REPRODUCTIONRESEARCH miR-361-3p regulates FSH by targeting FSHB in a porcine anterior pituitary cell model Rui-Song Ye*, Meng Li*, Chao-Yun Li, Qi-En Qi, Ting Chen, Xiao Cheng, Song-Bo Wang, Gang Shu, Li-Na Wang, Xiao-Tong Zhu, Qing-Yan Jiang, Qian-Yun Xi and Yong-Liang Zhang Chinese National Engineering Research Center for Breeding Swine Industry, SCAU-Alltech Research Joint Alliance, Guandong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, China Correspondence should be addressed to Q-Y Xi or Y-L Zhang; Email: [email protected] or [email protected] *(R-S Ye and M Li contributed equally to this work) Abstract FSH plays an essential role in processes involved in human reproduction, including spermatogenesis and the ovarian cycle. While the transcriptional regulatory mechanisms underlying its synthesis and secretion have been extensively studied, little is known about its posttranscriptional regulation. A bioinformatics analysis from our group indicated that a microRNA (miRNA; miR-361-3p) could regulate FSH secretion by potentially targeting the FSHB subunit. Herein, we sought to confirm these findings by investigating the miR-361-3p-mediated regulation of FSH production in primary pig anterior pituitary cells. Gonadotropin-releasing hormone (GnRH) treatment resulted in an increase in FSHB synthesis at both the mRNA, protein/hormone level, along with a significant decrease in miR-361-3p and its precursor (pre-miR-361) levels in time- and dose-dependent manner. Using the Dual-Luciferase Assay, we confirmed that miR-361-3p directly targets FSHB. Additionally, overexpression of miR-361-3p using mimics significantly decreased the FSHB production at both the mRNA and protein levels, with a reduction in both protein synthesis and secretion. Conversely, both synthesis and secretion were significantly increased following miR-361-3p blockade. To confirm that miR-361-3p targets FSHB, we designed FSH-targeted siRNAs, and co-transfected anterior pituitary cells with both the siRNA and miR-361-3p inhibitors. Our results indicated that the siRNA blocked the miR-361-3p inhibitor-mediated upregulation of FSH, while no significant effect on non-target expression. Taken together, our results demonstrate that miR-361-3p negatively regulates FSH synthesis and secretion by targeting FSHB, which provides more functional evidence that a miRNA is involved in the direct regulation of FSH. Reproduction (2017) 153 341–349 Introduction microRNAs (miRNAs) are small non-coding RNAs (−22 nt) that regulate gene expression at the posttranscription The gonadotropin FSH, which is secreted by the level by suppressing translation or degrading their target anterior pituitary gland, plays an essential role in animal mRNA (Ambros 2004, Karginov et al. 2010). The role of reproduction: it promotes antral follicle development miRNAs in pituitary hormone regulation is increasingly in females and stimulates spermatogenesis in the evident, for example, miR-26b was shown to be Sertoli cells in males (Bernard et al. 2010). FSH is a involved in growth hormone regulation by targeting heterodimeric glycoprotein composed of a unique β LEF-1 (Zhang et al. 2010), while miR-375 was found subunit (FSHB) and a common subunit ( -GSU) that α α to regulate pituitary pro-opiomelanocortin (POMC) is shared by chorionic gonadotropin (CG), LH and expression (Zhang et al. 2013). To date, several studies TSH. Many aspects of the transcriptional regulation have reported either direct or indirect role for miRNAs in of FSH synthesis and secretion have been revealed in the regulation of gonadotropins. Gonadotrope-specific recent decades. For example, FSH is mainly under the deletion of Dicer, an endoribonuclease responsible control of GnRH and TGF- signaling (Wurmbach et al. β for miRNA processing, results in severe suppression 2001, Thackray et al. 2010). Recently, adiponectin was of gonadotropin production and fertility defects shown to play an important role in FSH secretion during (Wang et al. 2014). Let-7b/c was found to enhance the estrous cycle (Kiezun et al. 2014). However, little the stability of the common subunit -GSU indirectly is known about the posttranscriptional regulation of α through downregulation of KSRP (Repetto et al. 2012). FSH production. © 2017 Society for Reproduction and Fertility DOI: 10.1530/REP-16-0373 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/24/2021 05:36:48AM via free access 10.1530/REP-16-0373 342 R-S Ye, M Li and others miR-200b/miR-429 was found to promote LH secretion 100 IU/mL penicillin, 100 μg/mL streptomycin and 2 mg/mL by targeting ZEB1 (Hasuwa et al. 2013), while it was BSA. The glands were then pooled and minced to produce a reported that miR-325-3p inhibits LH synthesis and suspension in the same medium and centrifuged for 10 min at secretion by targeting LHβ (Nemoto et al. 2012). 