MicroRNA Bta-miR-24-3p Suppressed Galectin-9 Expression through TLR4/NF-ĸB Signaling Pathway in LPS-Stimulated Bovine Endometrial Epithelial Cells.

Ayodele Olaolu Oladejo,Xiaoyu Ma,Jie Yang,Shengyi Wang,Zuoting Yan,Xiaohu Wu,Wenxiang Shen,Wei Jiang,Yanan Lv,Bereket Habte Imam,Yajuan Li,Xuezhi Ding

doi:10.3390/cells10123299

Abstract

Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment.

Highlights

The inflammatory model was established by bovine endometrial epithelial cells (BEECs) stimulation with LPS for 24 h, and the mRNA expression level of bta-miR-24-3p and LGALS9 genes was evaluated
BEECs were stimulated with different concentrations of LPS and induced by 3 μg/mL of LPS at different times
LGALS9 expression was significantly increased, and bta-miR-24-3p was significantly downregulated; it affirmed that both LGALS9 gene and bta-miR-24-3p are incriminated immune-inflammatory responses during endometritis

Summary

Introduction

Endometritis is an infection of the postpartum uterus common in postpartum dairy cows. After resolution of clinical condition without treatment, endometritis lengthens the interval between parturition and the first insemination and delays conception, resulting in higher culling rates for reproductive insufficiency [3,4,5]. Many pregnancy failures in natural and assisted reproductive technology pregnancies can be linked to insufficient endometrial receptivity, which is described as the physiological condition of the uterus when fertilized embryo growth and implantation are possible for the maintenance of pregnancy [6]. The presence of subclinical endometritis in a higher incidence of repeated breeder cows (RBC) is the highest known risk factor for the infections, leading to a delayed resumption of postpartum ovarian cycles and insemination failure [5,7,8]. Previous studies have demonstrated that a cellular immune response in the endometrium, occurring during subclinical endometritis, might play a role in subfertility/infertility in RBC [9,10]

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