The Novel Competing Endogenous Long Noncoding RNA SM2 Regulates Gonadotropin Secretion in the Hu Sheep Anterior Pituitary by Targeting the Oar-miR-16b/TGF-β/SMAD2 Signaling Pathway

Abstract

Pituitary gonadotropins play a pivotal role in reproduction. Long noncoding RNAs (lncRNAs) have been identified as important regulators in the hypothalamic−pituitary−ovarian (HPO) axis associated with reproduction. However, the contributions of lncRNAs to pituitary gonadotropin secretion remain largely unknown. Therefore, this work was performed to uncover the functional mechanisms of the novel lncRNA TCONS_00083279 (lncRNA SM2) and its potential targeting pathway oar-miR-16b/TGF-beta/SMAD2, which is associated with gonadotropin secretion in sheep pituitary cells. In the present study, the lncRNA SM2 showed high expression levels in the sheep pituitary gland, and it was located in both the nucleus and the cytoplasm of pituitary cells. lncRNA SM2 knockdown inhibited pituitary cell proliferation and FSH and LH secretion. The function of the lncRNA SM2 was sponged by oar-miR-16b, and this regulated the growth and gonadotropin secretion of pituitary cells by modulating SMAD2, as shown by the dual-luciferase reporter assay. FSH and LH levels were both upregulated by SMAD2 overexpression. Moreover, the levels of the lncRNA SM2, SMAD2 and TGFR1, as well as FSH and LH, in sheep pituitary cells increased significantly under gonadotropin-releasing hormone (GnRH) stimulation (p < 0.05). This work illustrates that the lncRNA SM2 regulates gonadotropin secretion in the Hu sheep anterior pituitary by targeting the oar-miR-16b/TGF-β/SMAD2 signaling pathway, providing a valuable resource for understanding the molecular mechanisms underlying sheep reproduction.

Keywords: TGF-β/SMAD2; gonadotropin; lncRNA SM2; oar-miR-16b; pituitary cells.

Figure 1

Characterization of a candidate transcript in Hu sheep. (A) The coding ability of lncRNA SM2 was predicted using Coding Potential Assessment Tool (CPAT). (B) Chromosomal localization of lncRNA SM2. (C,D) Expression of lncRNA SM2 in each group. (E) lncRNA SM2 localization in Hu sheep pituitary cells; scale bar, 50 μm. All data are shown as the mean ± SEM of at least three replicates. Different letters indicate significant differences between groups, p < 0.05.

Figure 2

LncSM2 knockdown inhibited the secretion of gonadotropins in pituitary cells. (A) Morphology of pituitary cells under a microscope. The left picture shows F1 generation pituitary cells, and the right picture shows F2 generation pituitary cells. (B) Identification of sheep pituitary cells by FSHβ and LHβ marker gene immunofluorescence staining. Scale bar, 100 μm. (C) mRNA expression of lncRNA SM2 in sheep pituitary cells treated with si-lncRNA SM2-2592, si-lncRNA SM2-3415 and si-lncRNA SM2-3794 was determined by qPCR. (D) FSH and LH secretion was detected by ELISA. (E,F) mRNA and protein expression of FSHβ and LHβ in pituitary cells in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 3

Effect of lncRNA SM2 on proliferation and apoptosis in pituitary cells. (A,B) Pituitary cells’ proliferation was assayed by EDU. Scale bars, 100 μm. (C,D) mRNA and protein expression of PCNA in pituitary cells in each group. (E,F) Pituitary cell apoptosis was detected by flow cytometry. (G,H) mRNA and protein expression of cell apoptosis-related genes in pituitary cells. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 4

lncRNA SM2 acts as a ceRNA and sponge to oar-miR-16b in the pituitary cells of Hu sheep. (A) RNAhybrid and miRanda predicted miRNA targets lncRNA SM2 and SMAD2. (B,C) Expression of oar-miR-16b, lncRNA SM2 and SMAD2 in each group by RT-qPCR. (D,E) SMAD2 protein level was decreased and increased in pituitary cells transfected with oar-miR-16b mimics and inhibitor by Western blot. (F,G) Schematic depicting the interactions of oar-miR-16b with wild-type lncRNA SM2 or SMAD2 and mutant lncRNA SM2 or SMAD2. The targeting relationship between lncRNA SM2 or SMAD2 and oar-miR-16b was detected by dual-luciferase reporter gene assay. (H,I) Expression of SMAD2 in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 5

Effect of oar-miR-16b level on pituitary cell proliferation and apoptosis. (A) Expression of oar-miR-16b in each group by RT-qPCR. (B,C) Pituitary cell proliferation was detected by EDU. Scale bars, 100 μm. (D) Protein expression of PCNA in pituitary cells. (E,F) Pituitary cell apoptosis was detected by flow cytometry. (G) Protein expression of cell apoptosis-related genes in pituitary cells. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 6

SMAD2 overexpression and knockdown affected the secretion of gonadotropins in pituitary cells. (A–D) mRNA and protein expression of SMAD2 in pituitary cells in each group. (E) FSH and LH secretion was detected by ELISA. (F,G) mRNA and protein expression of FSHβ and LHβ in pituitary cells in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 7

Effect of SMAD2 on proliferation and apoptosis in pituitary cells. (A,B) Pituitary cell proliferation was detected using EDU. Scale bars, 100μm. (C–E) mRNA and protein expression of PCNA in pituitary cells in each group. (F,G) Pituitary cell apoptosis was assayed by flow cytometry. (H,I) mRNA and protein expression of cell apoptosis-related genes in pituitary cells in each group. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).

Figure 8

lncRNA SM2 regulated the secretion of gonadotropins by TGFR1/SMAD2 signaling pathways in the pituitary cells of Hu sheep. (A) FSH and LH secretion was detected after stimulation with GnRH by ELISA. (B–D) mRNA expression of GnRHR, lncRNA SM2, SMAD2 and TGF family in pituitary cells in each group by RT-qPCR. (E) mRNA expression of TGFR1 in pituitary cells in each group by RT-qPCR. (F) Proposed model of lncRNA SM2 regulation of pituitary cell state in Hu sheep. All data are shown as the mean ± SEM of at least three replicates (* p < 0.05; ** p < 0.01).