| Prolactin is an important reproductive hormone secreted by the pituitary gland that promotes mammary gland development and growth,stimulates and maintains lactation,and plays a key role in the reproductive process of animal growth and development.Prolactin is also a gonadotropin with the function of promoting luteinization.Also,prolactin plays an important regulatory role in improving axonal myelin formation,fetal neurogenesis,promoting the formation of lung surface active substances,and immune tolerance during pregnancy.Prolactin is currently used clinically for screening of male infertility and hyperprolactinemia,and as an aid in the diagnosis of pituitary tumors,hypothalamic lesions,and hyperaldosteronism.Prolactin secretion is regulated by a variety of hormones and growth factors,mainly dopamine,thyrotropin-releasing hormone(TRH)and epidermal growth factor.Among them,TRH,as a prolactin-releasing factor,can influence the transcription factor PREB of prolactin to regulate prolactin gene expression,and also induce prolactin secretion through several different signaling pathways cascade regulation.However,there is still a lack of evidence whether TRH can regulate PRL synthesis and secretion at the post-transcriptional level.It has been found that miRNAs are involved in a variety of physiological processes in animals,such as hormone secretion,immune regulation and organogenesis.The pituitary gland is the most important endocrine organ in living organisms,and miRNAs have been shown to play a key role in the regulation of pituitary hormones.However,there is a lack of systematic studies on the regulatory role of TRH on the pituitary transcriptome,and the role of miRNAs in TRH regulation of PRL synthesis and secretion lacks experimental support.Therefore,resolving the role of miRNAs in the TRH-induced PRL release process is important for exploring the molecular mechanism of PRL synthesis.In this study,we first investigated the changes of PRL synthesis and secretion in rat pituitary gland after TRH administration,and m RNA sequencing was performed on TRH-treated cells.Based on the constructed differential gene expression profiles,bioinformatics analysis was performed to screen the genes involved in the regulation of PRL by TRH.To elucidate how miRNAs are involved in the regulation of PRL by TRH,Targetscan,Miranda and RNAhybrid native software were used to jointly predict miRNAs that might target the 3’UTR of PRL m RNA.miRNAs that might act on PRL genes were screened by a dual luciferase reporter system,followed by cell transfection,RT-q PCR and WB experiments were performed to verify the role of miRNAs in the regulation of PRL synthesis and secretion by TRH.The results showed that:1.We successfully constructed differential gene expression profiles before and after TRH treatment,and screened 590 differentially expressed genes,of which 102 genes were up-regulated and 488 genes were down-regulated.2.The KEGG signaling pathway enrichment analysis,GO enrichment analysis and protein interaction analysis of the differentially expressed genes revealed that TRH regulates PRL secretion by multiple pathways.3.miR-126a-5p in GH3 cells and rat pituitary cells could inhibit the expression of prolactin gene and thus affect prolactin secretion.4.TRH can promote prolactin synthesis and secretion by reducing the expression of miR-126a-5p and attenuating its inhibitory effect on PRL m RNA expression.In this study,we constructed differential gene expression profiles before and after TRH treatment,and screened genes that can regulate prolactin synthesis and secretion at the transcriptional level,providing data to support the molecular mechanism of TRH in the regulation of pituitary function.The role of miRNAs in the regulation of prolactin synthesis and secretion by TRH was analyzed,and the molecular mechanism of PRL secretion by TRH was complemented at the epigenetic level,which enriched the pathways of PRL regulation by TRH. |
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