Figure 5

Suppression of the autophagy in NRVMs via the miR-16-5p-mediated transcriptional suppression of SESN1. (a) Target/binding site of SESN1 and miR-16-5p, and Sesn1 mutation sequence. (b) Luciferase assay; SESN1 contains a target gene of mir-16-5p. Left side; Sesn1-3ʹUTR-wild type, Right side; Sesn1-3ʹUTR-mutant, Gray bar; miR-16-5p mimic (n = 4), Light gray bar; miR-mimic control (n = 4), (c) Representative blots of SESN1, phosphorylated-mTOR, mTOR, and β-actin protein expression in NRVMs. Left side; normoxia. Center; hypoxia. Right side; hypoxia with miR-16-5p mimic transfection. (d) Fold change of SESN1 activation in NRVMs under hypoxia with (n = 14) and without miR-16-5p mimic transfection (n = 12); the baseline refers to NRVMs under normoxia (n = 13). (e) Fold change of mTOR-phosphorylation in NRVMs under hypoxia with (n = 6) and without miR-16-5p mimic transfection (n = 5); the baseline refers to NRVMs under normoxia (n = 6). Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p transfection. (f) Representative images of autophagosomes in NRVMs (All; n = 4) [monodansylcadaverine (MDC); green]; the nuclei were stained with DAPI [blue]. Left side; normoxia. Center; hypoxia. Right side; hypoxia with miR-16-5p transfection. The upper panel is a low magnitude image. The lower panel is an expanded image of autophagosomes and DAPI-positive cells. Yellow arrow; representative autophagosome-positive cells. Scale bar = 50 μm. (g) The ratio of autophagosome positive NRVMs (All; n = 4). Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p transfection. The percentage of autophagosome positive NRVMs in all NRVMs is represented. Western blotting (h) and analysis of the protein expression of LC3B-II (i) in NRVMs (normo n = 3, the others; n = 4; respectively) transfected with miR-16-5p mimic was also evaluated. Left bar; normoxia. Center bar; hypoxia. Right bar; hypoxia with miR-16-5p mimic transfection.