Chen Jian, Cai Tanxi, Zheng Chunwei, Lin Xiwen, Wang Guojun, Liao Shangying, Wang Xiuxia, Gan Haiyun, Zhang Daoqin, Hu Xiangjing, Wang Si, Li Zhen, Feng Yanmin, Yang Fuquan, Han Chunsheng
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
The Key Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Nucleic Acids Res. 2017 Apr 20;45(7):4142-4157. doi: 10.1093/nar/gkw1287.
miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.
微小RNA（miRNAs）在哺乳动物精子发生过程中发挥着重要作用。然而，大多数miRNAs在精子发生中的功能及其潜在机制仍不清楚。在此，我们报道miR-202在小鼠精原干细胞（SSCs）中高度表达，并且受胶质细胞源性神经营养因子（GDNF）和视黄酸（RA）的反向调控，这两个因子是SSC自我更新和分化的关键因素。我们使用诱导型CRISPR-Cas9在培养的SSCs中敲除miR-202，发现敲除后的SSCs启动了过早分化，同时干细胞活性降低，有丝分裂和凋亡增加。通过基于iTRAQ的蛋白质组学分析和RNA测序鉴定了靶基因，这些基因富含细胞周期调节因子和RNA结合蛋白。发现Rbfox2和Cpeb1是miR-202的直接靶标，并且Rbfox2而非Cpeb1对于SSCs分化为减数分裂细胞至关重要。因此，已经确定了一个由GDNF和RA的信号分子、miR-202和多种下游效应器组成的SSC命运调控网络。
