Pituitary miRNAs target GHRHR splice variants to regulate GH synthesis by mediating different intracellular signalling pathways.

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.

Key pituitary miRNAs mediate the expression of pig GHRHR splice variants by regulating splice factors.

The growth hormone releasing hormone receptor (GHRHR) is well documented in organism growth and its alternative splicing may generate multiple functional GHRHR splice variants (SVs). Our previous study has demonstrated the key pituitary miRNAs (let-7e and miR-328-5p) in pig regulated the expression of GHRHR SVs by directly targeting to them. And according to recent reports, the interplay between miRNA-based silencing of mRNAs and alternative splicing of pre-mRNAs is a crucial post-transcriptional mechanism. In this study, SF3B3 and CPSF4 were firstly excavated as the splice factors that involved in the formation of GHRHR SVs mediated by let-7e and miR-328-5p through the comparation of the expression relations of GHRHR SVs, let-7e/miR-328-5p and SF3B3/CPSF4 in pituitary tissues between Landrace pigs and BaMa pigs, as well as the prediction of the target relations of let-7e/miR-328-5p with SF3B3 and/or CPSF4. SF3B3 and CPSF4 targeted by let-7e and miR-328-5p were further verified by performing dual-luciferase reporter assays and detecting the expression of target transcripts. Then the RT-PCR, RT-qPCR and Western blot assays were used to confirm SF3B3 and CPSF4 were involved in the formation of the GHRHR SVs, and in this process, let-7e and miR-328-5p mediated GHRHR SVs by regulating SF3B3 and CPSF4. Finally, the target site of SF3B3 on pre-GHRHR was on the Exon 12 to Exon14, while CPSF4 acted on the other fragments of the pre-GHRHR, which were explored by dual-luciferase reporter system preliminarily. To the best of our knowledge, this paper is the first to report the miRNAs regulate GHRHR SVs indirectly by splice factors.