ABSTRACT

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 GHRHR 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 GHRHR 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 GHRHR 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 GHRHR.

摘要

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，是机体生长、代谢和免疫调节的关键调节因子。猪 GHRHR 剪接变体 (SVs) 可以通过可变剪接通过可变 C 端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 SV（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 合成和细胞的抑制作用。初级垂体细胞增殖。GHRHR的 SV通过生物信息学分析预测 let-7e 和 miR-328-5p 靶向的基因，并通过执行双荧光素酶报告基因测定和检测目标转录物的表达进行验证。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，表明两种miRNA以不同的方式发挥了不同的作用。最后，结果表明GHRHR转录本编码的蛋白通过 NO/NOS 信号通路调控 GH，而 SV1 和 SV2 编码的蛋白通过 PKA/CREB ​​信号通路调控 GH，转染后信号通路的变化证实了这一点。GHRHR的表达载体SV 到 GH3 细胞。据我们所知，本文是第一个报道垂体 miRNA 通过靶向GHRHR的不同 SV 来调节 GH 合成的论文。