miR-125b调控红细胞脱核成熟的作用及其机制研究

To overcome the shortage of blood transfusions in the treatment of anemia and regular surgery, it is necessary to explore new source of red blood cells (RBCs). Although ex vivo derivation of RBCs from stem cells (embryonic stem cells or hematopoietic stem cells) is under investigation, there are still plenty of hurdles needed to be strided over before these RBCs show their clinical usages. Among these hurdles, enucleation remains one of the critical rate-limiting steps of in vitro RBC synthesis. We propose to take advantage of microRNA to improve the maturation and enucleation of erythrocytes, especially miR-125b. microRNAs are small RNA molecules binding to partially complementary sites in the 3'-UTR of target transcripts and repressing their expression, they have been shown as important regulators in stem cell commitment, cell proliferation and apoptosis. Provious studies from other groups and our lab showed that miR-125b is a pivotal regulator in hematopoietic stem cell homeostasis as well as proapoptosis/ anti-apoptosis gene and cell cycle regulator in different cancer cells depending on the intracellular environment. More importantly, we discovered the relationship between miR-125b and enucleation. Mature miR-125b was enriched in RBCs, and the overexpression of miR-125b improved the erythroid maturation and enucleation of erythroleukemia cells (K562, TF-1) and of proerythrocytes derived from cord blood. Bcl-2 and SMARCD2 might be the mediators of this function. Since the main models of enucleation are apoptosis and asymmetric cytokinesis, and miR-125b targets lots of proapoptosis/ anti-apoptosis genes and cell cycle regulators, we hypothesize that by targeting several apoptosis or/ and cell cycle regulators, miR-125b dosage control the extrusion of erythrocytes nuclei. In this study, we plan to confirm the positive effect of miR-125b in the maturation and enucleation of erythrocytes, explore the molecular mechanism underline, and improve the yield of ex-vivo derived RBCs in the long run. Firstly, miR-125b expression profile will be checked in different hematopoietic stem cells, erythroblasts, proerythrocytes and mature red blood cells. Then the function of miR-125b is going to be confirmed by gene LOF (loss of function) and GOF (gain of function) experiments. The anticipation is overexpression of miR-125b could improve the maturation and enucleation of primary erythroblasts and erythroleukemia cells. Third, targets of miR-125b will be predicted with bioinformatics analysis, and confirmed by target gene LOF/ GOF studies. Novel molecular imaging tool will be used in this step to discover the correlation between miR-125b, putative target genes and organelles movement. The study of novel miR-125b target genes which might be important regulators in erythrocyte maturation could further prompt ex-vivo RBCs production and meet the requirement of blood supply in the country.

诱导干细胞红系分化可能解决目前临床血供短缺的问题，但是如何提高红细胞成熟和脱核效率是该技术中一个急待解决的关键问题。我们试图利用miR-125b 来提高红细胞成熟的效率。已有研究显示miR-125b能通过靶向凋亡相关基因调节造血干细胞的增殖和动态平衡。我们前期的研究发现，miR-125b 具有促红细胞成熟和脱核的潜能，在成熟红细胞中有miR-125b 的富集，而在成红细胞或红白血病细胞系中过表达miR-125b 也能促进细胞的成熟和脱核，抑制其表达则出现相反效果，Bcl-2和SMARCD2可能参与了这个过程。本课题将通过转基因实现miR-125b 功能的缺失或获得（LOF/GOF）确定其对红细胞脱核的作用，进一步利用信息学和靶基因LOF/GOF 确定在红细胞脱核成熟过程中发挥关键作用的miR-125b 靶基因，研究脱核的细胞及分子机制，最终提高体外诱导红细胞成熟的效率，满足临床应用需求。

诱导干细胞红系分化可能解决目前临床血供短缺的问题，但是如何提高红细胞成熟和脱核 效率是该技术中一个急待解决的关键问题。已有研究显示miR-125b能通过靶向凋亡相关基因调节造血干细胞的增殖和动态平衡，而我们前期的研究发现，miR-125b 具有促红细胞成熟和脱核的潜能，因此本研究中我们试图利用miR-125b 来提高红细胞成熟的效率，并对其作用机制做深入分析。本项目研究发现，在成熟红细胞中有miR-125b 的富集，而在成红细胞或红白血病细胞系中过表达miR-125b 也能促进细胞的成熟和脱核，抑制其表达则出现相反效果；真性红细胞增多症病人中外周血有核红细胞比例增加，与之相对应的，外周血miR-125b的表达也低于正常人；在小鼠骨髓腔中注射miR-125b inhibitor，将导致骨髓、脾脏及外周血中有核红细胞的比例增加；这些发现均提示miR-125b参与红细胞的脱核调控。在miR-125b促红细胞成熟脱核的过程中，我们观测到线粒体膜电位的下降，细胞及细胞核的收缩，但红细胞的凋亡水平未发生明显改变；进一步分析miR-125b作用的信号通路，利用生物信息学和靶基因LOF/GOF 确定在红细胞脱核成熟过程中发挥关键作用的miR-125b靶基因，最终确定，Bcl-2和SMARCD2可能参与了红细胞的脱核调控，Bcl-2通过改变线粒体膜电位，活化凋亡相关基因caspase3，进一步活化细胞骨架调节蛋白Rock1，调节细胞骨架，促进红细胞脱核；SMARCD2则通过直接调节细胞核的凝集状态促进红细胞成熟脱核。对鼠源及人源红系祖细胞转染miR-125b，移植体内，均能观查到红细胞成熟的提前，并在小鼠体内检测到成熟的人源红细胞。这类体外制备的红细胞未来有望用于输血，满足临床应用的需求。