Abstract: Objective To investigate the role of miR-21 in airway immunologic dysfunction induced by cold air irritation.
Methods Immortalized human airway epithelial cell lines BEAS-2B and 16HBE cells were cultured in air-liquid phases. The
differential expressions of endogenous miR-21, miR-164, and miR-155 in the cells induced by cold air exposure for different
time were detected by real-time PCR. The reporter plasmid containing wild-type or mutated 3’UTR of TLR-4 were constructed
and co-transfected into BEAS-2B cells or 16HBE cells together with miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, or
miR-21 inhibitor control. Following the transfection, dual luciferase reporter assay was performed to verify the action of
miR-21 on TLR-4. miR-21 mimic, miR-21 mimic control, miR-21 inhibitor, and miR-21 inhibitor control were transfected via
lipofectamine 2000 in BEAS-2B or 16HBE cells that were subsequently exposed to a temperature at 37 ℃ or cold irritation
(30 ℃), and the protein levels of TLR-4/MyD88 were detected by Western blotting. Results Cold irritation caused a timedependent
up-regulation of miR-21 in both BEAS-2B and 16HBE cells (P<0.05) without obviously affecting the expressions of
miR-164 and miR-155. Dual luciferase reporter assay demonstrated a direct combination of miR-21 and its target protein
TLR-4. The synthesis levels of TLR-4/MyD88 protein were decreased in miR-21 mimic group even at a routine culture
temperature (P<0.05), as also seen in cells with cold irritation (P<0.05). Treatment with the miR-21 inhibitor partially attenuated
cold irritation-induced down-regulation of TLR-4/MyD88 protein (P<0.05). Conclusion Cold air irritation-induced airway
immunologic dysfunction is probably associated with TLR-4/MyD88 down-regulation by an increased endogenic miR-21.