MicroRNA-451a overexpression induces accelerated neuronal differentiation of Ntera2/D1 cells and ablation affects neurogenesis in microRNA-451a-/- mice

MiR-451a is best known for its role in erythropoiesis and for its tumour suppressor features. Here we show a role for miR-451a in neuronal differentiation through analysis of endogenous and ectopically expressed or silenced miR-451a in Ntera2/D1 cells during neuronal differentiation. Furthermore, we compared neuronal differentiation in the dentate gyrus of hippocampus of miR-451a-/- and wild type mice. MiR-451a overexpression in lentiviral transduced Ntera2/D1 cells was associated with a significant shifting of mRNA expression of the developmental markers Nestin, βIII Tubulin, NF200, DCX and MAP2 to earlier developmental time points, compared to control vector transduced cells. In line with this, accelerated neuronal network formation in AB.G.miR-451a transduced cells, as well as an increase in neurite outgrowth both in number and length was observed. MiR-451a targets genes MIF, AKT1, CAB39, YWHAZ, RAB14, TSC1, OSR1, POU3F2, TNS4, PSMB8, CXCL16, CDKN2D and IL6R were, moreover, either constantly downregulated or exhibited shifted expression profiles in AB.G.miR-451a transduced cells. Lentiviral knockdown of endogenous miR-451a expression in Ntera2/D1 cells resulted in decelerated differentiation. Endogenous miR-451a expression was upregulated during development in the hippocampus of wildtype mice. In situ hybridization revealed intensively stained single cells in the subgranular zone and the hilus of the dentate gyrus of wild type mice, while genetic ablation of miR-451a was observed to promote an imbalance between proliferation and neuronal differentiation in neurogenic brain regions, suggested by Ki67 and DCX staining. Taken together, these results provide strong support for a role of miR-451a in neuronal maturation processes in vitro and in vivo.

miR-451a最为人熟知的功能是参与红细胞生成（erythropoiesis）以及发挥肿瘤抑制作用。本研究通过分析神经元分化过程中Ntera2/D1细胞内源性、过表达或沉默的miR-451a，揭示了其在神经元分化中的作用。此外，我们对比了miR-451a基因敲除（miR-451a-/-）与野生型（wild type）小鼠海马齿状回的神经元分化情况。与对照载体转染的细胞相比，慢病毒（lentiviral）转染的Ntera2/D1细胞中过表达miR-451a，可使发育标志物巢蛋白（Nestin）、βIII微管蛋白（βIII Tubulin）、NF200、双皮质素（DCX）及微管相关蛋白2（MAP2）的mRNA表达显著提前至更早的发育时间点。与此一致，AB.G.miR-451a转染的细胞中神经元网络形成加速，同时观察到神经突的数量与长度均显著增加。此外，在AB.G.miR-451a转染的细胞中，miR-451a的靶基因包括MIF、AKT1、CAB39、YWHAZ、RAB14、TSC1、OSR1、POU3F2、TNS4、PSMB8、CXCL16、CDKN2D及IL6R，这些基因的表达要么持续下调，要么呈现表达谱偏移。在Ntera2/D1细胞中通过慢病毒敲低内源性miR-451a的表达，会导致神经元分化进程减缓。野生型小鼠海马发育过程中，内源性miR-451a的表达会上调。原位杂交（in situ hybridization）结果显示，野生型小鼠齿状回的颗粒下区与门区可见着色强烈的单个细胞；而miR-451a基因敲除会导致神经发生脑区的增殖与神经元分化失衡，该现象可通过Ki67与DCX染色得到证实。综上，上述结果为miR-451a在体外与体内的神经元成熟过程中发挥的作用提供了强有力的实验支撑。