Li D, Ji Y, Wang F, Wang Y, Wang M, Zhang C, Zhang W, Lu Z, Sun C, Ahmed M F, He N, Jin K, Cheng S, Wang Y, He Y, Song J, Zhang Y, Li B
Jiangsu Province Key Laboratory of Animal Breeding and Molecular Design, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China.
Department of Animal and Avian Sciences, University of Maryland, College Park, MD, 20742, USA.
Anim Genet. 2017 Apr;48(2):191-204. doi: 10.1111/age.12510. Epub 2016 Nov 14.
Regulation of crucial lncRNAs involved in differentiation of chicken embryonic stem cells (ESCs) to spermatogonia stem cells (SSCs) was explored by sequencing the transcriptome of ESCs, primordial germ cells (PGCs) and SSCs with RNA-Seq; analytical bioinformatic methods were used to excavate candidate lncRNAs. We detected expression of candidate lncRNAs in ESCs, PGCs and SSCs and forecasted related target genes. Utilizing wego, david and string, function and protein-protein interactions of target genes were analyzed. Finally, based on string analysis, interaction diagrams and relevant signaling pathways were established. Our results indicate a total of 9657 lncRNAs in ESCs, PGCs and SSCs, with 3549 defined as significantly different. We screened 20 candidate lncRNAs, each demonstrating a greater than eight-fold difference in |logFC| value between groups (ESCs vs. PGCs, ESCs vs. SSCs and PGCs vs. SSCs) or specifically expressed in an individual cell type. qRT-PCR results indicated that expression tendencies of candidate lncRNAs were consistent with RNA-Seq. Fifteen cis and four trans target genes were forecasted. Based on wego and string analyses, we found lnc-SSC1, lnc-SSC5, lnc-SSC2 and lnc-ESC2 negatively regulated target genes SUFU, EPHA3, KLF3, ARL3 and TRIM8, whereas SHH, NOTCH, TGF-β, cAMP/cGMP and JAK/STAT signaling pathways were promoted, causing differentiation of ESCs into SSCs. Our findings represent a preliminary unveiling of lncRNA-associated regulatory mechanisms during differentiation of chicken ESCs into SSCs, filling a research void in male germ cell differentiation related to lncRNA. Our results also provide basic information for improving in vitro induction systems for differentiation of chicken ESCs into SSCs.
通过RNA-Seq对鸡胚胎干细胞（ESCs）、原始生殖细胞（PGCs）和精原干细胞（SSCs）的转录组进行测序，探索参与鸡胚胎干细胞向精原干细胞分化的关键长链非编码RNA（lncRNAs）的调控机制；运用生物信息学分析方法挖掘候选lncRNAs。我们检测了候选lncRNAs在ESCs、PGCs和SSCs中的表达情况，并预测了相关的靶基因。利用wego、david和string软件分析靶基因的功能和蛋白质-蛋白质相互作用。最后，基于string分析建立了相互作用图和相关信号通路。我们的结果表明，ESCs、PGCs和SSCs中共有9657个lncRNAs，其中3549个被定义为差异显著。我们筛选出20个候选lncRNAs，每组（ESCs与PGCs、ESCs与SSCs、PGCs与SSCs）之间的|logFC|值差异均大于8倍，或在单个细胞类型中特异性表达。qRT-PCR结果表明，候选lncRNAs的表达趋势与RNA-Seq一致。预测了15个顺式和4个反式靶基因。基于wego和string分析，我们发现lnc-SSC1、lnc-SSC5、lnc-SSC2和lnc-ESC2对靶基因SUFU、EPHA3、KLF3、ARL3和TRIM8具有负调控作用，而SHH、NOTCH、TGF-β、cAMP/cGMP和JAK/STAT信号通路被激活，从而促使ESCs分化为SSCs。我们的研究结果初步揭示了鸡ESCs向SSCs分化过程中lncRNA相关的调控机制，填补了lncRNA在雄性生殖细胞分化方面的研究空白。我们的结果也为改进鸡ESCs向SSCs分化的体外诱导系统提供了基础信息。
