miR-148b Regulates Proliferation and Differentiation of Neural Stem Cells via Wnt/β-Catenin Signaling in Rat Ischemic Stroke Model

Abstract

Stroke is the second leading cause of death worldwide. Stroke induced proliferation and differentiation of neural stem cells (NSCs) that have been proven to participate in ischemic brain repair. However, molecular mechanisms that regulate neurogenesis have not been fully investigated. MicroRNAs play an important role in the neurological repairing process and impact stroke recovery outcome. MiRNA-148b has been reported to regulate cell proliferation in tumor cells, but its role in NSCs after ischemic stroke remains unknown. Here, we found an overexpression of MiRNA-148b in subventricular zone (SVZ) of rat ischemic brain. In original cultured ischemic NSCs, transfection of MiRNA-148b mimic or inhibitor could suppress or enhance the expression of Wnt-1, β-catenin, and Cyclin D1, hence effected wnt/β-catenin signaling. MiRNA-148b inhibitor promoted NSCs proliferation and differentiation into newborn neural and astrocytes, and this action could be silenced with knockdown of Wnt-1. In middle cerebral artery occlusion (MCAo) rats, injection of MiRNA-148b inhibitor could reduce ischemic lesion volume and improve neurological function outcome. Collectively, our data suggest that MiRNA-148b suppressed wnt/β-catenin signaling attenuates proliferation and differentiation of neural stem cells, these findings shed new light on the role of MiRNA-148b in the recovery process during the stroke and contribute to the novel therapy strategy.

Keywords: Wnt/β-catenin signaling; ischemic stroke; microRNA-148b; neurogenesis; sub-ventricular zone.

Figure 1

miR-148a/b expression levels changed after MCAo. (A) Image of TTC staining of rat brain section after MCAo. SVZ (marked with black rectangle) in ischemic hemisphere and the corresponding area from sham group rats were isolated respectively for qRT-PCR test; (B) miR-148a/b expression levels were measured in sham and MCAo rat SVZ area. Mean ± SE, ^*p < 0.05, n = 3/group.

Figure 2

miR-148b directly targeted Wnt1 in HEK 293T cells. (A) Schematic diagram shows wild-type or mutated miR-148b binding site in Wnt1 3′UTR fragment; (B) Luciferase reporter assay data of wild type or mutant 3′ UTR of Wnt1 with miR-148b mimic-con or miR-148b mimic. mean ± SE, ^**p < 0.01, n = 3/group.

Figure 3

Effects of miR-148b on Wnt/β-catenin signaling activity. (A) miR-148b expression levels in ischemic NSCs transfected with miR-148b mimic or miR-148b-mimic-con; (B) miR-148b expression levels in ischemic NSCs transfected with miR-148b inhibitor or miR-148b inhibitor-con; (C–E). Western blot analysis of Wnt-1, β-catenin, Cyclin D1, NeuroD1, and Prox1 expression contents and their quantitative data in ischemic NSCs transfected with miR-148b mimic, miR-148b inhibitor and their negative controls. mean ± SE, ^*p < 0.05, ^**p < 0.01, n = 3/group.

Figure 4

miR-148b involved in neurogenesis via regulating Wnt/β-catenin signaling activity. (A,B) Immunostaining images and quantitative data of BrdU, Tuj1 and GFAP positive cells in ischemic NSCs transfected with miR-148b inhibitor-con, miR-148b inhibitor, or miR-148b inhibitor with siRNA Wnt-1; (C,D). Western blot analysis of Wnt-1, β-catenin, DCX and NG2 expression and their quantitative data in ischemic NSCs transfected with miR-148b inhibitor, miR-148b inhibitor-con and siRNA Wnt-1. mean ± SE, ^*p < 0.05, ^**p < 0.01. Scale bar = 5 μm.

Figure 5

LV-148b inhibitor reduced brain infarction volume and improved neurological functional recovery after MCAo. (A) Effect of LV-148b inhibitor on infraction volume of rat brains after MCAo; Brain coronal sections stained with H&E show infarction from rats treated with saline, LV-GFP or LV-148b inhibitor after MCAo. (B) Quantitation of infarct volumes in all experimental groups. n = 10/group, mean ± SE, ^*p < 0.05. (C–E) The adhesive-removal test, foot-fault test and mNSS scores test were performed at days1, 4, 7, and 14 after MCAo applied. n = 10/group, mean ± SE, ^*p < 0.05.

Figure 6

LV-148b inhibitor improved neurogenesis in rats SVZ after MCAo. (A) Immunofluorescence staining for DCX (red), NeuN (green), and GFAP (green) at day 14 after ischemia. Nuclei were counterstained with DAPI (Blue). Scale bar = 50 μm. (B) Data are represented as the number of positive cells in five view fields for each section, and three section for each animal. (n = 10/group. mean ± SE, ^*p < 0.05, ^**p < 0.01vs. LV-GFP group); (C) The relative miR-148b expression level in LV-148b and LV-GFP inhibitor injected rat brains SVZ (n = 5/group. mean ± SE, ^**p < 0.01).