Extracellular Vesicle-Derived microRNA-410 From Mesenchymal Stem Cells Protects Against Neonatal Hypoxia-Ischemia Brain Damage Through an HDAC1-Dependent EGR2/Bcl2 Axis

Abstract

Hypoxia-ischemia brain damage (HIBD) is a neurological disorder occring in neonates, which is exacerbated by neuronal apoptosis. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) have been proposed as a promising strategy for treating or preventing ischemia-related diseases. However, their mechanisms in HIBD remain unclear. Thus, we aimed to address the role of EV-derived microRNA (miR)-410 in HIBD. Neonatal HIBD mouse model was constructed using HI insult, from which neurons were isolated, followed by exposure to oxygen glucose deprivation (OGD). EVs were isolated from human umbilical cord (hUC)-derived MSCs. In silico analyses, dual-luciferase reporter gene and chromatin immunoprecipitation assays were adopted to determine relationships among miR-410, histone deacetylase 1 (HDAC1), early growth response protein 2 (EGR2), and B cell lymphoma/leukemia 2 (Bcl2). The functional roles of EV-derived miR-410 were determined using loss- and gain-of functions experiments, and by evaluating neuronal viability, cell-cycle distribution and neuronal apoptosis in vitro as well as modified neurological severity score (mNSS), edema formation, and cerebral infarction volume in vivo. hUC-MSCs-derived EVs protected against HIBD in vivo and inhibited the OGD-induced neuronal apoptosis in vitro. miR-410 was successfully delivered to neurons by hUC-MSCs-EVs and negatively targeted HDAC1, which inversely mediated the expression of EGR2/Bcl2. Upregulation of EV-derived miR-410 promoted the viability but inhibited apoptosis of neurons, which was reversed by HDAC1 overexpression. EV-derived miR-410 elevation reduced mNSS, edema formation, and cerebral infarction volume by increasing EGR2/Bcl2 expression through downregulating HDAC1 expression in vivo. In summary, EV-derived miR-410 impeded neuronal apoptosis by elevating the expression of EGR2/Bcl2 via HDAC1 downregulation, thereby providing a potential strategy for treating or preventing HIBD.

Keywords: Bcl2; early growth response 2; extracellular vesicle; histone deacetylase 1; microRNA-410; neonatal hypoxia-ischemia brain damage.

FIGURE 1

Human umbilical cord-MSCs-derived EVs prevent HIBD in neonatal mice. HIBD mice were treated with PBS, EVs-free supernatant (EFS) or MSCs-derived EVs (MSCs-EVs) (n = 12/group) to simulate the action on HIBD in neonatus. (A) The morphology or EVs observed using a transmission electron microscope (100 nm). (B) The size and distribution of EVs determined by NanoSight. (C) The protein expression of Alix, TSG101, GM130, and CD63 in EVs assessed by immunoblotting. (D) The distribution of red-labeled (PKH26) EVs in the hippocampus was determined using immunofluorescence (400×). The nuclei were stained blue by DAPI. (E) mNSS in mice following injections of PBS, EFS, or MSCs-EVs. (F) The edema formation in mice after different treatments. (G) The cerebral infarction volume in mice determined by TTC staining. (H) The neuron morphology determined using H&E staining (200×). *p < 0.05 vs. sham-operated mice; #p < 0.05 vs. HIBD mice treated with PBS. Unpaired t-test was applied for the comparisons between two groups and one-way ANOVA followed by Tukey’s multiple comparisons was used for comparisons among three or more groups.

FIGURE 2

Human umbilical cord-MSCs-derived EVs inhibit the OGD-induced neuronal apoptosis in vitro. OGD-exposed neurons were treated with PBS, MSCs-EVs or EFS. (A) The isolated neurons evaluated using immunofluorescence (400×) (NeuN: green; DAPI: blue). (B) The content of LDH released from neurons at 0, 24, and 48 h prior to OGD. (C) Neuronal cell survival rate determined by MTT assay. (D) Neuronal cell-cycle distribution determined using flow cytometry. (E) Neuronal cell apoptosis determined using flow cytometry. (F) The red-labeled (PKH26) EVs in neurons determined using immunofluorescence (500×). *p < 0.05 vs. untreated neurons; #p < 0.05 vs. neurons treated with PBS. Unpaired t-test was applied for the comparisons between two groups and one-way 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.

