MEG3 knockdown inhibits autophagy and alleviates CI/RI.
The right ventricle of mice was transfected with si-MEG3 or si-NC, and then the CI/RI mouse model was established. Sham group: mice did not undergo MCAO procedure, but the other surgery was the same as that performed on the MCAO group; MCAO group: mice underwent MCAO surgery; MCAO+si-NC group: mice were injected with a mixture of si-NC and Lipofectamine RNAiMAX through the right cerebral ventricle, and then underwent the MCAO procedure; MCAO+si-MEG3 group: mice were injected with a mixture of MEG3 and Lipofectamine RNAiMAX through the right cerebral ventricle, and then underwent the MCAO procedure. (A, B) Brain tissue was stained with TTC, and the infarct volume (marked with yellow lines) was measured (n = 3 mice/group). (C, D) The expression of MEG3 and miR-181c-5p was quantified by qRT-PCR (n = 4 mice/group) and normalized to the Sham group. (E, F) The levels of autophagy-specific proteins ATG7, Beclin1, and LC3II/I were examined by western blotting (n = 4 mice/group). The target protein expression was normalized to the Sham group. (G, H) TUNEL assay was used to evaluate cell death in mouse brain tissues (scale bar: 3 μm; original magnification 400×). After CI/RI, cell death in the brain tissue was significantly increased and si-MEG3 injection reversed the cell death. Arrows indicate dead cells. Data are expressed as mean ± SD. **P < 0.01, ***P < 0.001, vs. Sham group; #P < 0.05, ##P < 0.01, vs. MCAO + si-NC group (one-way analysis of variance followed by the least significant difference post hoc test). ATG7: Autophagy-related gene 7; CI/RI: cerebral ischemia-reperfusion injury; LC3: light chain 3; MEG3: maternally expressed gene 3; qRT-PCR: quantitative real-time PCR; TTC: 2,3,5-triphenyltetrazolium chloride; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.