Prmt7-piRNAs通路对猪精原干细胞增殖分化的调控机制研究

Pig spermatogonial stem cells (SSCs) are the foundation of spermatogenesis and characterized by the ability to self-renew and differentiate into spermatids, ultimately, forming spermatozoa. Better understanding the mechanism underlying pig SSCs proliferation and differentiation has great significance for pig genetics, breeding and reproduction. It has been shown that protein arginine methyltransferase (Prmt) involved in the regulation of Piwi-interacting RNAs (piRNAs) biogenesis, a class of newly discovered small non-coding RNAs (26-31 nt in length) which were specifically expressed in gonad. Both Prmts and piRNAs play critical roles in stem cell maintenance and male germ cell development. Our former study discovered that Prmt7 was able to replace Sox2 in combination with Oct4 and Klf4 to reprogram mouse embryonic fibroblasts into induced pluripotent stem cells. Therefore, we propose the scientific hypothesis: Prmt7 regulates the proliferation and differentiation of pig SSCs through affecting piRNA biogenesis. To this end, we plan to overexpress or knock down Prmt7 expression in pig SSCs followed by examining the piRNA biogenesis, proliferation and differentiation of pig SSCs via qPCR, Western blot, Northern blot, small RNA sequencing, and bioinformatic analysis, to uncover the biological function of Prmt7-piRNAs pathway in regulating the proliferation and differentiation of pig SSCs, which would further provide new theoretical information for the molecular mechanisms of pig spermatogenesis, as well as technological support for the application of pig SSCs.

猪精原干细胞（Spermatogonial stem cells, SSCs）的增殖与分化是猪精子发生的基础，探索其调控机制对猪遗传育种与繁殖具有重要意义。蛋白精氨酸甲基转移酶（Protein arginine methyltransferase，Prmt）调控生殖腺特异表达的piRNA（一类非编码小RNA）生成，二者均参与干细胞维持和雄性生殖细胞发育；申请人前期工作发现Prmt7能取代Sox2诱导多能干细胞形成，因此提出科学假设：Prmt7通过影响piRNA生成而调控猪SSCs增殖分化。项目拟通过在猪SSCs中过表达、干扰Prmt7，利用qPCR、Western和Northern杂交、测序及生物信息学等技术，探讨piRNA生成和猪SSCs增殖分化变化，从而解析Prmt7-piRNAs通路对猪SSCs增殖分化的调控作用，为猪精子发生的调控机制提供理论基础，也为猪SSCs的应用提供技术支持。

精原干细胞是精子发生的基础，探索其调控机制对动物遗传育种与繁殖具有重要意义。蛋白精氨酸甲基转移酶（Protein arginine methyltransferase，Prmt）能够调控生殖腺特异表达的piRNA（piwi-interacting RNA）生成，且Prmt和piRNA均参与干细胞维持和雄性生殖细胞发育。因此，为探究Prmt7-piRNAs通路在精原细胞中的调控作用，本项目分离获得了小鼠和猪的精原干细胞，解析了白消安对精原干细胞自噬的作用及其分子机制，检测了Prmt7在小鼠和猪多个组织中的表达，解析了Prmt7在精原细胞中的作用及调控机制，以及Prmt7对精原细胞mRNA，miRNA和piRNA的调控。发现白消安通过AKT和p53信号通路中的mTOR抑制精原干细胞自噬；Prmt7在心、肝、脾、肺、肾、肌肉和睾丸中广泛表达；干扰Prmt7表达抑制精原细胞增殖，出现S期阻滞；RNA测序分析发现287个差异基因（|log2FoldChange|>=1和padj<0.05）、48个差异miRNA（padj<0.05）、7个差异piRNA cluster（padj<0.05），并对差异基因、差异miRNA的靶基因、及产生差异piRNA的来源基因进行了GO功能富集和KEGG通路分析；分析后发现一个在睾丸中特异性高表达的Akt亚型，即Akt3，不仅是差异基因，也是差异miRNA的靶基因富集通路中的基因，于是推测Prmt7可能是通过Akt3参与调控精原细胞的增殖等过程；干扰及补救实验结果显示Akt激动剂SC79能够恢复Prmt7抑制剂SGC3027对CCNE1和CDK2的抑制作用，说明Prmt7很可能是通过Akt3作用于精原细胞的增殖等过程。项目结果为猪精原干细胞相关研究提供了新的理论基础，也为精原干细胞的应用提供了技术支持。

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