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. 2021 Jan 11;8:579236. doi: 10.3389/fcell.2020.579236

FIGURE 6.

FIGURE 6

The upregulation of miR-410 encapsulated from EVs inhibits apoptosis but promotes the viability of neurons via EGR2/Bcl2 upregulation by repressing HDAC1. (A) The expression of miR-410 in EVs determined by RT-qPCR in EVs infected with a lentiviral vector containing antagomir NC or miR-410 antagomir. (B) HDAC1 was targeted by EV-derived miR-410 verified using dual-luciferase reporter gene assay. (C) The expression of miR-410 in neurons determined by RT-qPCR in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, or miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. (D) The mRNA expression of HDAC1, EGR2, and Bcl2 in neurons determined using RT-qPCR in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. (E) The protein expression of HDAC1, acetyl-histone 3, EGR2, Bcl2 and cleaved-Caspase-3/pro-Caspase-3 in neurons determined by immunoblotting in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, or miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. (F) The HDAC1 and acetyl-histone 3 expression in the promoter region of EGR2 analyzed using ChIP assay in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, or miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. (G) Neuronal cell survival rate determined by MTT assay in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir. (H) Neuronal cell-cycle distribution determined using flow cytometry in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, or miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. (I), Neuronal cell apoptosis determined using flow cytometry in EVs exposed to OGD, infected with antagomir-NC, miR-410 antagomir, miR-410 antagomir + sh-NC or miR-410 antagomir + sh-HDAC1. *p < 0.05 vs. neurons treated with mock-EV; #p < 0.05 vs. OGD-exposed neurons treated with antagomir-NC-EV; &p < 0.05 vs. OGD-exposed neurons infected with miR-410 antagomir-EV and sh-NC. ANOVA followed by Tukey’s multiple comparisons was used for comparisons among more than two groups. Repeated measures ANOVA was conducted to compare data at different time points, followed by Bonferroni’s post hoc test.