ABSTRACT
OBJECTIVE: To observe the effect of transfected microRNA-146a (miR-146a) on expression of tumor necrosis factor-alpha ( TNF-alpha ) in alveolar macrophages, and to analyze its regulatory mechanism in the inflammatory response of alveolar macrophages. METHODS: Cy.11113 labeled with 25, 50, 100 nmol/L of Pre-miR", respectively, were transfected into rat alveolar macrophages NR8383 cultured in vitro. The highest transfection efficiency was selected to he the experimental concentration. NR8383 cells were divided into two groups: transfected group was tranfected with 50 nmol/L Pre-miR1111 miR-146a precursor, and control group with SO nmol/L Cym3 labeled Pre-miR1" as the negative control. The mRNA expression of miR-146a of cells was detected hy real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Then cells were stimulated with lipopolysaccharide (LPS, 1 mg/L) for 6 hours. The production of TNF-alpha protein in the supernatant of cells was assayed by enzyme-linked immunosorhent assay (ELISA) , and the expression of TNF-alpha mRNA of cells was detected hy RT-qPCR. RESULTS: Transfection rate was highest in the 50 nmol/L Cy11113 labeled Pre-miR"' cells, and it reached 80%. Compared with control group (set at 1), the expression of miR-146a increased by (24.55 ±6.14) folds in transfected alveolar marrophages (P<0.01). After the cells were stimulated with LPS, the production of TNF-alpha protein (ng/L) in the supernatant of cell was decreased from 616.6 ± 42.3 to 211.5 ± 30.4 (P<0.01), and the expression of TNF-alpha mRNA was decreased by (47 ± 6) % (P<0.05) in transfected alveolar macrophages when compared with that of the control. CONCLUSIONS: Transfection alveolar macrophages with miR-146a precursors could down-regulate the expression of TNF-alpha. It is therefore suggests that up-regulation of miR-146a can inhibit inflammatory responses as induced hy LPS, in alveolar macrophages.
