Kinetics Of MiR-146a Expression In Lipopolysaccharide-induced Alveolar Macrophages And Its Relationship With TNF-a

Objective:To determine kinetics of miR-146a(microRNA-146a) and TNF-a expression in lipopolysaccharide(LPS)-induced alveolar macrophages(AM) at different times and their relations, exploring regulative effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods:NR8383 alveolar macrophages seeded at 1×106cell/mL in a 6 well plate, and stimulated with 1μg/mL of LPS after 90min. Cells were harvested and supernatant were collected at 0,3,6 or 12 h after incubation. The expression of miR-146a and TNF-a mRNA of cells were detected by Real-time qPCR and the production of TNF-a protein in the supernatant of cells were assayed by enzyme-linked immunosorbent assay (ELISA). Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-a mRNA.Results:1. The expression of miR-146a increased continuously at 6h or 12h after LPS challenge (6h:5.33±0.81 fold,12h:8.21±1.19 fold, P<0.01), and it showed an upward tendency;2. The expression of TNF-a mRNA peaked at 3h after LPS challenge (67.48±24.52 fold,P<0.01), and then decreased gradually;3. The production of TNF-a in the supernatant of cell is significantly increased at 3h after LPS challenge(359.80±57.54pg/mL, P< 0.01), and peaked at 12h(729.22±50.40pg/mL, P<0.01);4. The expression levels of miR-146a are negatively correlated with TNF-a(r=-0.895, P<0.01).Conclusion:The kinetics of miR-146a and TNF-a expression in LPS-induced AM were the unique expression pattern and miR-146a inversely correlated with TNF-αmRNA, predicting miR-146a may be involved in the regulation of the NR8383 alveolar macrophage inflammatory response.