Effect And Mechanism Of The Key Pituitary MiRNAs On GHRHR Splice Variants Expression And GH Synthesis In Pig

Study on the regulation of growth and development of animal is a hotspot in animal husbandry,and the regulation of animal growth is finally realized through the neuroendocrine growth axis of hypothalamus-pituitary-target organs.The pituitary growth hormone(GH)is mainly secreted by the pituitary anterior,and its physiological function is very extensive,which can obviously promote the growth of bone,cartilage and other tissues,and is closely related to the growth,metabolism and immunity of animals.The GHRH/GHRHR-mediated GH synthesis and release is one of the important factors affecting the GH level in vivo.Therefore,there is great significance to study the factors mediating GHRHR expression when exploring the GH regulation.micro RNA(miRNA)is a class of non-coding single-stranded RNA molecules encoded by endogenous genes,which are involved in the regulation of post-transcriptional gene expression.A large number of studies have shown that miRNAs in the pituitary are closely related to the regulation of animal growth,while its regulation on GH is mostly limited to the miRNAs regulate its expression by targeting GH directly,and the indirect effects and the new pathways of the miRNAs regulate GH synthesis through its upstream receptor GHRHR have not been reported.Therefore,the aim of this study was to explore the deeper molecular mechanism of the key pituitary miRNAs influence the GH synthesis by regulating GHRHR.Three alternative splice variants(SVs)of the pig GHRHR gene(GHRHR,GHRHR SV1 and GHRHR SV2)are annotated in the NCBI database.In this study,firstly,the existence and expression of the three SVs of GHRHR were detected in the pituitary tissues of Landrace pigs and Bama Xiang pigs,which have significant different growth and development rates,and results showed that all the three transcripts were existent and the expression of GHRHR and GHRHR SV2 was significantly higher in Landrace pigs than that in Bama Xiang pigs(p<0.05).Then,based on the microarray results of miRNAs in pituitary tissues of Landrace and Bama Xiang pigs reported in the previous study and the bioinformatics analysis,let-7e and miR-328-5p were screen out with a high expression abundance,and with significant expression differences,as well as the potential to regulate GH synthesis by regulating the expression of different SVs of GHRHR,moreover,the expression of the two miRNAs in Bama Xiang pigs was significantly higher than that in Landrace pigs(p<0.05).The effect of miRNAs on the expression of GHRHR SVs and the dual-luciferase reporter system further clarified the regulation effects of let-7e and miR-328-5p on GHRHR SVs by directly targeting to them.Furthermore,study indicated that the interplay between miRNA-based gene silencing of m RNAs and alternative splicing of pre-m RNAs is a crucial post-transcriptional mechanism.Based on the RNA-seq results of Landrace and Ba Ma pigs,we screened splicing factors with differential expression and potential targeting relationships with let-7e and miR-328-5p,focusing on the two splicing factors,SF3B3 and CPSF4.Then the differential expression of the GHRHR SVs,SF3B3,CPSF4,let-7e and miR-328-5p were first confirmed in the pituitary tissues of the same batch Landrace and Ba Ma pigs,results indicated that SF3B3 and CPSF4 have the same trend with the expression difference of GHRHR SVs between the two pig breeds,while they exhibited opposite expression trend with the let-7e and miR-328-5p,suggesting the regulation relationships of the miRNAs and splice factors on the GHRHR SVs.At last,the effect of miRNAs on the expression of SF3B3 and CPSF4 and the dual-luciferase reporter system further clarified the targeted regulation effect of let-7e on CPSF4,and the targeted regulation of both SF3B3 and CPSF4 was observed by miR-328-5p.Based on let-7e and miR-328-5p that regulated its expression by directly targeting either SF3B3 or CPSF4.To explore whether let-7e and miR-328-5p mediate GHRHR SVs by regulating SF3B3 and CPSF4,this study first examined the effects of SF3B3 and CPSF4 on GHRHR SVs,showing that SF3B3 alters the expression ratio of different SVs during GHRHR SVs formation,while CPSF4 promotes the splicing of all SVs.Under this basis,we further examined the indirect effects of these two miRNA on GHRHR SVs through either SF3B3 or CPSF4.The results showed that the GHRHR reduced by SF3B3 overexpression was restored by the inhibitory effect of miR-328-5p on SF3B3,the increase of GHRHR SV1 or GHRHR SV2 by SF3B3 overexpression was partially offset by the inhibitory effect of miR-328-5p.A portion of GHRHR increased by CPSF4 overexpression was reduced by the inhibitory effects of miR-328-5p and let-7e,while GHRHR SV2 increased by CPSF4 overexpression was also decreased by the inhibitory effect of both miR-328-5p and let-7e on CPSF4.That is,miR-328-5p and let-7e were indirectly involved in the regulation of GHRHR SVs through the splicing factors SF3B3 and CPSF 4 involved in the alternative splicing of GHRHR SVs.To further clarify whether SF3B3 and CPSF4 are involved in the splicing of the three alternative splice variants of the GHRHR,a mini-GHRHR luciferase reporter vector containing the 3′-terminal exons(exons 12 to 14)and part of the intron was constructed according to the three transcripts of the GHRHR with the differences in the 3′ terminus.The dual luciferase activity detected by overexpression and knockdown of SF3B3 and CPSF4 indicated that only SF3B3 affected mini-GHRHR splicing,indicating the target site of SF3B3 on the pre-GHRHR is in exon 12 to exon 14,while CPSF4 does not act on this fragment.It has been reported that different splice variants of GHRHR may lead to the activation difference of the downstream signaling pathway,so the effect of let-7e and miR-328-5p on the downstream signaling pathway of GHRHR was further examined.According to our results,let-7e is involved in the NOS/NO signaling pathway,while miR-328-5p is involved in the PKA/CREB signaling pathway mostly.Based on let-7e and miR-328-5p could target different GHRHR SVs and it could affect the activation of different signaling pathways downstream of the GHRHR,we speculated that different SVs of GHRHR may activate different signaling pathways.Thus we examined the effects of the three SVs of GHRHR on the downstream signaling pathways,results showed that GHRHR mainly regulated GH through the NOS/NO signaling pathway,and GHRHR SV1 and SV regulated GH through the PKA/CREB signaling pathway.In conclusion,this study analyzed the role and molecular mechanism of the key pituitary miRNAs regulated GHRHR SVs by directly targeting them and indirectly regulated the GHRHR SVs by targeting splicing factors,which provided new data for elucidating the precise mechanism of pituitary miRNAs regulating animal growth.