利用基因组编辑技术CRISPR/Cas9研究SRSF2 P95H杂合性突变在MDS发病中的作用

As the most frequently recurrent somatic mutation, spliceosome mutation is largely specific and fundamental mutation and plays an important role in the pathogenesis of MDS. SRSF2 is an important component of spliceosome, and SRSF2 P95H is a dominant mutation which is significantly associated with specific disease phenotype and poor prognosis. So the SRSF2 mutation is a representative spliceosome mutation. SRSF2 P95H is the most common form of the mutation. By now, the molecular mechanisms of the mutation induceing specific phenotypes and resulting in poor prognosis are not clear. Therefore, on the basis of the preliminary work, we plan to apply a genome editing technique (CRISPR/Cas9) to construct a SKM-1 MDS cell line with heterozygous type SRSF2 P95H mutation. The effects of SRSF2 mutation on hematopoiesis will be observed by using the cell line and Srsf2 P95H/wt mice model. The RNA-seq combined with bioinformatics methods will be used to identify the changes of splicing manner and screen the major differentially spliced genes. The expression of screened candidate genes will be validated in cell line model and bone marrow samples of Srsf2 P95H/wt mouse and MDS patients, the function of the candidate genes will be validated by inducing or knockdown expression with lentivirus vector in SKM-1 cell line loaded SRSF2 P95H/wt mutation. So it will be very valuable to explore the role of SRSF2 mutation in MDS pathogenesis and screen novel therapeutic targets.

剪接体突变在骨髓增生异常综合征（MDS）中检出率最高，是基础性和特异性的突变，在MDS发病中发挥重要作用。SRSF2是剪接体的重要组分，其突变率高，与MDS表型相关，能提示不良预后，是代表性的剪接体突变，P95H是最常见的突变形式。但SRSF2突变致MDS特殊表型及不良预后的机制不明，研究甚少。因此，本项目拟在前期工作基础上，用基因组编辑技术CRISPR/Cas9建立SRSF2 P95H/wt MDS细胞系模型，结合Srsf2 P95H/wt小鼠模型，观察SRSF2突变对造血的影响。用RNA-seq及生物信息学方法，鉴定SRSF2突变对剪接功能的影响，筛选差异剪接基因；在细胞系、小鼠模型和患者造血干细胞中验证其表达；在细胞模型中用慢病毒载体诱导或沉默候选基因表达，明确SRSF2下游分子靶点/通路。这对于探讨剪接体突变在MDS发病中的作用机制、寻找新的治疗靶点都具有重要的理论及现实意义。

骨随增生异常综合征（MDS）是一种血液系统肿瘤。多数MDS患者造血干细胞存在基因突变在其发生发展中起着重要作用。剪接体（SRSF2,SF3B1等）突变与MDS表型及预后相关，探讨剪接体突变及其下游分子，对明确MDS发病机制，寻找治疗靶点意义重大。.我们用CRISPR/Cas9构建负载SRSF2 P95H的SKM-1细胞系，观察细胞生物学行为改变，RNA-seq寻找下游靶点，用B6J.B6NTac(SJL)-Srsf2tm1.1Oaw/J小鼠和B6.Cg-Tg(Vav1-icre)A2Kio/J小鼠构建负载SRSF2 P95H的Vav1-icre Srsf2 P95H/wt小鼠模型，对备选靶点进行功能验证。.成功构建了负载SRSF2 P95H的SKM-1细胞系和负载SRSF2 P95H的Vav1-icre Srsf2 P95H/wt小鼠模型；负载SRSF2 P95H的SKM-1细胞系增殖降低，Vav1-icre Srsf2 P95H/wt小鼠模型短期未见血象变化，但RNA-seq结合文献检索，发现多种基因通路存在变化。通过降低EZH2表达，可使H3K27me3和H3K4me3组蛋白表达降低， H3K27me3功能失活，抑制HOXA9的表达抑制MDS细胞的增殖。.综上，SRSF2 P95H突变可致多种下游事件；通过降低EZH2表达，可以抑制MDS细胞的增殖。这不仅进一步明确了MDS的发病机制，寻找了潜在的治疗靶点，并且突变细胞模型及动物模型的建立，为探索研究MDS建立了一稳定的技术平台，为下一步研究打下了坚实的基础。