Zhou Zuyang, Li Kunyu, Liu Jiannan, Zhang Hui, Fan Yekai, Chen Yulin, Han Haiyin, Yang Junqi, Liu Yufang
College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan, China.
School of Landscape and Ecological Engineering, Hebei University of Engineering, Handan, China.
Front Vet Sci. 2022 Apr 18;9:833946. doi: 10.3389/fvets.2022.833946. eCollection 2022.
The growth and development of skeletal muscle is a physiological process regulated by a variety of genes and signaling pathways. As a posttranscriptional regulatory factor, circRNA plays a certain regulatory role in the development of animal skeletal muscle in the form of a miRNA sponge. However, the role of circRNAs in muscle development and growth in goats is still unclear. In our study, apparent differences in muscle fibers in Wu'an goats of different ages was firstly detected by hematoxylin-eosin (HE) staining, the circRNA expression profiles of muscles from 1-month-old (mon1) and 9-month-old (mon9) goats were screened by RNA-seq and verified by RT-qPCR. The host genes of differentially expressed (DE) circRNAs were predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG) of host genes with DE circRNAs were performed to explore the functions of circRNAs. The circRNA-miRNA-mRNA networks were then constructed using Cytoscape software. Ten significantly differentially expressed circRNAs were also verified in the mon1 and mon9 groups by RT-qPCR. Luciferase Reporter Assay was used to verify the binding site between circRNA and its targeted miRNA. The results showed that a total of 686 DE circRNAs were identified between the mon9 and mon1 groups, of which 357 were upregulated and 329 were downregulated. Subsequently, the 467 host genes of DE circRNAs were predicted using Find_circ and CIRI software. The circRNA-miRNA-mRNA network contained 201 circRNAs, 85 miRNAs, and 581 mRNAs; the host mRNAs were associated with "muscle fiber development" and "AMPK signaling pathway" and were enriched in the FoxO signaling pathway. Competing endogenous RNA (ceRNA) network analysis showed that novel_circ_0005314, novel_circ_0005319, novel_circ_0009256, novel_circ_0009845, novel_circ_0005934 and novel_circ_0000134 may play important roles in skeletal muscle growth and development between the mon9 and mon1 groups. Luciferase Reporter Assay confirmed the combination between novel_circ_0005319 and chi-miR-199a-5p, novel_circ_0005934 and chi-miR-450-3p and novel_circ_0000134 and chi-miR-655. Our results provide specific information related to goat muscle development and a reference for the goat circRNA profile.
骨骼肌的生长发育是一个受多种基因和信号通路调控的生理过程。环状RNA（circRNA）作为一种转录后调控因子，以微小RNA（miRNA）海绵的形式在动物骨骼肌发育中发挥一定的调控作用。然而，circRNA在山羊肌肉发育和生长中的作用仍不清楚。在我们的研究中，首先通过苏木精-伊红（HE）染色检测了不同年龄武安山羊肌肉纤维的明显差异，通过RNA测序筛选了1月龄（mon1）和9月龄（mon9）山羊肌肉的circRNA表达谱，并通过逆转录定量聚合酶链反应（RT-qPCR）进行了验证。预测了差异表达（DE）circRNA的宿主基因，并对含有DE circRNA的宿主基因进行了基因本体（GO）分析和京都基因与基因组百科全书分析（KEGG），以探索circRNA的功能。然后使用Cytoscape软件构建circRNA-miRNA-mRNA网络。还通过RT-qPCR在mon1和mon9组中验证了10个显著差异表达的circRNA。使用荧光素酶报告基因检测法验证circRNA与其靶向miRNA之间的结合位点。结果显示，在mon9和mon1组之间共鉴定出686个DE circRNA，其中357个上调，329个下调。随后，使用Find_circ和CIRI软件预测了DE circRNA的467个宿主基因。circRNA-miRNA-mRNA网络包含201个circRNA、85个miRNA和581个mRNA；宿主mRNA与“肌纤维发育”和“AMPK信号通路”相关，并在FoxO信号通路中富集。竞争性内源RNA（ceRNA）网络分析表明，新型_circ_0005314、新型_circ_0005319、新型_circ_0009256、新型_circ_0009845、新型_circ_0005934和新型_circ_0000134可能在mon9和mon1组之间的骨骼肌生长发育中发挥重要作用。荧光素酶报告基因检测法证实了新型_circ_0005319与chi-miR-199a-5p、新型_circ_0005934与chi-miR-450-3p以及新型_circ_0000134与chi-miR-655之间的结合。我们的研究结果提供了与山羊肌肉发育相关的具体信息，并为山羊circRNA图谱提供了参考。
