Endometrial Epithelial Cell Apoptosis Is Inhibited by a ciR8073-miR181a-Neurotensis Pathway during Embryo Implantation

Abstract

Development of the receptive endometrium (RE) from the pre-receptive endometrium (PE) is essential for embryo implantation, but its molecular mechanisms have not been fully understood. In this study, lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA networks were constructed to explore the functions of potential competing endogenous RNAs (ceRNA) during the development of RE in dairy goats. We observed that circRNA8073 (ciR8073) decreased the levels of miR-181a by acting as a miRNA sponge. This effect indirectly increased the expression of neurotensin in endometrial epithelial cells (EECs). Neurotensin then inhibited EEC apoptosis by increasing the expression of BCL-2/BAX in favor of BCL-2 via the MAPK pathway and also induced increased expression of leukemia-inhibitory factor, cyclo-oxygenase 2, vascular endothelial growth factor A, and homeobox A10. We have thus identified a ciR8073-miR181a-neurotensin pathway in the endometrium of dairy goats. Through this pathway, ciR8073 functions as a ceRNA that sequesters miR-181a, thereby protecting neurotensin transcripts from miR-181a-mediated suppression in EECs.

Keywords: EECs; ceRNA; ciR8073-miR181a-NTS; dairy goats; endometrial epithelial cells; receptive endometrium.

Figure 1

The ceRNA Network Centered on miR-181a and ciR8073 in Endometrium of Dairy Goats (A) The differentially expressed targets of miR-181a; (B) the ceRNA network centered on ciR8073. Red hexagon, ciR8073; yellow diamond, NTS; green triangle, miRNAs; red circle, circRNAs; blue circle, lncRNAs; pink circle, mRNAs.

Figure 2

miR-181a Was Significantly Decreased in RE Compared with PE (A) The miR-181a was expressed in various tissues of dairy goats; (B) the miR-181a levels in endometrium in PE and RE. The miR-181a levels were measured by stem-loop qRT-PCR and normalized to U6; the values are shown as means ± SD (n = 3); **p < 0.01, *p < 0.05. (C) The NC for in situ hybridization; the miR-181a in the endometrium in PE (D) and RE (E) were detected by in situ hybridization. UC, uterine cavity; LE, luminal epithelium; GE, glandular epithelium; SC, stroma cell. Original magnification ×200; scale bars, 100 μm.

Figure 3

NTS Was Significantly Increased in RE Compared with PE (A) The NTS was expressed in various tissues of dairy goats; (B) the NTS levels in endometrium in PE and RE. The NTS levels were measured by qRT-PCR and normalized to GAPDH; the values are shown as means ± SD (n = 3). **p < 0.01, *p < 0.05. (C and D) The NC for immunohistochemical staining in PE (C) and RE (D); NTS levels were detected by immunohistochemical staining in the endometrium in PE (E) and RE (F). LE, luminal epithelium; GE, glandular epithelium; SC, stroma cell. Original magnification ×100; scale bars, 50 μm.

Figure 4

E2 and P4 Regulated the Expression Levels of miR-181a and NTS in EECs (A) The levels of miR-181a; (B) the levels of NTS mRNA. The values are shown as means ± SD (n = 3).

Figure 5

miR-181a Downregulated the Expression Level of NTS via the 3′ UTR (A) Schematic diagram illustrating the design of luciferase reporters with the WT-NTS 3′ UTR (Wt-NTS) or the site-directed mutant NTS 3′ UTR (Mu-NTS). The nucleotides in red represent the “seed sequence” of miR-181a; the mutation nucleotides are in gray. (B) The NTS-3′ UTR luciferase reporter vectors were co-transfected with miR-181a mimic (or negative control) into 293T cells; luciferase assay was performed 24 h after transfection. (C) miR-181a downregulated the NTS mRNA levels in EEC; NTS mRNA levels were measured by qRT-PCR and normalized to GAPDH. (D) NTS protein levels in EECS were measured by WB, and the densitometry was normalized to the β-actin density from the same lane. Each experiment was repeated three times in triplicate; the results are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

