脐带间充质干细胞外泌体lncRNA-UCA1通过SRSF3/c-Myc轴调控脐血CD34细胞自我更新的机制研究

脐血是急需移植或无合适供者的恶性血液病患者重要的替代移植物，但因单份脐血中造血干祖细胞（HSPCs）数量相对不足而应用受限。扩增HSPCs作为突破瓶颈的重要方法，是当今世界范围内的研究热点与难点，但目前尚未真正实现，而造血干细胞（HSCs）自我更新机制的阐明是实现HSPCs扩增的前提。本研究在前期工作的基础上，在细胞及动物水平上进一步验证UCA1通过SRSF3/c-Myc轴调控HSCs的自我更新，进而围绕分子交互位点探寻直接调控机制。本研究分为3个部分：（1）在脐血CD34细胞中过表达或沉默UCA1，或在沉默UCA1的同时过表达SRPK2，通过细胞及动物水平的正反回复实验验证UCA1/SRSF3/c-Myc轴对HSCs的调控作用；（2）RNA pull-down实验及RNA免疫共沉淀（RIP）实验双向验证UCA1与SRPK2、pSRSF3存在直接结合，阐明UCA1的分子脚手架作用；（3）对UCA1构建分段截短突变体，用RNA pull-down实验验证UCA1与SRPK2、pSRSF3的结合位点，进而构建结合位点缺失突变体（△UCA1），与野生型UCA1相比较，通过细胞及动物实验验证结合位点在分子间交互及HSCs自我更新调控中发挥的重要作用。本研究将验证UCA1通过介导SRPK2对SRSF3的磷酸化，影响c-Myc转录后剪接进而发挥对HSCs自我更新的调控作用；将明确UCA1通过脚手架模式与SRPK2及pSRSF3直接结合，三者构成的复合体共同调控c-Myc前体mRNA的剪接；将阐明UCA1与SRPK2及pSRSF3相结合的核酸序列区域，并验证结合位点对分子间交互及HSCs自我更新的重要作用。本研究原创性地提出了UCA1/SRSF3/c-Myc信号轴，并深入探明了其调控HSCs自我更新的直接分子机制，为HSPCs的体外扩增提供了新的理论依据。

Umbilical cord blood (UCB) is an important alternative source of transplant for patients who are in urgent need of hematopoietic stem cell (HSC) transplantation or do not have suitable donors. UCB has the advantages of rapid availability, relatively less human leukocyte antigen (HLA) restriction and lower incidence of graft-versus-host disease (GVHD). However, the low finite number of hematopoietic stem and progenitor cells (HSPCs) that can be collected from a placenta limits its application. Expansion of HSPCs is an important method to break through the bottleneck, but it has not yet been realized. It is mainly because the mechanism of HSC self-renewal regulation has not yet been elucidated. We previously found that UCB CD34 cells tend to maintain self-renewal when co-cultured with Wharton's jelly mesenchymal stem cells (WJ-MSCs) in hypoxic conditions compared to normoxic co-culture or suspension culture in hypoxia. And we clarified the relevant mechanisms at the level of cytokines and signal pathways which related to self-renewal. We further explore the mechanisms at the level of exosomes and found that the exosomes of WJ-MSCs cultured in hypoxia maintained the proportion of UCB CD34 cells better than normoxic exosomes, and the content of long non-coding RNA urothelial carcinoma associated 1 (UCA1) in hypoxic exosomes is higher than that in normoxic exosomes. Furthermore, we proved that UCA1 in exosomes could be taken up by UCB CD34 cells. Subsequently, we discovered that the phosphorylation of serine-arginine splicing factor 3 (SRSF3), as well as the expression of c-Myc which is an important regulator of HSCs self-renewal, are regulated by UCA1. When added hypoxic WJ-MSCs exosomes containing UCA1 together with serine-arginine protein kinase 2 (SRPK2) inhibitor to the culture medium, the content of phosphorylated form of SRSF3 (pSRSF3) and the expression of c-Myc were down-regulated in UCB CD34 cells. As above, UCA1 may regulate HSCs self-renewal through SRSF3/c-Myc axis. Since pSRSF3 is more susceptible to be dephosphorylated by phosphatase, UCA1 may combine with SRSF3 and SRPK2 through the scaffold mode to form a complex, promote the phosphorylation of SRSF3, and inhibit the binding of pSRSF3 to phosphatase at the same time. Based on the previous work, we will further explore how UCA1 regulates the self-renewal of HSCs through the SRSF3/c-Myc axis. First, we will overexpress or silence UCA1, or overexpress SRPK2 while silencing UCA1 in cord blood CD34 cells, and verify the regulation of UCA1/SRSF3/c-Myc axis on HSCs through in vivo and in vitro experiments. Second, we will use RNA pull-down and RNA immunoprecipitation (RIP) experiments to prove that UCA1 directly binds with SRPK2 and pSRSF3, and clarify the molecular scaffolding effect of UCA1. Furthermore, we will construct a segmented truncation mutant of UCA1, use RNA pull-down experiments to explore the binding sites of UCA1 and SRPK2, pSRSF3, and then construct a binding site deletion mutant (△UCA1), and compare it with wild-type UCA1 through in vitro and in vivo experiments to prove that the binding sites play an important role in molecular interactions and HSCs self-renewal regulation. This study will verify that UCA1 mediates the phosphorylation of SRSF3 by SRPK2, affects the post-transcriptional splicing of c-Myc and plays a regulatory role in HSCs self-renewal; and UCA1 directly binds to SRPK2 and pSRSF3 through the scaffolding mode, and the complex regulates the splicing of c-Myc precursor mRNA; and the nucleic acid sequence region where UCA1 binds to SRPK2 and pSRSF3; furthermore, it will clarify the important role of the binding site on intermolecular interaction and HSCs self-renewal. This study originally proposed the UCA1/SRSF3/c-Myc signal axis, and thoroughly explored its direct molecular mechanism regulating the self-renewal of HSCs, and provided a new theoretical basis for the expansion of HSPCs in vitro.