿瘤组织凋亡细胞百分比显著上调；免疫组化结果显示P5091处理组的巨核系表面标志物CD41显著上调，GSN表达上调。总之，我们的研究第一次揭示了USP7在MDS中的作用，同时表明USP7/GSN轴可能是MDS潜在的治疗标靶。