Figure 6

lncR915 Was Not a Target of miR-181a in EECs (A) Schematic diagram illustrating the design of luciferase reporters with the Wt-lncR915 or the site-directed mutant (Mu-lncR915). The nucleotides in red represent the “seed sequence” of miR-181a, and the mutation nucleotides are in gray. (B) The lncR915 luciferase reporter vectors were co-transfected with miR-181a mimic (or negative control) into 293T cells; luciferase assay was performed 24 h after transfection. (C) miR-181a did not regulate the lncR915 levels in EECs; lncR915 levels were measured by qRT-PCR and normalized to GAPDH. (D) lncR915 did not regulate the miR-181a levels in EECs. (E) lncR915 did not regulate the NTS mRNA levels in EECs. Each experiment was repeated three times in triplicate, and the results are represented as mean ± SD (n = 3).

Figure 7

Decreased Levels of miR-181a Served as a miRNA Sponge in EECs (A) Schematic diagram illustrating the design of luciferase reporters with the Wt-ciR8073 or the site-directed mutant (Mu-ciR8073). The nucleotides in red represent the “seed sequence” of miR-181a, and the mutation nucleotides are in gray. (B) The ciR8073 or its mutation luciferase reporter vectors were co-transfected with miR-181a mimic (or negative control) into 293T cells; luciferase assay was performed 24 h after transfection. (C) miR-181a decreased the ciR8073 levels in EECs; the levels were measured by qRT-PCR and normalized to GAPDH. (D) The miR-181a levels in EECs were overexpressed or mutational ciR8073 (Mu-ciR8073). (E) The NTS mRNA levels in EECs were overexpressed or mutational ciR8073 (Mu-ciR8073). (F) The NTS mRNA levels in EECs were overexpressed ciR8073 and miR-181a. (G) NTS protein levels in EECs were overexpressed ciR8073 and miR-181a; the densitometry was normalized to the β-actin density from the same lane. Each experiment was repeated three times in triplicate. *p < 0.05, **p < 0.01.

Figure 8

miR-181a Promoted EECs Apoptosis In Vitro (A) The effect of miR-181a on the proliferation of EECs that were measured by MTT. Values were shown as means ± SD (n = 7). The cell cycle (B) and apoptosis (C) analysis of EECs were detected with FCM. (D) The levels of BCL-2, BAX, MAPK pathway proteins, and endometrial receptivity markers (LIF, COX2, VEGFA, HOXA10, and OPN) were measured by WB after the EECs were treated with miR-181a. Densitometry was normalized to the β-actin density from the same lane; **p < 0.01, *p < 0.05.

Figure 9

NTS Inhibited EECs Apoptosis In Vitro Overexpressed NTS markedly promoted proliferation of EECs (A), did not change the cell cycle (B), and inhibited EEC apoptosis (C). siRNA-NTS significantly inhibited proliferation of EECs (D), did not change the cell cycle (E), and dramatically induced EEC apoptosis (F). Values were showed as means ± SD (n = 7). The cell cycle (B and E) and apoptosis (C and F) analysis of EECs were detected with FCM. **p < 0.01, *p < 0.05.

Figure 10

NTS Increased the Expression Levels of BCL-2/BAX and Increased LIF, COX2, VEGFA, and HOXA10 in EECs In Vitro The levels of apoptosis-related proteins (A) (BCL-2, BAX, Caspase-8, Caspase-3, FAS, SP1, and PTN), MAPK pathway proteins (B), and endometrial receptivity markers (C) (LIF, COX2, VEGFA, HOXA10, and OPN) were measured by WB when the NTS was overexpressed in EECs. Densitometry was normalized to the β-actin density from the same lane. **p < 0.01, *p < 0.05.

Figure 11

Proposed Network of ciR8073-miR181a-NTS in the Endometrium of Dairy Goats ciR8073 regulates EECs by functioning as a ceRNA, which sequesters miR-181a, thereby relieving its repressive effect on NTS.