FIGURE 3

Extracellular vesicle-derived miR-410 from hUC-MSCs inhibits the OGD-exposed neuronal apoptosis. OGD-exposed neurons treated with equal amounts of purified EVs, together with either 20 μg/μL proteinase K or RNase A supplemented with 0.05% Triton X-100 in panels (A–F). OGD-exposed neurons were introduced with PBS, MSCs-EVs or EFS in panel (G). (A) Gel electrophoresis showing the RNase digested EVs were depleted of RNAs compared with proteinase and control treatment. (B) Silver staining (right) showing that after proteinase treatment, EVs were degraded thoroughly. (C) The intact structure of EVs following enzyme treatment determined by NanoSight. (D) Neuronal cell survival rate determined by MTT assay. (E) Neuronal cell-cycle distribution determined using flow cytometry. (F) Neuronal cell apoptosis determined using flow cytometry. (G) The expression of miR-410 in neurons determined by RT-qPCR following treatment of MSCs-EVs. *p < 0.05 vs. MSCs-EVs or OGD-exposed neurons introduced with MSCs-EVs or neurons. One-way 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.

FIGURE 4

In silico analyses of miR-410 in regulating the HDAC1/EGR2/Bcl2 axis. (A) The Venn diagram of downstream target genes of mouse miR-410 predicted by DIANA TOOL, miRDB, miRWalk, StarBase, TargetScan, and microRNA database. (B) The binding sites between miR-410 and HDAC1 in mice and human determined by StarBase. The above rectangle represents the binding site for mice (mmu), and the lower rectangle represents the binding site for humans (hsa). (C) Volcano plot of gene expression dataset GSE23160. Green dots indicate significantly downregulated genes and red dots indicate significantly upregulated genes. (D) The Venn diagram of intersection among co-expressed genes, the DEGs and human transcription factors from the database hTFtarget. (E) The targeting relationship between EGR2 and Bcl2 predicted by hTFtarget.

FIGURE 5

Upregulation of miR-410 inhibits apoptosis but enhances the viability of OGD-exposed neurons by elevation of EGR2/Bcl2 axis via HDAC1 repression. OGD-exposed neurons were treated with lentiviral vectors containing miR-410 agomir, and/or HDAC1 overexpression. (A) HDAC1 was targeted by miR-410 in HEK293T cells determined by dual-luciferase reporter gene assay. (B) The expression of miR-410 in neurons determined by RT-qPCR. (C) Neuronal cell survival rate determined by MTT assay. (D) Neuronal cell-cycle distribution determined by flow cytometry. (E) Neuronal cell apoptosis determined by flow cytometry. (F) The mRNA expression of HDAC1, EGR2, and Bcl2 in neurons determined by RT-qPCR. (G) The protein expression of HDAC1, EGR2, cleaved-Caspase-3/pro-Caspase-3, Bcl2 and acetyl-histone 3 determined by immunoblotting. (H) The enrichment of HDAC1 and acetyl-histone 3 at the promoter region of EGR2 determined by ChIP. *p < 0.05 vs. untreated neurons; #p < 0.05 vs. OGD-exposed neurons infected with agomir-NC; &p < 0.05 vs. OGD-exposed neurons infected with miR-410 agomir and NC. Unpaired t-test was applied for comparisons between two groups. One-way 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.

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.

FIGURE 7

Extracellular vesicle-derived miR-410 attenuates HIBD via EGR2/Bcl2 upregulation by downregulating HDAC1 in neonatal mice. HIBD mice were injected with PBS, MSCs-EV, lentiviral vectors containing miR-410 antagomir-EV or antagomir-NC-EV (n = 12/group). (A) mNSS in mice after different treatment. (B) The edema formation in mice after different treatments. (C) The cerebral infarction volume in mice determined by TTC staining after different treatments. (D) The neuron morphology in mice determined using H&E staining (200×). (E) The expression of miR-410, HDAC1, EGR2, and Bcl2 determined using RT-qPCR. (F) The expression of cleaved-Caspase-3/pro-Caspase-3, HDAC1, EGR2, BCL2, and acetyl-histone 3 determined by immunoblotting. *p < 0.05 vs. sham-operated mice; #p < 0.05 vs. HIBD mice treated with PBS; &p < 0.05 vs. HIBD mice infected with lentiviral vector containing antagomir-NC-EVs. Unpaired t-test was applied for the comparisons between two groups and one-way ANOVA followed by Tukey’s multiple comparisons was used for comparisons among more than two groups.

FIGURE 8

The graphical summary of the function and mechanism of EV-derived miR-410 in HIBD. miR-410 is transferred from hUC-MSCs to neurons via EVs. miR-410 negatively targets HDAC1, and HDAC1 downregulation at the promoter of EGR2 elevates the expression of EGR2/Bcl2 axis, thereby enhancing viability and inhibiting apoptosis of neurons.