| ObjectiveTo explore the effect and possible mechanisms of miRNA-221-3p on mast cell secretion of IL-4MethodAfter stimulation with lipopolysaccharide (LPS) on P815, miRNA-221-3p (abbreviated as miR-221 below) expression was detected by RT-PCR. P815 cells then were divided into blank control group, miR-221 high expression group (transfected with lentiviral vector), miR-221 silencing group (transfected with inhibitor) and Lip2000 group. ELISA were used to measure supernatant concentration of IL-4 on different miR-221 expression levels.By luciferase reporter gene, gene chip technology and Western Blot technology, the target gene of miRNA-221 and signaling pathway involved in increased mast cell secretion of IL-4 were preliminary predicted. For further verification, target gene expression was increased and the pathway was inhibited to observe changes in the expression level of IL-4 in miR-221 high expression group.ResultsIn LPS stimulated P815, miR-221 expression increased. IL-4 level in the miR-221 high expression group was higher than that in the control group, with statistical significance. IL-4 level in miR-221 silencing group was lower than that in the control group (P< 0.05).Bioinformatics analysis, luciferase reporter gene and Western Blot technology confirmed that PTEN is the target genes of miR-221. Compared to miR-221 high expression group, the concentration of IL-4 in miR-221+PTEN high expression group supernatant was reduced (P< 0.05). In miR-221 high expression group, the gene chip result suggested Toll like receptors (TLR) singaling pathway expression increased, and Western Blot confirmed the phosphate-P38 protein levels was higher than in normal group.The concertration of IL-4 in miR-221+P38 signal pathway inhibitor group was reduced (P< 0.05).ConclusionmiR-221 can stimulate mast cells secret of IL-4, inhibition of miR-221 can decrease IL-4 secretion. The PTEN and phosphorylation P38 protein play a role in regulation of miR-221 on P815 IL-4 secretion. |
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