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. 2022 Feb 16;2022:8603427. doi: 10.1155/2022/8603427

Figure 4.

Figure 4

miR-155-5p in CPE-derived EVs inhibits Rheb to promote neuronal autophagy and apoptosis. (a) Target genes of miR-155-5p were predicted by analyzing the miRDB, TargetScan, and RNA22 databases. (b) The binding sites of miR-155-5p and Rheb in humans, rats, and mice were obtained from the TargetScan analysis. (c) Crosstalk between miR-155-5p and Rheb was determined by luciferase assay. (d) Relative miR-155-5p level in cocultured cell models was determined by RT-qPCR. (e) Phosphorylation levels of mTOR, S6, and 4E-BP1 in the cell model after transfection were confirmed by western blotting. (f) Expressions of autophagy-related proteins in the cell model after transfection and coculture were measured by western blotting. (g) Activity of neurons treated with OGD/R after transfection and coculture was measured by CCK-8 assay. (h) Levels of total caspase3 and cleaved-caspase3 proteins in the cell model after coculture were detected by western blotting. (i) The number of TUNEL+ neurons exposed to OGD/R after coculture was determined by TUNEL staining. ∗p < 0.05 vs. mimic-NC+Rheb-3′ UTR-WT cotreated neurons, mimic-NC-transfected neurons, controls, oe-NC-transfected neurons or oe-NC+PBS-cotreated neurons. #p < 0.05 vs. neurons cotreated with inhibitor-NC or oe-Rheb+EV and mimic-NC (n = 3).