miR-150调控红细胞膜蛋白生成对红系终末分化的作用和机制研究

Formation of erythrocyte membrane skeleton is a critical part of erythropoiesis because this enables erythrocyte to circulate in capillary to fulfill its oxygen delivery function. However, the regulatory mechanism for the formation of this unique membrane skeleton is not well studied. We have recently discovered that the formation of erythrocyte membrane is characterized by the increased expression of major red cell membrane proteins such as α-spectrin and Band 3 during terminal erythrocyte differentiation. However, deep sequencing analysis revealed that the transcripts for these two proteins are stabilization. Furthermore, miRNA array analysis showed that miR-150 decreased more than 20 folders during terminal erythrocyte differentiation and bioinformatics software found miR-150 targets the 3′UTR of α-spectrin and Band 3 mRNA. Luciferase-based reporter assay documented the direct binding of miR-150 to the 3′UTR of these two gene mRNA in Hela cells. Based on these findings, we hypothesize that miR-150 (which plays an important role in lineage commitment during hematopoiesis) may modulate terminal erythroid differentiation by directly targeting α-spectrin and Band 3 expression. In the application, we will explore the effect of anti-miRNA-150 combine with erythropoietin on K562 cells differentiation and its underlying mechanism. The successful accomplishment of our proposed studies will reveal the role of miR-150 in erythropoiesis and may provide insights into the diagnosing and treating of various hemolytic anemias.

红细胞膜蛋白是成熟红细胞行使功能的重要元件，但膜蛋白生成调控机制尚不明确。申请人首次发现红细胞重要结构和功能蛋白α-spectrin、Band 3在红系终末分化阶段显著增加；Deep sequence结果显示两个膜蛋白分子mRNA相对恒定；miRNAs array 结果显示miR-150呈阶段性下降20多倍；生物信息学表明miR-150靶向α-spectrin和Band 3 mRNA的3′UTR，在Hela细胞中荧光素酶实验发现miR-150与这两个分子mRNA 的3′UTR有结合。申请人据此提出在造血系统分化中发挥关键节点作用的miR-150在翻译后水平调控α-spectrin和Band3表达并影响红系终末分化；进而探讨anti-miR-150协同红系诱导分化剂诱导红白血病K562细胞向红系分化的作用和机制。本研究将揭示miR-150在红系发育中的功能，为白血病治疗提供新策略。

本课题在红系分化终末阶段发现miR-150表达逐渐降低，为了研究miR-150在红系分化终末阶段的作用，在hemin诱导的K562红系分化体系中过表达或者抑制miR-150，通过qRT-PCR检测发现，过表达miR-150组的HBB、HBE和HBG水平均有降低，同时通过联苯胺染色检测到过表达miR-150后血红蛋白表达阳性率显著降低，流式细胞术检测也发现过表达miR-150后CD235a的表达低于对照组；反之，转染miR-150 inhibitor，hemin诱导的K562红系分化进程加快，相关指标与过表达miR-150时表现相反。另外，通过在EPO诱导的CD34+细胞红系分化体系中进行miR-150的过表达和抑制实验，在诱导分化后的第6天（进入了红系分化终末阶段）开始处理细胞并在第10天检测，结果显示miR-150在抑制红系终末分化上与K562中效果类似。这些研究结果证实，miR-150能够抑制红系分化终末阶段的进程，低表达的miR-150能保证红系分化终末阶段顺利进行。研究中我们通过荧光素酶报告基因的实验证实了一个miR-150的靶基因EPB41和Band3，4.1R和Band3是红细胞膜蛋白的主要组成成分，在红细胞膜稳定性和功能的完整性中发挥重要作用，但我们进一步的研究发现4.1R本身并不能调控红系分化的进程。. 过表达miR-150后K562细胞增殖明显减慢，通过细胞计数和软琼脂集落形成实验证明了miR-150能够抑制K562细胞的增殖，为了明确其机制我们在K562细胞中转染miR-150 mimics后进行表达谱芯片检测，经分析发现ErbB-MAPK-p38 和 ErbB-PI3K-AKT两条通路中的基因表达差异明显，结合生物信息学预测miR-150的靶基因，我们发现PIK3CB 3′UTR上含有miR-150的预测靶点，通过荧光素酶报告基因实验证实了PIK3CB是miR-150的靶分子。鉴于PI3K-AKT通路与细胞增殖关系密切，我们认为miR-150通过靶向PIK3CB抑制PI3K的表达从而抑制了K562细胞的增殖。.综上，本研究发现miR-150能够抑制红系终末分化进程，EPB41和Band3是miR-150的靶分子；miR-150能够通过靶向PIK3CB抑制PI3K的表达从而抑制了K562细胞的增殖。