Exosomal miR-320e through wnt2targeted inhibition of the Wnt/β-catenin pathway allevisate cerebral small vessel disease and cognitive impairment

Figure 1 Construction and bioinformatics analysis of differentially expressed miRNA libraries. A: Volcano diagram showing that miRNA-320e was significantly downregulated; B: High-throughput sequencing of plasma exosomes showed that the expression of miR-320e was significantly downregulated; C: Bioinformatics suggested the biological processes involved in differentially expressed miRNAs. Further information was shown in the Supplementary Material.

Figure 2 miR-320e could bind to Wnt2 to inhibit the Wnt/β-catenin pathway. A: The results of the dual-luciferase reporter gene experiment; B: Binding sequence between Wnt2 mRNA and miR-320e; C and D: Protein blots were listed in the column. After cotransfection of the miR-320e mimic, western blotting showed that Wnt2, FZD2, Axin2, GSK3β, and β-catenin expression increased compared with that of the control group. After cotransfection of the miR-320e inhibitor, western blotting showed that Wnt2, FZD2, Axin2, GSK3β, and β-catenin expression decreased compared with that of the control group; E: Cell image after cotransfection of cells. The image above showed transfection with miR-320e mimic and the one below showed transfection with miR-320e inhibitor. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 3 Representative protein blots of Wnt2, FZD2, Axin2, GSK3β, and β-catenin expression under hydrogen peroxide. A and B: The Western-Blot results showed that Wnt2 expression in the hydrogen peroxide group increased compared to that in the control group; C: The PCR results showed that Wnt2 expression in the hydrogen peroxide group increased compared to that in the control group; D-F: Expression of Wnt2, FZD2, Axin2, GSK3β, and β-catenin expression after treating with WIF1 and R-Spondin under hydrogen peroxide were similar and further enhanced, respectively, compared to that in the groups without hydrogen peroxide; G and H: The PCR results showed that GSK3β in the WIF + H₂O₂ group increased, and the other mRNA expression levels remained similar. PCR: Polymerase chain reaction. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 4 miR-320e inhibited the Wnt/β-catenin pathway through targeting Wnt2. A and B: Cell transfection images were shown; C-E: The Western-Blot results showed that the expression of Wnt2, FZD2, Axin2, GSK3β, and β-catenin in the miR-320e mimics group was inhibited compared to that in the control group, while this inhibition was effectively blocked by Wnt2 silencing compared to the siRNA control; F and G: The results of real-time PCR showed similar trends to the protein levels of Wnt2, FZD2, Axin2, GSK3β, and β-catenin with overexpressed miR-320e and knockdown of Wnt2. PCR: Polymerase chain reaction. ^aP < 0.05.

Figure 5 The expression of miR-320e in the exosomes secreted by the miR-320e mimic-transfected cells was significantly higher than that in the mimic NC group and the blank control. A: HUVECs were transfected using miR-320e mimics and the expression of miR-320e was detected. B: Lipid membrane of HUVECs marked by DiI; C: Microplate reader analysis showed that exosomes were also marked by DiI; D: As shown by using marked exosomes harvested from HUVECs and cocultured with HASMCs, HUVECs also showed red fluorescence; E-G: Western blotting and PCR showed that Wnt2 and β-catenin expression in the Exo-mimic group decreased compared to that of the control group. HUVECs: Human umbilical vein endothelial cells; HASMCs: Human aortic vascular smooth muscle cells; PCR: Polymerase chain reaction. ^aP < 0.05, ^bP < 0.01.