Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University , Changchun, China.
Guangdong Provincial Key Laboratory of Animal Nutritional Regulation, National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Laboratory of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University , Guangzhou, China.
RNA Biol. 2020 Dec;17(12):1754-1766. doi: 10.1080/15476286.2020.1778295. Epub 2020 Jun 19.
Growth hormone (GH), whose synthesis and release are mainly regulated by intracellular signals mediated by growth hormone-releasing hormone receptor (GHRHR), is one of the major pituitary hormones and critical regulators of organism growth, metabolism, and immunoregulation. Pig GHRHR splice variants (SVs) may activate different signalling pathways via the variable C-terminal by alternative splicing, and SVs have the potential to change microRNA (miRNA) binding sites. In this study, we first confirmed the existence of pig GHRHR SVs (i.e., GHRHR, GHRHR SV1 and SV2) and demonstrated the inhibitory effects of critical pituitary miRNAs (i.e., let-7e and miR-328-5p) on GH synthesis and cell proliferation of primary pituitary cells. The SVs of targeted by let-7e and miR-328-5p were predicted via bioinformatics analysis and verified by performing dual-luciferase reporter assays and detecting the expression of target transcripts. The differential responses of let-7e, and miR-328-5p to GH-releasing hormone and the changes in signalling pathways mediated by GHRHR suggested that let-7e and miR-328-5p were involved in GH synthesis mediated by GHRHR SVs, indicating that the two miRNAs played different roles by different ways. Finally, results showed that the protein coded by the transcript regulated GH through the NO/NOS signalling pathway, whereas that coded by SV1 and SV2 regulated GH through the PKA/CREB signalling pathway, which was confirmed by the changes in signalling pathways after transfecting the expression vectors of SVs to GH3 cells. To the best of our knowledge, this paper is the first to report pituitary miRNAs regulate GH synthesis by targeting the different SVs of .
生长激素（GH）的合成和释放主要受生长激素释放激素受体（GHRHR）介导的细胞内信号调节，是主要的垂体激素之一，是调节机体生长、代谢和免疫调节的关键因素。猪 GHRHR 剪接变异体（SVs）可能通过可变 C 端的不同拼接激活不同的信号通路，并且 SVs 有可能改变 microRNA（miRNA）结合位点。在本研究中，我们首先证实了猪 GHRHR SVs（即 GHRHR、GHRHR SV1 和 SV2）的存在，并证明了关键垂体 miRNA（即 let-7e 和 miR-328-5p）对 GH 合成和原代垂体细胞增殖的抑制作用。通过生物信息学分析预测了 let-7e 和 miR-328-5p 的靶 SVs，并通过双荧光素酶报告基因检测和靶转录本的表达检测进行了验证。let-7e 和 miR-328-5p 对 GH 释放激素的不同反应以及 GHRHR 介导的信号通路的变化表明，let-7e 和 miR-328-5p 参与了 GHRHR SVs 介导的 GH 合成，表明这两种 miRNA 通过不同的方式发挥不同的作用。最后，结果表明，转录本编码的蛋白质通过 NO/NOS 信号通路调节 GH，而 SV1 和 SV2 编码的蛋白质通过 PKA/CREB 信号通路调节 GH，这通过转染 GH3 细胞的 SVs 表达载体后信号通路的变化得到了证实。据我们所知，本文首次报道了垂体 miRNA 通过靶向 GHRHR 的不同 SVs 调节 GH 合成。
