Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury

Abstract

MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2?,7?-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020.

Keywords: brain injury; cell migration; cell proliferation; endothelial cell; hypoxia-inducible factor-1 alpha; metallothionein 2; microRNA; neovascularization; neurons; vascular endothelial growth factor.

Figure 1

Downregulation of miR-491-5p improves the recovery of neurological function in mice after TBI. (A) Quantitative polymerase chain reaction analysis of miR-491-5p levels in the mouse cortex in each group preoperatively and 1, 3, 7, 14, and 21 days after TBI (n = 4). (B) NSS scoring was performed preoperatively and 1, 3, 7, 14, and 21 days after TBI (n = 8). (C) Latency spent in quadrant 4 during the cued learning performance phase of the Morris water maze test 16–20 days after TBI (n = 8). (D) Time spent in quadrant 4 when the platform was removed from the Morris water maze test 21 days after TBI (n = 8). (E) Length of the swimming tracks in quadrant 4 after the platform was removed from the Morris water maze test 21 days after TBI (n = 8). (F) Time traveling across the previous platform location after the platform was removed from the Morris water maze test 21 days after TBI (n = 8). Data are expressed as the mean ± SD (one-way analysis of variance followed by Tukey’s post hoc test). *P < 0.05, **P < 0.01, vs. CCI group; #P < 0.05, ##P < 0.01, vs. sham group. CCI: Controlled cortical injury; NSS: neurological severity scores; TBI: traumatic brain injury.

Figure 2

Downregulation of miR-491-5p promotes neovascularization and the recovery of cerebral blood flow around the injury site 21 days after TBI in mice. (A) Lectin-positive microvasculature around the injury site after TBI in mice (left) and quantification (right); lectin is a marker of the microvasculature (red); DAPI is a marker of the cell nucleus (blue). Scale bar: 100 µm. (B) Laser speckle images of cortical blood perfusion in each group at or around the injury site after TBI in mice (left) and quantification (right): The intensity of the red signal is proportional to the amount of cerebral blood perfusion; the circle represents the area that we detected. Data are expressed as the mean ± SD (n = 8; one-way analysis of variance followed by Tukey’s post hoc test). **P < 0.01. CCI: Controlled cortical injury; DAPI: 4′,6-diamidino-2-phenylindole; TBI: traumatic brain injury.

Figure 3

Downregulation of miR-491-5p promotes neuronal survival around the injury site 21 days after TBI in mice. NeuN-positive cells around the injury site (left) and quantification (right). NeuN is a marker of neurons (green); DAPI is a marker of cell nuclei (blue). Data are expressed as the mean ± SD (n = 8; one-way analysis of variance followed by Tukey’s post hoc test). **P < 0.01. CCI: Controlled cortical injury; DAPI: 4′,6-diamidino-2-phenylindole; NeuN: neuronal nuclei.

Figure 4

Downregulation of miR-491-5p improves cell viability, decreases apoptosis, and alleviates oxidative stress. (A) Quantitative polymerase chain reaction for the detection of miR-491-5p expression levels in each group (n = 4). (B) CCK-8 assay was used to detect cellular viability in each group (n = 6). (C) Flow cytometry was used to detect cell apoptosis in each group and (E) quantified. The lower left quadrant represents normal cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents mid-late apoptotic cells, and the upper left quadrant represents necrotic cells (n = 6). (D) ROS levels in each group and (F) quantification: The intensity of green fluorescence is proportional to the level of intracellular oxidative stress (n = 6). Data are expressed as the mean ± SD (one-way analysis of variance followed by Tukey’s post hoc test). *P < 0.05, **P < 0.01. INH: MiR-491-5p inhibitor; INH-NEG: miR-491-5p inhibitor negative control; MIC: miR-491-5p mimic; MIC-NEG: miR-491-5p mimic negative control; OGD: oxygen-glucose deprivation; ROS: reactive oxygen species.

Figure 5

MT2 is a target mRNA of miR-491-5p, and the downregulation of miR-491-5p increases the expression of MT2. (A) ENCORI, miRDB, and miRWalk databases were used to predict the target mRNAs of miR-491-5p. (B) The dual-luciferase reporter assay results verified the binding activity between miR-491-5p and the 3’UTR of MT2 mRNA (n = 4). (C) Western blot assay for the detection of MT2 and HIF-1α expression in each group and quantifications: β-actin was used to normalize protein expression levels. (D and E) The ordinates of “MT2” or “HIF-1α” represents the protein expression level of MT2 and HIF-1α (n = 5). Data are expressed as the mean ± SD (B: Student’s t-test; D and E: one-way analysis of variance followed by Tukey’s post hoc test). **P < 0.01. HIF-1α: Hypoxia-inducible factor 1α; INH: MiR-491-5p inhibitor; MT2: metallothionein 2; OGD: oxygen-glucose deprivation.

Figure 6

Downregulation of miR-491-5p promotes BMEC migration and tube formation through an MT2-dependent HIF-1α pathway. (A–C) Images of the cell scratch assay in each group at different time points and quantifications: The miR-491-5p inhibitor increased the migration area compared with the OGD group at 24 and 48 hours. (D and F) Representative images of the Transwell assay in each group and quantifications. Scale bars: 50 µm. (E and G) Tube formation images in each group and quantifications. Data are expressed as the mean ± SD (n = 6; one-way analysis of variance followed by Tukey’s post hoc test). **P < 0.01. BMEC: Brain microvascular endothelial cell; DMSO: dimethyl sulfoxide; HIF-1a: hypoxia-inducible factor 1a; INH: miR-491-5p inhibitor; LW6: HIF-1a inhibitor; MT2: metallothionein 2; OGD: oxygen-glucose deprivation.

Figure 7

Downregulation of miR-491-5p activates the HIF-1α/VEGF/MAPK pathway in an MT2-dependent manner. (A) Western blot assay for the detection of MT2, HIF-1α, VEGF, p-ERK, ERK, p-p38MAPK, p38MAPK, MMP-9, and cyclin D1 protein expression in each group and quantifications (B–H), β-actin was used to normalize the protein expression. Data are expressed as the mean ± SD (n = 5; one-way analysis of variance followed by Tukey’s post hoc test). **P < 0.01. DMSO: Dimethyl sulfoxide; ERK: extracellular-signal-regulated protein kinase; HIF-1α: hypoxia-inducible factor 1α; INH: miR-491-5p inhibitor; LW6: inhibitor of HIF-1α; MMP-9: matrix metalloproteinase-9; OGD: oxygen-glucose deprivation; p38MAPK: p38-mitogen activated protein kinase; p-ERK: phosphorylated extracellular-signal-regulated protein kinase; p-p38MAPK: phosphorylated p38-mitogen activated protein kinase; VEGF: vascular endothelial growth factor.