In Vitro Analysis of LPS-Induced miRNA Differences in Bovine Endometrial Cells and Study of Related Pathways

Abstract

Lipopolysaccharide (LPS) is one of the main factors inducing endometritis in dairy cows. However, the specific pathogenesis of LPS-induced endometritis in dairy cows is not fully understood. The objective of this study was to establish an in vitro endometritis model using LPS-induced bovine endometrial epithelial (BEND) cells. BEND cells were treated with LPS of different concentrations and times. The cell-counting kit-8 (CCK-8) was used to detect the cell survival rate after LPS treatment, and quantitative real-time PCR (RT-qPCR) was used to detect the expression of control group and LPS-treated group of inflammatory factors interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). The results showed that the survival rate of endometrial epithelial cells stimulated by 5 μg/mL LPS for 6 h was 75.13%, and the expression of inflammatory factors was significantly increased. Therefore, 5 μg/mL LPS for 6 h could be selected as a suitable model for the study of inflammation. In addition, miRNA sequencing and target gene prediction was performed on normal and LPS-treated BEND cells. Among twenty-one differentially expressed miRNAs, six miRNAs were selected and their expression levels were detected by RT-qPCR, which were consistent with the sequencing results. Twenty-one differentially expressed miRNAs collectively predicted 17,050 target genes. This study provides a theoretical basis for further investigation of the pathogenesis of endometritis.