Effects of swine microRNA mimics on lipopolysaccharide (LPS) induced inflammatory changes in 3D4/21 cells. (5th December 2022)

• Record Type: Journal Article
• Authors: Swain, Trevon; Chavez, Chris; Myers, Michael J.
• Is Part Of: Research in veterinary science. Volume 150(2022)
• Page Start: 115
• Page End: 121
• Publication Date: 2022-12-05
• Subjects: Inflammation -- miRNA mimics -- Swine; Veterinary medicine -- Periodicals; Veterinary Medicine -- Periodicals; Médecine vétérinaire -- Périodiques; Médecine vétérinaire -- Recherche -- Périodiques; Diergeneeskunde; 636.089
• DOI: 10.1016/j.rvsc.2022.06.017
• Languages: English
• ISSNs: 0034-5288

Abstract

Abstract: There have been limited studies focused on validation of swine microRNAs (miRNA) with mRNA targets. The objective of this study was to validate a defined set of targets using artificial miRNA mimics transfected into cell lines to confirm specific targets of endogenous miRNAs after administration of Escherichia coli lipopolysaccharide (LPS). Sixteen hours after mimic transfection of 3D4/21 cell lines, the cells were stimulated with 1 μg/ml LPS or phosphate-buffered saline (PBS). The cells were harvested and collected at 0, 1, 3, and 8 h post administration. The selected genes DAD1, IL8, and ESR, which are involved in known pathways of inflammation. and are predicted or validated human targets of either miR-146a, let-7a, or miR-22-3p. These were then evaluated by quantitative real-time-PCR (qRT-PCR) to verify microRNA-mRNA interaction in swine. Using the ROX reference dye, mRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization against the PBS control group. DAD1 and ESR1 were negatively regulated by miR-22-3p and miR-146a-5p, respectively in 3D4/21 cells after LPS stimulation. However, miR-146a-5p may play an indirect positive regulatory role of both DAD1 and IL8 mRNA expression. Furthermore, we found an inverse relationship between LPS stimulation compared with the let-7a-5p overexpression with DAD1. Our inflammation study provides new evidence on the roles and predicted targets of miR-146a, let-7a, and miR-22-3p in swine. Highlights: The swine macrophage cell line 3D4/21 were treated with lipopolysaccharide and artificial miRNA to validate a subset of swine miRNAs. miR-146a, let-7a, and miR-22-3p were evaluated for their ability to regulate DAD1, IL8, and ESR mRNA expression DAD1 and ESR1 were negatively regulated by miR-22-3p and miR-146a-5p respectively. miR-146a-5p appears to play an indirect positive regulatory role of both DAD1 and IL8 mRNA expression. There is an inverse relationship between lipopolysaccharide stimulation compared with the let-7a-5p overexpression with DAD1.