西藏牛和三江牛大脑组织中低氧适应相关circRNAs的比较分析

doi:10.11843/j.issn.0366-6964.2020.03.009

摘要/Abstract

摘要： 旨在从circRNA层面加深哺乳动物对高原低氧环境适应性的认识，探索西藏牛和三江牛大脑组织中差异表达的circRNA及相关调控网络，为研究circRNA在西藏牛低氧适应性方面的分子调控机制奠定理论基础。本研究分别采集3头4.5岁健康、雌性西藏牛和三江牛的大脑组织作为试验样本，构建cDNA文库进行高通量测序，利用生物信息学方法对宿主基因进行GO和KEGG分析，预测差异表达circRNA下游靶向基因，构建circRNA-miRNA和circRNA-miRNA-mRNA可视化调控网络，辅以RNaseR酶抗性检测和实时荧光定量PCR（qRT-PCR）验证测序数据的可靠性。结果，在西藏牛和三江牛的6个样本中，共筛选获得858个显著差异的circRNAs，其中上调表达394个，下调表达464个。其宿主基因共参与49个功能亚类，注释到31条显著富集的信号通路（P<0.05），主要涉及MAPK信号通路、谷氨酸能突触、Rap1信号通路、磷脂酶D信号通路及cGMP-PKG信号通路等生物学过程。靶基因预测结果显示，350个上调和364个下调差异表达circRNAs分别靶向结合492和508个miRNAs。其中，miRNA靶点数最多的是novel_circ_017825和novel_circ_046762，说明这两个circRNAs可能在西藏牛脑功能调控方面发挥重要作用。circRNA-bta-miR-2284z-mRNA调控网络展示了bta-miR-2284z与circRNA、mRNA间的靶向关系，推测上述circRNAs可能通过充当bta-miR-2284z海绵的方式，间接影响西藏牛脑组织中相关靶向mRNA的表达水平。随机挑选6个circRNA进行RNaseR酶抗性试验和qRT-PCR验证，表达趋势与测序结果一致，说明所获得的circRNA为环状转录本。本研究利用高通量测序技术获得了西藏牛和三江牛大脑组织中circRNA的表达谱及信号通路，并初步构建了circRNA-miRNA-mRNA网络互作模型，为后续进一步探索circRNA参与西藏牛低氧适应性的生物学过程和分子功能，研究circRNA在大型哺乳动物低氧适应性方面的调控机制提供了可靠的数据支持。

关键词: 西藏牛, 三江牛, 低氧适应性, 转录组, circRNA

Abstract: The aim of this study was to enhance the understanding of mammals' adaptability to high altitude hypoxia environment at the circRNA level and explore the differentially expressed circRNA and related regulatory networks in brain of Zang cattle and Sanjiang cattle, which would lay the theoretical foundation for further researching the molecular regulation mechanism of circRNA in hypoxia adaptation in Zang cattle. In this study, the brain tissues of three 4.5-year-old female healthy Zang cattles and Sanjiang cattles were sampled to construct the cDNA libraries for high-throughput sequencing. Bioinformatics methods were used to analyze host genes of circRNA by GO and KEGG, and predict the target genes of differentially expressed circRNA. At the same time, the circRNA-miRNA and circRNA-miRNA-mRNA visualization regulatory networks were constructed. Moreover, the reliability of sequence data was verified by RNaseR enzyme resistance assay and real-time quantitative PCR (qRT-PCR). The results showed that 858 significantly differentially expressed circRNAs were filtered in 6 samples of Zang cattle and Sanjiang cattle, included 394 up-regulated circRNAs and 464 down-regulated circRNAs. The host genes of these differentially expressed circRNAs were involved in 49 functional subclasses, involved in 31 significantly enriched signaling pathways (P<0.05), mainly included MAPK signaling pathway, glutamatergic synapse, Rap1 signaling pathway, phospholipase D signaling pathway, cGMP-PKG signaling pathway, etc.. The result of target gene prediction revealed that 350 up-regulated and 364 down-regulated differentially expressed circRNAs were able to target 492 and 508 miRNAs, respectively. Among them, the novel_circ_017825 (235) and novel_circ_046762 (231) had the most target points to miRNAs, indicated that novel_circ_017825 and novel_circ_046762 might play an important role in the function regulation of Zang cattle brain. The circRNA-bta-miR-2284z-mRNA regulatory network showed the targeting relationship between bta-miR-2284z and circRNA, mRNA. It was speculated that the above circRNAs might indirectly affect the related targeted mRNA expression levels in Zang cattle brain by acting as bta-miR-2284z sponge. In addition, the RNaseR enzyme resistance test and qRT-PCR were performed on the randomly selected 6 circRNAs. The expression trend was consistent with the sequence results, which indicated that these obtained circRNAs were the circular transcript.The expression profile and signaling pathways of circRNA in brain tissues of Zang cattle and Sanjiang cattle were obtained by high-throughput sequencing technology, and the interaction model of circRNA-miRNA-mRNA network was initially constructed, which provided reliable data for further researching the biological process and molecular function of circRNA in Zang cattle hypoxia adaptability, as well as the regulation mechanism of circRNA in hypoxia adaptability in large mammals.

Key words: Zang cattle, Sanjiang cattle, hypoxia adaptability, transcriptome, circRNA

中图分类号:

S823.2

邓磊, 柴志欣, 王嘉博, 王会, 王吉坤, 武志娟, 信金伟, 钟金城, 姬秋梅. 西藏牛和三江牛大脑组织中低氧适应相关circRNAs的比较分析[J]. 畜牧兽医学报, 2020, 51(3): 490-504.

DENG Lei, CHAI Zhixin, WANG Jiabo, WANG Hui, WANG Jikun, WU Zhijuan, XIN Jinwei, ZHONG Jincheng, JI Qiumei. Comparative Analysis of Hypoxia-Adapted circRNAs in Brain Tissues of Zang Cattle and Sanjiang Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 490-504.

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