Sequencing of microRNA in spermatogonial stem cell enriched cell populations.

MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughout sequencing, we profiled the expression of miRs in the Thy1+ testis cell population, which is highly enriched for SSCs, and the Thy1- cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, -146a, -465a-3p, -465b-3p, -465c-3p, and -465c-5p are preferentially expressed in the Thy1+ SSC-enriched population, as compared to Thy1- somatic cells, and we further observed that Thy1+ SSC-enriched testis cells and SSC-enriched cultured germ cells share remarkably similar miR expression profiles. Spermatogonial Stem Cell enriched cell populations (freshly isolated and short-term cultured) and somatic cell populations were isolated from C57B/L6 mouse donors and subjected to small RNA isolation and sequencing.微小RNA（MicroRNAs，miRs）在众多组织系统的基因表达调控中发挥关键作用，其功能涵盖自我更新、细胞分化、增殖与凋亡的调控。然而，单个miRs在精原干细胞（spermatogonial stem cell，SSC）稳态调控中的功能重要性尚未得到研究。本研究通过高通量测序技术，对高度富集精原干细胞的Thy1阳性睾丸细胞群，以及主要由睾丸体细胞构成的Thy1阴性细胞群中的miRs表达谱进行了分析；此外，我们还对同样富集精原干细胞的体外培养生殖细胞中的miRs全局表达谱展开了检测。研究结果表明，相较于Thy1阴性体细胞群，miR-21与miR-34c、-182、-183、-146a、-465a-3p、-465b-3p、-465c-3p及-465c-5p在Thy1阳性精原干细胞富集群中呈偏好性表达；进一步观察发现，新鲜分离的Thy1阳性精原干细胞富集睾丸细胞与体外培养的精原干细胞富集生殖细胞，二者的miRs表达谱极为相似。本研究从C57BL/6小鼠供体中分离得到精原干细胞富集的细胞群（含新鲜分离样本与短期培养样本）及体细胞群，并对其进行小分子RNA提取与测序。