SF3B1基因调节Bcl-x可变剪接参与骨髓增生异常综合征-RARS红系无效造血的研究

Myelodysplastic syndromes(MDS)-refractory anemia with ring sideroblasts (RARS) is mainly characterized by ineffective erythropoiesis in erythroblasts. Our previous study had demonstrated the mutations in splicing factor 3b subunit 1 (SF3B1) were the most frequently identified genetic abnormalities in RARS and may act as a loss of function. Subjects with SF3B1 mutations often had more severely ineffective erythropoiesis compared to subjects without mutation. However, many questions remain unanswered in linking SF3B1 dysfunction to MDS biology like the downstream targets and the exact mechanisms contribute to RARS ineffective erythropoiesis. We found that RARS with SF3B1 mutations had reduced Bcl-xL expression, an alternative pre-mRNA splicing product of Bcl-x which actes as a main medicator of ineffective erythroiesis in RARS. So, we will use in induced erythroid differentiation system of k562, normal bone marrow CD34+ progenitors and RARS primary cell to identify ①whether SF3B1 can directly regulate Bcl-x alternative splicing② the effect of downregulation of SF3B1 on RARS ineffective erythropoiesis , and ③whether downregulation of SF3B1 regulate RARS ineffective erythropoiesis through Bcl-x alternative splicing. This project will provide new insights into the pathogenesis and treatment of MDS-RARS as expected, and has great significance for fully clarifying the role of SF3B1 in ineffective erythropoiesis.

红系无效造血是骨髓增生异常综合征（MDS）-难治性贫血伴环状铁粒幼红细胞增多（RARS）主要病理特征。前期研究发现MDS-RARS患者存在剪接复合体基因SF3B1高频功能减退性突变，突变型患者红系无效造血更显著，但具体作用机制不明确。我们首次发现导致MDS-RARS红系无效造血重要基因Bcl-x可变剪接产物Bcl-xL在SF3B1突变患者中表达显著减低，提示SF3B1突变可能通过对Bcl-x异常可变剪接参与MDS-RARS红系无效造血。本课题拟在向红系分化的k562细胞系和正常人骨髓CD34+细胞、MDS-RARS原代细胞中多层面上研究：①SF3B1直接调控Bcl-x 可变剪接；②SF3B1低表达导致红系无效造血；③SF3B1通过Bcl-x异常剪接参与红系无效造血，以阐述SF3B1突变在MDS-RARS红系无效造血中作用机制,为MDS-RARS发病机制研究及治疗提供新途径。

近期研究已证实SF3B1突变为参与骨髓增生异常综合征-RARS特征性分子生物事件，具有该突变的患者预后良好。本研究通过k562细胞系及正常人CD34＋细胞下调SF3B1基因表达，发现红系祖细胞增殖减慢，凋亡增加，红系终末阶段分化受阻。RNA-seq data 提示SF3B1基因下调参与RNA剪接异常发生，其关键作用靶基因为Bcl-x 。在SF3B1下调的CD34+细胞中二次转染Bcl-x异常剪接产物Bcl-xL，红系祖细胞凋亡显著减少，细胞增殖增加，从而明确SF3B1基因通过调控Bcl-x可变剪接产物参与MDS-RARS 无效造血。本研究为临床上MDS-RARS患者治疗提供新的治疗靶点，对于阐明MDS-RARS患者无效造血机制具有重要意义.

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