Ye Rui-Song, Li Meng, Li Chao-Yun, Qi Qi-En, Chen Ting, Cheng Xiao, Wang Song-Bo, Shu Gang, Wang Li-Na, Zhu Xiao-Tong, Jiang Qing-Yan, Xi Qian-Yun, Zhang Yong-Liang
Chinese National Engineering Research Center for Breeding Swine IndustrySCAU-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.
Chinese National Engineering Research Center for Breeding Swine IndustrySCAU-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
Reproduction. 2017 Mar;153(3):341-349. doi: 10.1530/REP-16-0373. Epub 2016 Dec 20.
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.
促卵泡生成素（FSH）在人类生殖过程中发挥着重要作用，包括精子发生和卵巢周期。虽然其合成和分泌的转录调控机制已得到广泛研究，但其转录后调控却知之甚少。我们团队的一项生物信息学分析表明，一种微小RNA（miRNA；miR-361-3p）可能通过靶向FSHB亚基来调节FSH分泌。在此，我们试图通过研究miR-361-3p对原代猪垂体前叶细胞中FSH产生的调节作用来证实这些发现。促性腺激素释放激素（GnRH）处理导致FSHB在mRNA、蛋白质/激素水平上的合成增加，同时miR-361-3p及其前体（pre-miR-361）水平呈时间和剂量依赖性显著下降。使用双荧光素酶测定法，我们证实miR-361-3p直接靶向FSHB。此外，使用模拟物过表达miR-361-3p在mRNA和蛋白质水平上均显著降低了FSHB的产生，蛋白质合成和分泌均减少。相反，在阻断miR-361-3p后，合成和分泌均显著增加。为了证实miR-361-3p靶向FSHB，我们设计了靶向FSH的小干扰RNA（siRNA），并将垂体前叶细胞与siRNA和miR-361-3p抑制剂共转染。我们的结果表明，siRNA阻断了miR-361-3p抑制剂介导的FSH上调，而对非靶向表达无显著影响。综上所述，我们的结果表明miR-361-3p通过靶向FSHB对FSH合成和分泌起负调控作用，这为miRNA参与FSH的直接调节提供了更多功能证据。
