Dataset Information

Roles of differential expression of microRNA-21-3p and microRNA-433 in FSH regulation in rat anterior pituitary cells.

ABSTRACT: Follicle-stimulating hormone (FSH) secreted by adenohypophyseal cells plays an important role in the regulation of reproduction, but whether microRNAs (miRNAs) regulate the secretion of FSH remains unclear. In the present study, we predicted and screened miRNAs that might act on the follicle-stimulating hormone beta-subunit (FSHb) gene of rats using the TargetScan program and luciferase reporter assays, and the results identified two miRNAs, miR-21-3p and miR-433. We then transfected these miRNAs into rat anterior adenohypophyseal cells and assessed the FSHb expression levels in and FSH secretion by the transfected cells through quantitative PCR and ELISA. The results showed that both miR-21-3p and miR-433 down-regulated the expression levels of FSHb and resulted in the decrease of the secretion of FSH compared with the control group, and treatment with miR-21-3p and miR-433 inhibitors up-regulated the expression levels of FSHb and resulted in the increase of the secretion of FSH. Taken together, our results indicate that miR-21-3p and miR-433 can down-regulate the expression of FSHb by directly targeting the FSHb 3'UTR in rat primary pituitary cells. Our findings provide evidence that miRNAs can regulate FSHb expression and further affect the secretion of FSH and might contribute to the use of miRNAs for the regulation of animal reproduction.

SUBMITTER: Han DX

PROVIDER: S-EPMC5482676 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Roles of differential expression of microRNA-21-3p and microRNA-433 in FSH regulation in rat anterior pituitary cells.

Han Dong-Xu DX Sun Xu-Lei XL Xu Ming-Qiang MQ Chen Cheng-Zhen CZ Jiang Hao H Gao Yan Y Yuan Bao B Zhang Jia-Bao JB

Oncotarget 20170501 22

Follicle-stimulating hormone (FSH) secreted by adenohypophyseal cells plays an important role in the regulation of reproduction, but whether microRNAs (miRNAs) regulate the secretion of FSH remains unclear. In the present study, we predicted and screened miRNAs that might act on the follicle-stimulating hormone beta-subunit (FSHb) gene of rats using the TargetScan program and luciferase reporter assays, and the results identified two miRNAs, miR-21-3p and miR-433. We then transfected these miRNA ...[more]

PMID: 28402262

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Project description:Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3'-untranslated region (3'-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSH? mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSH? mRNA 3'-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.

Project description:Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/?-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.

Dataset Information

Roles of differential expression of microRNA-21-3p and microRNA-433 in FSH regulation in rat anterior pituitary cells.

Publications

Roles of differential expression of microRNA-21-3p and microRNA-433 in FSH regulation in rat anterior pituitary cells.

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