Fig. 5: Histamine increases miR-206 and miR-216b to repress hypoxia-induced autophagy.

a H9c2 cells were treated with 10 μM histamine. After 12 h, cells were harvested to determine the level change of miRNAs using RT-PCR. b H9c2 cells were transfected with miR-206 and miR-216b antagomirs together (miR-206/216b Anta) or negative controls (Anta-NC) for 8 h and then treated with 10 μM histamine under hypoxia for 12 h. LC3 and p62 were analyzed by western blotting at 3d after surgery. Representative photos are shown. c H9c2 cells were transfected with GFP-LC3 accompanied by miR-206 and miR-216b antagomirs (miR-206/216b Anta) or negative controls (Anta-NC). After 8 h, cells were treated with 10 μM histamine under hypoxia for 12 h. The percentage of cells with GFP-LC3 puncta was quantified. d H9c2 cells were transfected as b. Quantitative analysis of apoptosis detected by TUNEL assay is shown. e Cells were treated with 10 μM Histamine with 1 μM H1R antagonist (pyrilamine) or 50 μM H2R antagonist (cimetidine) for 12 h. The levels of miR-206 and miR-216b were determined by RT-PCR. f H9c2 cells were treated by 10 μM histamine alone or with 1 μM pyrilamine under hypoxia. After 12 h, miR-206 and miR-216b mimics mixture (miR-206/216b mimics) were transfected into cells for another 12 h. LC3 and p62 were analyzed by western blotting. Representative photos of LC3 and p62 immunoblot are shown at the above panels. Quantitative analysis of apoptosis detected by TUNEL assay is shown at the below panels. For detecting autophagy flux assay, GFP-LC3 plasmids were transfected into cells for 8 h prior to other treatment. The percentage of cells with GFP-LC3 puncta was quantified as the middle panel. *P < 0.05; **P < 0.01