Effect of MiR-100-5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

Abstract

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR-100-5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR-100-5p was significantly inhibited in the receptive phase (RE) rather than in the pre-receptive phase (PE). Overexpression of miR-100-5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR-100-5p, HOXA1, was confirmed by 3'-UTR assays. Meanwhile, the product of HOXA1 mRNA RT-PCR increased in the RE more than that in the PE. The HOXA1-siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR-100-5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ-9110 which acted as a sponge for miR-100-5p reversed the relevant biological effects of miR-100-5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ-9110/miR-100-5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR-100-5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR-100-5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR-100-5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ-9110/miR-100-5p/HOXA1 axis in vivo.

Keywords: HOXA1; Circ-9110; dairy goat; endometrial stromal cells; miR-100-5p.

FIGURE 1

MiR‐100‐5p inhibited ESCs proliferation and induced ESCs apoptosis in vitro. Note: (A) The miR‐100‐5p expression levels in endometrium PE and RE. (B) Efficiency of miR‐100‐5p transfection in the ESCs. (C) ESCs viability after miR‐100‐5p transfection (D) ESCs were transfected with NC inhibitor or miR‐100‐5p inhibitor, and cell proliferation was assessed using the cell counting kit‐8(CCK‐8) assay. (E) Cell proliferation indices were assessed after treatment with EdU. Scale bar = 100 μm. (F) ESCs were transfected with NC, miR‐100‐5p, NC inhibitor or miR‐100‐5p inhibitor, and the apoptosis analysis of ESCs was performed with FCM. (G) ESCs were transfected with NC, miR‐100‐5p, NC inhibitor or miR‐100‐5p inhibitor, and cell phases were analysed by FCM. (H) Caspase3, BCL2 and BAX protein levels in the ESCs that were transfected with NC, miR‐100‐5p, NC inhibitor or miR‐100‐5p inhibitor. Protein levels were normalized to β‐actin from the same lane. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01; * indicates that p < 0.05

FIGURE 2

HOXA1 promoted ESCs proliferation inhibited ESCs apoptosis in vitro. Note: (A) HOXA1 levels in endometrium PE and RE. (B) HOXA1 was expressed in various dairy goat tissues. (C) ESCs were transfected with pcDNA3.1, pcDNA3.1(+)‐HOXA1, NC or HOXA1‐siRNA, and cell viability was assessed using the cell counting kit‐8(CCK‐8) assay. (D) Cell proliferation indices were assessed after treatment with EdU. Scale bar = 100 μm. (E) ESCs were transfected with pcDNA3.1, pcDNA3.1(+)‐HOXA1, NC or HOXA1‐siRNA, and the apoptosis analysis of ESCs was performed with FCM. (F) ESCs were transfected with pcDNA3.1, pcDNA3.1(+)‐HOXA1, NC, or HOXA1‐siRNA, and cell phases were analysed by FCM. (G) The Caspase3, BCL2 and BAX protein levels in the ESCs that were transfected with pcDNA3.1, pcDNA3.1(+)‐HOXA1, NC or HOXA1‐siRNA. The protein levels were normalized to β‐actin from the same lane. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01; * indicates that p < 0.05

FIGURE 3

Circ‐9110 acted as a miRNA sponge and thereby decreased miR‐100‐5p levels in the ESCs Note: (A) Schematic diagram illustrating the design of luciferase reporters with the WTCirc‐9110 or site‐directed mutant (MUT‐Circ‐9110). The nucleotides in red represent the ‘seed sequence’ of miR‐100‐5p. (B) The luciferase reporter assay of 293T cells co‐transfected with WTCirc‐9110 or MUT‐Circ‐9110 and miR‐100‐5p mimic, NC, miR‐100‐5p inhibitor or NC inhibitor. (C) Circ‐9110 decreased miR‐100‐5p mRNA level in the ESCs. (D) The levels of Circ‐9110 after transfection with Circ‐9110 (pc2.1‐Circ‐9110) or Circ‐9110 siRNAs. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01; * indicates that p < 0.05

FIGURE 4

Circ‐9110 promoted ESCs proliferation inhibited ESCs apoptosis in vitro. Note: (A) Circ‐9110 levels in endometrium PE and RE. (B) ESCs were transfected with pc2.1, pc2.1‐Circ‐9110, pc2.1‐Circ‐9110+miR‐100‐5p, NC, siRNA1 or siRNA2, and cell viability was assessed using the cell counting kit‐8(CCK‐8) assay. (C, D) Cell proliferation indices were assessed after treatment with EdU. (E1) ESCs were transfected with pc2.1, pc2.1‐Circ‐9110 or pc2.1‐Circ‐9110+miR‐100‐5p, and the apoptosis analysis of ESCs was performed with FCM. (E2) ESCs were transfected with NC, siRNA1 or siRNA2, and the apoptosis analysis of ESCs was performed with FCM. (F1) ESCs were transfected with pc2.1, pc2.1‐Circ‐9110 or pc2.1‐Circ‐9110+miR‐100‐5p, and cell phases were analysed by FCM. (F2) ESCs were transfected with NC, siRNA1 or siRNA2, and the cell phases were analysed by FCM. (G1) Caspase3, BCL2 and BAX protein levels in ESCs transfected with pc2.1, pc2.1‐Circ‐9110 or pc2.1‐Circ‐9110+miR‐100‐5p. (G2) Caspase3, BCL2 and BAX protein levels in ESCs that were transfected with NC, siRNA1 or siRNA2. Protein levels were normalized to β‐actin. Scale bar = 100 μm. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01; * indicates that p < 0.05

FIGURE 5

miR‐100‐5p, HOXA1 and Circ‐9110 regulated the PI3K/AKT/mTOR and ERK1/2 signal pathways in the ESCs. Note: (A) p‐PI3K, PI3K, p‐AKT, AKT, p‐MTOR, MTOR, p‐ERK and ERK protein levels in the ESCs transfected with miR‐100‐5p mimic or miR‐100‐5p inhibitor. (B) p‐PI3K, PI3K, p‐AKT, AKT, p‐MTOR, MTOR, p‐ERK and ERK protein levels in the ESCs transfected with pcDNA3.1(+)‐HOXA1 or HOXA1‐siRNA. (C) p‐PI3K, PI3K, p‐AKT, AKT, p‐MTOR, MTOR, RAS, p‐ERK and ERK protein levels in the ESCs transfected with pc2.1, pc2.1‐Circ‐9110 or pc2.1‐Circ‐9110+miR‐100‐5p. (D) p‐PI3K, PI3K, p‐AKT, AKT, p‐MTOR, MTOR, RAS, p‐ERK and ERK protein levels in the ESCs transfected with NC, siRNA1 or siRNA2. Protein levels were normalized to β‐actin. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01

FIGURE 6

MiR‐100‐5p reduced embryo implantation in vivo. Note: Surface morphology of the endometrium under a scanning electron microscope. (A) Mice were injected with the miR‐100‐5p agomir. Scale bar: 2 μm (B) Mice were injected with the agomir NC. Scale bar: 2 μm (C) Mice uterus was injected with miR‐100‐5p agomir and agomir NC at day 3 of pregnancy, and, observed on day 9. (D) Number of embryo implantation was significantly decreased in the uterine horn injected with miR‐100‐5p agomir. The values are shown as mean ± SEM for three individuals. ** indicates that p < 0.01