2000 rpm. After removal of the supernatant, the pellet containing A recent study showed that miR-132/212 is involved the tissue fragments was incubated at 37°C in DMEM/F12 in GnRH-induced FSH expression by targeting SIRT1 containing 0.25% trypsin-EDTA (Life Technologies) and 0.25% and subsequently reducing the FOXO1-mediated collagenase type α in a flask with constant stirring for 30 min. inhibition of FSHB transcription (Lannes et al. 2015). The enzyme-digested pituitary suspension was then centrifuged In addition, Dang et al. showed that decrease of miR- at 2000 rpm for 5 min, the supernatant was discarded and the cell pellet was resuspended in DMEM/F12 supplemented with 22-3p is associated with FSH increase in the plasma antibiotics and BSA, filtered through a 75- m nylon mesh to of Han Chinese patients with premature ovarian μ remove undigested tissue and cell aggregates, and centrifuged et al. failure (Dang 2015). However, it is still unknown at 2000 rpm for 10 min. The supernatant was discarded and whether miRNAs can directly regulate FSH synthesis or the cell pellet was washed twice in DMEM/F12 supplemented secretion by targeting FSHB. miR-361-3p, an X-linked with antibiotics and BSA and suspended at 3 × 105 live cells/mL miRNA, is detected in reproductive organs, including in DMEM/F12 supplemented with antibiotics and 10% FBS testis (Wu et al. 2014), ovary (Sontakke et al. 2014) (Life Technologies). Finally, six well plates were seeded with and exosome of follicular fluid (Sohel et al. 2013). 2 mL/well of the cell suspension and cultured at 37°C in a Additionally, miR-361-3p was frequently differentially humidified, 5% CO2 atmosphere. expressed in multiple reproduction processes including GnRH induction of FSH secretion (Ye et al. 2013), fetal ovarian development change (Veiga-Lopez et al. 2013) GnRH treatment and atretic follicle (Sontakke et al. 2014), which are When 80–90% confluent, the pig anterior pituitary cells closely linked to FSH regulation. Most importantly, were serum-starved for 6 h in DMEM/F12 supplemented a bioinformatics study from our group predicted that with antibiotics and BSA, and then incubated for 12 h or miR-361-3p may be involved in FSH secretion by 24 h in fresh starvation medium supplemented with 100 nM targeting FSHB mRNA (Ye et al. 2013). Accordingly, in GnRH (Okada et al. 2003, Yuen et al. 2009, Ye et al. 2013) the present study, we hypothesized that miR-361-3p (Gonadoliberin I; AnaSpec, Fremont, CA, USA). For the dose- regulates FSH by targeting FSHB, and examined the dependent assay (10, 25, 100 nM), pituitary cell were treated relationship between miR-361-3p and FSHB 3ʹ-UTR for 12 h. using the luciferase reporter assay, and investigated intracellular FSHB synthesis and extracellular FSH Anterior pituitary tissues collection secretion following overexpression or knockdown of miR-361-3p in pig anterior pituitary cells. Moreover, the Eight anterior pituitary tissues were collected from eight male miR-361-3p binding specificity to the target FSHB was pigs (13–16 weeks). In brief, pituitary glands were removed and further confirmed by co-transfection with miR-361-3p the anterior lobe was immediately dissected from each pituitary inhibitors and siRNAs. gland. Eight anterior pituitary glands were immediately frozen in liquid nitrogen until RNA extraction. Total RNAs were used to quantify FSHB mRNA and miR-361-3p expression by qRT- Materials and methods PCR analysis. Animals The animal experiments were conducted in accordance with the FSHB mRNA 3ʹ-UTR plasmid construction Review of Welfare and Ethics of Laboratory Animals guidelines Based on the miR-361-3p seed sequence, wild-type (WT) approved by the Guangdong Province Administration Office or seed-mutated (MT) and seed-deleted (DT) FSHB 3ʹ-UTR of Laboratory Animals, and all experiments were conducted sequences, where the binding site (CTGGGG) of miR-361-3p as described in the animal protocol (SCAU-AEC-2010-0416) was mutated or deleted, respectively, were chemically approved by the Institutional Animal Care and Use Committee synthesized (Sangon, Shanghai, China). The sequences are as of South China Agricultural University. follows: WT sense oligo, 5ʹ-CTAGAGGAGGAGCTCCAGGAA TGCAGAGTGCTGGGGCCTCAGTCCTATCACCACTCGTT-3ʹ; Primary culture of porcine anterior pituitary cells antisense, 5ʹ-AACGAGTGGTGATAGGACTGAGGCCCCAGCA CTCTGCATTCCTGGAGCTCCTCCT-3ʹ; MT sense oligo, 5ʹ-CTA Six healthy 7-day-old male piglets (Landrace) were killed, and GAGGAGGAGCTCCAGGAATGCAGAGTGgatatcCCTCAGTC the primary anterior pituitary cell culture