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Jinglin Zhang, Jie Gao, Di Zhang, Hui Liu, Kemian Gou, and Sheng Cui

Prolactin (PRL) is a pituitary hormone that regulates multiple physiological processes. However, the mechanisms of PLR synthesis have not been fully elucidated. The aims of the present study were to study the functions and the related mechanisms of miR-375 regulating PRL synthesis. We initially found that miR-375 mainly expressed in the lactotrophs of mouse pituitary gland. To identify the function of miR-375 in the pituitary gland, the miR-375 knockout mice were generated by using Crispr/Cas9 technique. The results showed that miR-375 knockout resulted in the decline of pituitary PRL mRNA and protein levels by 75.7% and 60.4% respectively, and the serum PRL level reduced about 46.1%, but had no significant effect on FSH, LH and TSH. Further, we identified that Estrogen receptor 1 (alpha) (Esr1) was a downstream molecule of miR-375. The real-time PCR and western blot results showed that ESR1 mRNA and protein levels markedly decreased by 40.9% and 42.9% in the miR-375 knockout mouse pituitary, and these were subsequently confirmed by the in vitro study using transfections of miR-375 mimics and inhibitors in pituitary lactotroph GH4 cells. Further, Rasd1 was predicted by bioinformatic tools and proved to be the direct target of miR-375 in lactotrophs using dual-luciferase reporter assay. Rasd1-siRNA transfection results revealed the negative effect of Rasd1 in regulating ESR1. Collectively, the results presented here demonstrate that miR-375 positively modulates PRL synthesis through Rasd1 and Esr1, which are crucial for understanding the regulating mechanisms of pituitary hormone synthesis.

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Xin Li, Hongjiao Li, Di Zhang, Guojin Xu, Jinglin Zhang, and Sheng Cui

MicroRNA-7 (miR-7) is an important modulator of a plenty of gene expressions and the interrelated biological processes, highly expressed in porcine pituitary. Norepinephrine (NE), acting as an important neurotransmitter or/and a hormone secreted excessively under stress, affects the synthesis and secretion of various hormones, including pituitary follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are the key hormones which regulate sexual maturation and reproductive functions. However, the relationship among NE, miR-7 and gonadotropin needs to be elucidated. The aim of this study was to identify whether miR-7 involved in the NE-adrenoceptor signaling pathway affects the synthesis and secretion of FSH and LH in porcine pituitary. Our results showed that the NE intracerebroventricular injection increased pituitary miR-7 level and the synthesis and secretion of FSH and LH in porcine, whereas the inhibition of either endogenous miR-7 or β-adrenergic receptors hindered the rise of FSH and LH synthesis induced by NE in cultured primary porcine anterior pituitary cells. Further, we identified the molecular type of β-adrenergic receptors and the signaling pathway in porcine pituitary, and we found that NE played its roles relying on adrenoceptor beta 2 (β2AR) and the RAF/MEK/ERK1/2 signaling pathway. The phosphorylation of ERK1/2 upregulated miR-7 level which subsequently enhanced FSH and LH synthesis by targeting to Golgi glycoprotein 1 (GLG1). These suggest that miR-7 mediates NE’s effect on promoting FSH and LH synthesis in porcine pituitary.