Figure 4.

MEG3 regulates cell autophagy during OGD/R through miR-181c-5p/ATG7.

HT22 cells were transfected or co-transfected with MEG3 overexpression plasmid (MEG3), miR-181c-5p mimic, and control (pcDNA3.1 + NC mimic). Then, the cells were reoxygenated for 48 hours after OGD treatment for 6 hours. pcDNA3.1 + NC mimic group: cells were co-transfected with empty plasmid (pcDNA3.1) and NC mimic; MEG3: cells were transfected with MEG3 overexpression plasmid; miR-181c-5p mimic group: cells were transfected with miR-181c-5p mimic; MEG3+ miR-181c-5p group: cells were co-transfected with MEG3 overexpression plasmid and miR-181c-5p mimic. (A, B) The levels of autophagy-specific proteins ATG7, Beclin1, and LC3II/I were examined by western blotting. The target protein expression was normalized to the pcDNA3.1 + mimic group. (C, D) MDC staining was used to measure cell autophagy (arrows) (scale bar: 100 μm; original magnification 200×). After transfection with miR-181c-5p mimic, cell autophagy was significantly decreased. Overexpression of miR-181c-5p reversed the effect of MEG3 overexpression on autophagy. (E) Cell viability was measured by the CCK8 assay. All data are representative of three independent experiments (mean ± SD). *P < 0.05, **P < 0.01, ***P < 0.001, vs. pcDNA3.1 + mimic group; #P < 0.05, ###P < 0.001, vs. MEG3 group (one-way analysis of variance followed by the least significant difference post hoc test). ATG7: Autophagy-related gene 7; CCK8: Cell Counting Kit-8; LC3: light chain 3; MDC: monodansylcadaverine; MEG3: maternally expressed gene 3; OD: optical density; OGD/R: oxygen and glucose deprivation/reoxygenation.