Inflammatory intensity and stimulus type induce distinct miRNA regulatory programs in bovine endometrial epithelial...

Endometrial immune homeostasis is essential for uterine recovery and fertility in postpartum dairy cows, whereas dysregulated inflammation can lead to cytological endometritis (CE) and impaired reproductive performance. Although microRNAs (miRNAs) regulate inflammatory responses, how inflammatory intensity and origin shape miRNA programs in the bovine endometrium remains unclear. In this study, bovine endometrial epithelial cells (BEECs) were exposed to graded inflammatory stimuli, including low- and high-dose lipopolysaccharide (LPS; 10 and 100 μg/mL) and interleukin-1β (IL-1β; 100 ng/mL), followed by small RNA sequencing. A total of 407 miRNAs were retained, among which 16 showed dose-dependent patterns and were classified as early-responsive, threshold-induced, or high-intensity-associated repression groups. Nine miRNAs were early-responsive, whereas six were activated only under high-intensity stimulation. Notably, bta-miR-12053 showed repression under high-dose LPS. In total, 24 inflammation-responsive miRNAs were identified, including 13 with consistent regulation between LPS and IL-1β, and 5 and 6 showing LPS- and IL-1β-biased responses. Integration with in vivo datasets showed selective and time-dependent recapitulation of inflammation-responsive miRNAs, with bta-miR-155 elevated at both day 7 and day 21 and bta-miR-146a elevated at day 21. Predicted targets of miRNAs upregulated under stronger inflammatory stimulation showed enrichment toward downregulated transcripts in CE and were associated with pathways including regulation of the actin cytoskeleton, MAPK signaling, cell adhesion, chemokine signaling, NF-κB signaling, and mTOR signaling. High-intensity-associated miRNAs were enriched toward the disease-associated end of in vivo profiles. Collectively, inflammatory intensity drives shifts in miRNA programs, while stimulus origin shapes specificity. These miRNAs may link excessive inflammation to impaired endometrial immune homeostasis and help identify cows at risk of CE.