miR-142-3p調控脊椎動物造血干細胞的形成和分化
過去關于造血發生的研究主要集中在信號和轉錄因子，然而近期的研究熱點主要是包括microRNAs的表觀遺傳調控。本文研究者發現在斑馬魚和小鼠中，miR-142-3p在造血干細胞（HSCs)中特異性表達。敲除斑馬魚的miR-142a-3p基因導致主動脈-性腺-中腎(AGM)區的HSCs數量降低，同時胸腺中的T細胞數量也降低。從機制上來講，miR-142a-3p通過抑制干擾素調節因子7（irf7）介導的炎癥信號通路來調節HSCs的形成和分化。此外，研究者還證實miR-142-3p在小鼠的AGM區HSCs的形成中也發揮了重要的作用，這也暗示了它在脊椎動物中扮演著一個高度保守的角色。該研究還揭示了miR-142a-3p通過抑制irf7信號通路在HSCs的形成和發生過程中起關鍵作用。本研究中Agilent ZebrafishOligo Microarrays服務由上海伯豪提供。
本研究是由中國科學院動物研究所生物膜與膜生物工程國家重點實驗室與北京307醫院青藤轉化醫學中心腫瘤學實驗室共同合作完成，文章的通訊作者是中科院動物所的劉峰研究院。
研究者首先以斑馬魚為研究對象，進行一系列功能學實驗，發現miR-142a-3p在造血細胞中特異性表達，推測它可能在造血中起了重要的作用。為進一步的證實以上推論，研究者通過敲除miR-142a-3p基因，進行了一系列loss of function的實驗，實驗數據表明miR-142a-3p是HSC形成和T細胞分化過程中不可或缺的因子。進一步研究發現，敲除miR-142a-3p基因影響了造血內皮細胞，進而延緩了最早的HSCs細胞在胚胎中出現的時間。
為進一步的深入研究miR-142a-3p對HSC的形成和分化的生物學影響，研究者比較了對照組和miR-142a-3p基因敲除組的胚胎，分別提取受精后2天（2dpf）和受精后4天（4dpf）的對照組、突變組AGM區的RNAs，表達譜芯片檢測后，發現有70個基因在2dpf和4dpf的胚胎中上調(Figure 4B)，采用Pictar和TargetScan預測miR-142a-3p的靶基因，獲得4個候選基因，進一步研究發現其中irf7可能是miR-142a-3p在HSCs中作用的最主要的靶基因。后期，研究者通過qPCR、Western blotting等一系列實驗證實irf7是miR-142a-3p的直接靶基因，并非間接的。
那么irf7下游又調控什么呢？為了使整個故事更加完整，研究者試圖探究更多，最終發現irf7與Gcsfr-Nitric Oxide (NO)炎癥信號通路可能相關。同時，他們還發現miR-142a-3p在小鼠造血中也發揮了重要的作用。
Figure 4 irf7 is a direct target of miR-142a-3p. (A) Heat map analysis of gene expression in controls and miR-142a-3p morphantsat 2 dpf and 4 dpf. (B) Fold changes of upregulated or downregulated genes at 2 dpf and 4 dpf in miR-142a-3p morphants comparedto 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 containingWT 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 tocontrols (left panel). Quantitative analysis of the western blotting results is shown in right panel.
原文出處：
miR-142-3p regulates the formation and differentiation ofhematopoietic 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 zebrafishand 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-celldefects in the thymus. Mechanistically, miR-142a-3p regulates HSC formation and differentiation through the repressionof 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 HSCsby repressing irf7 signaling.
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