miR-142-3p调控脊椎动物造血干细胞的形成和分化

过去关于造血发生的研究主要集中在信号和转录因子，然而近期的研究热点主要是包括microRNAs的表观遗传调控。本文研究者发现在斑马鱼和小鼠中，miR-142-3p在造血干细胞（HSCs)中特异性表达。

敲除斑马鱼的miR-142a-3p基因导致主动脉-性腺-中肾(AGM)区的HSCs数量降低，同时胸腺中的T细胞数量也降低。从机制上来讲，miR-142a-3p通过抑制干扰素调节因子7（irf7）介导的炎症信号通路来调节HSCs的形成和分化。

此外，研究者还证实miR-142-3p在小鼠的AGM区HSCs的形成中也发挥了重要的作用，这也暗示了它在脊椎动物中扮演着一个高度保守的角色。该研究还揭示了miR-142a-3p通过抑制irf7信号通路在HSCs的形成和发生过程中起关键作用。本研究中Agilent Zebrafish Oligo Microarrays服务由上海伯豪提供。

本研究是由中国科学院动物研究所生物膜与膜生物工程国家重点实验室与北京307医院青藤转化医学中心肿瘤学实验室共同合作完成，文章的通讯作者是中科院动物所的刘峰研究院。

研究者首先以斑马鱼为研究对象，进行一系列功能学实验，发现miR-142a-3p在造血细胞中特异性表达，推测它可能在造血中起了重要的作用。

为进一步的证实以上推论，研究者通过敲除miR-142a-3p基因，进行了一系列loss of function的实验，实验数据表明miR-142a-3p是HSC形成和T细胞分化过程中不可或缺的因子。进一步研究发现，敲除miR-142a-3p基因影响了造血内皮细胞，进而延缓了最早的HSCs细胞在胚胎中出现的时间。

为进一步的深入研究miR-142a-3p对HSC的形成和分化的生物学影响，研究者比较了对照组和miR-142a-3p基因敲除组的胚胎，分别提取受精后2天（2dpf）和受精后4天（4dpf）的对照组、突变组AGM区的RNAs，表达谱芯片检测后。

发现有70个基因在2dpf和4dpf的胚胎中上调(Figure 4B)，采用Pictar和TargetScan预测miR-142a-3p的靶基因，获得4个候选基因，进一步研究发现其中irf7可能是miR-142a-3p在HSCs中作用的最主要的靶基因。后期，研究者通过qPCR、Western blotting等一系列实验证实irf7是miR-142a-3p的直接靶基因，并非间接的。

那么irf7下游又调控什么呢？为了使整个故事更加完整，研究者试图探究更多，最终发现irf7与Gcsfr-Nitric Oxide (NO)炎症信号通路可能相关。同时，他们还发现miR-142a-3p在小鼠造血中也发挥了重要的作用。

Figure 4 irf7 is a direct target of miR-142a-3p. (A) Heat map analysis of gene expression in controls and miR-142a-3p morphants at 2 dpf and 4 dpf. (B) Fold changes of upregulated or downregulated genes at 2 dpf and 4 dpf in miR-142a-3p morphants compared to controls. (C) An imperfect match of irf7 3′ UTR with miR-142a-3p as determined by a calculation using RNAHybrid. (D)Scheme of the PGL3 constructs containing irf7 WT and mutated 3′UTR. (E) Luciferase reporter activity of the reporter containing WT or mutated irf7 3′UTR when co-transfected with the miR-142a-3p duplex into HEK293T cells (mean ± SD, t-test, *P < 0.05, n= 3). (F) Western blotting images showing that irf7 protein level was decreased in duplex-injected embryos at 4 dpf, compared to controls (left panel). Quantitative analysis of the western blotting results is shown in right panel.

原文出处：

miR-142-3p regulates the formation and differentiation of hematopoietic stem cells in vertebrates.

Abstract: Previous studies on developmental hematopoiesis have mainly focused on signaling and transcription factors,while the appreciation of epigenetic regulation including that of microRNAs is recent. Here, we show that in zebrafish and mouse, miR-142-3p is specifically expressed in hematopoietic stem cells (HSCs). Knockdown of miR-142a-3p in zebrafish led to a reduced population of HSCs in the aorta-gonad-mesonephros (AGM) region as well as T-cell defects in the thymus. Mechanistically, miR-142a-3p regulates HSC formation and differentiation through the repression of interferon regulatory factor 7 (irf7)-mediated inflammation signaling. Finally, we show that miR-142-3p is also involved in the development of HSCs in mouse AGM, suggesting that it has a highly conserved role in vertebrates. Together, these findings unveil the pivotal roles that miR-142a-3p plays in the formation and differentiation of HSCs by repressing irf7 signaling.