College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Key Lab for Animal Biotechnology of Agriculture Ministry, Northwest A&F University, Yangling, Shaanxi, 712100, China.
Cell Prolif. 2013 Apr;46(2):223-31. doi: 10.1111/cpr.12013.
Recent lines of evidence have indicated that miR-34c can play important roles in regulation of the cell cycle, cell senescence and apoptosis of mouse and human tumour cells, spermatogenesis, and male germ-cell apoptosis. However, there is little information on the effects of miR-34c on proliferation and apoptosis of livestock male germ cells. The dairy goat is a convenient domestic species for biological investigation and application. The purpose of this study was to investigate the effects of miR-34c on apoptosis and proliferation of dairy goat male germline stem cells (mGSCs), as well as to determine the relationship between p53 and miR-34c in this species.
Morphological observation, miRNA in situ hybridisation (ISH), bromodeoxyuridine staining, flow cytometry, quantitative-RT-PCR (Q-RT-PCR) and western blotting were utilized to ascertain apoptosis and proliferation of mGSCs, through transfection of miR-34c mimics (miR-34c), miR-34c inhibitor (anti-miR-34c), miR-34c mimics and inhibitors co-transfected (mixture) compared to control groups.
Results manifested that miR-34c over-expression promoted mGSCs apoptosis and suppressed their proliferation. Simultaneously, a variety of apoptosis-related gene expression was increased while some proliferation-related genes were downregulated. Accordingly, miR-34c promoted apoptosis in mGSCs and reduced their proliferation; moreover, expression of miR-34c was p53-dependent.
This study is the first to provide a model for study of miRNAs and mechanisms of proliferation and apoptosis in male dairy goat germ cells.
最近的研究证据表明，miR-34c 可以在调控小鼠和人类肿瘤细胞的细胞周期、细胞衰老和凋亡、精子发生以及雄性生殖细胞凋亡中发挥重要作用。然而，关于 miR-34c 对家畜雄性生殖细胞增殖和凋亡的影响的信息很少。奶山羊是一种便于进行生物学研究和应用的家畜。本研究旨在探讨 miR-34c 对奶山羊雄性生殖干细胞（mGSCs）凋亡和增殖的影响，并确定该物种中 p53 和 miR-34c 之间的关系。
通过转染 miR-34c 模拟物（miR-34c）、miR-34c 抑制剂（anti-miR-34c）、miR-34c 模拟物和抑制剂混合物，利用形态学观察、miRNA 原位杂交（ISH）、溴脱氧尿苷染色、流式细胞术、定量 RT-PCR（Q-RT-PCR）和 Western blot 分析来确定 mGSCs 的凋亡和增殖情况，并与对照组进行比较。
结果表明，miR-34c 的过表达促进了 mGSCs 的凋亡并抑制了其增殖。同时，多种凋亡相关基因的表达增加，而一些增殖相关基因的表达下调。因此，miR-34c 促进了 mGSCs 的凋亡并减少了其增殖；此外，miR-34c 的表达依赖于 p53。
本研究首次为研究男性奶山羊生殖细胞中的 miRNA 增殖和凋亡机制提供了模型。
