Department of Animal Science, Tibet Agricultural and Animal Husbandry College, Nyingzhi, Tibet 860000, China.
Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Sciences and Technology, China Agricultural University, Haidian, Beijing 100193, China.
Gene. 2018 Jul 15;663:88-100. doi: 10.1016/j.gene.2018.04.036. Epub 2018 Apr 14.
To explore the expression profile of the cellular miRNAs in bovine ovarian granulosa cells responding to transforming growth factor-β1 (TGF-β1), the effect of TGF-β1 on cell proliferation was firstly investigated by CCK-8 method and the results showed that there was a significant inhibitory effect on bovine granulosa cell proliferation treated with 5/10 ng/mL human recombinant TGF-β1 for 24 h compared to the control (P < 0.05). Then, we performed high-throughput sequencing of two small RNA libraries prepared from cultured bovine granulosa cells stimulated with or without 10 ng/mL human recombinant TGF-β1. A total of 13,257,248 and 138,726,391 clean reads per library were obtained from TGF-β1 and control groups, respectively. There were 498 and 499 bovine-specific exist miRNAs (exist miRNAs), 627 and 570 conserved known miRNAs (known miRNAs), and 593 and 585 predicted novel miRNAs in TGF-β1 and control groups, respectively. A total of 78 miRNAs with significant differential expression, including 39 up-regulated miRNAs and 39 down-regulated miRNAs were identified in the TGF-β1 group compared with the control. Real-time quantitative PCR analyses of bta-miR-106a and bta-miR-1434-5p showed that their up-expressions were interrupted by SB431542, an inhibitor that blocks TGFβ1/Smad signaling, which supported the sequencing data. GO analysis showed involvement of the predicted genes of the differentially expressed miRNAs in a broad spectrum of cell biological processes, cell components, and molecular functions. KEGG pathway analysis of the predicted miRNA targets further indicated that these differentially expressed miRNAs are involved in various signaling pathways, such as Wnt, MAPK, and TGF-β signaling, which might be involved in follicular development. These results provide valuable information on the composition, expression, and function of miRNAs in bovine granulosa cells responding to TGF-β1, and will aid in understanding the molecular mechanisms of TGF-β1 in granulosa cells.
为了探索转化生长因子-β1（TGF-β1）作用于牛卵巢颗粒细胞时细胞微小 RNA（miRNA）的表达谱，首先用 CCK-8 法研究了 TGF-β1 对细胞增殖的影响，结果表明，与对照组相比，5/10ng/ml 人重组 TGF-β1 处理 24h 后对牛颗粒细胞增殖有显著抑制作用（P<0.05）。然后，我们对用或不用 10ng/ml 人重组 TGF-β1 刺激培养的牛颗粒细胞进行了两个小 RNA 文库的高通量测序。每个文库分别从 TGF-β1 组和对照组获得了 13257248 条和 138726391 条清洁reads。TGF-β1 组和对照组分别有 498 个和 499 个牛特异存在 miRNA（exist miRNAs）、627 个和 570 个保守已知 miRNA（known miRNAs）以及 593 个和 585 个预测新 miRNA。与对照组相比，TGF-β1 组有 78 个 miRNA 表达差异显著，其中 39 个上调，39 个下调。实时定量 PCR 分析显示，TGF-β1/Smad 信号通路阻断剂 SB431542 可阻断 bta-miR-106a 和 bta-miR-1434-5p 的上调表达，支持测序数据。GO 分析显示，差异表达 miRNA 的预测基因参与了广泛的细胞生物学过程、细胞成分和分子功能。差异表达 miRNA 靶基因的 KEGG 通路分析进一步表明，这些差异表达 miRNA 参与了各种信号通路，如 Wnt、MAPK 和 TGF-β 信号通路，这些信号通路可能参与了卵泡发育。这些结果为 TGF-β1 作用于牛颗粒细胞时 miRNA 的组成、表达和功能提供了有价值的信息，并将有助于理解 TGF-β1 在颗粒细胞中的分子机制。
