All experimental procedures were consistent with the Animal Use and Care of Medical Laboratory Animals from the Ministry of Public Health of People's Republic of China. Male adult Sprague-Dawley rats weighing 180–220 g were purchased from Animal Centre of Shandong University. Rats were subjected to middle cerebral artery occlusion (MCAo) as published protocol (Chen et al., 2001; Zhang L. et al., 2016). Briefly, rats were anesthetized with isoflurane (4% for induction and 2% for maintenance) in 70% N₂O and 30% O₂. Under the operating microscope, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. An 18-mm-long 4 ± 0 nylon filament with an expanded tip was gently advanced from the ECA into the lumen of the ICA. The tip of the filament was positioned at the origin of the middle cerebral artery (MCA).

Dual-Luciferase assay was performed follow by manufacture's instruction. Briefly, a segment of the 3′UTR of wild type Wnt1 gene (Wnt1-3′UTR) encompassing the miR-148b binding site was cloned in a pEZX-MT06 vector with Firefly/Renilla duo Luciferase reporter driven by a CMV promoter (Genecopoeia, USA). Point mutations of 3′UTR of Wnt1 gene (Wnt1-3′UTR-m) in miR-148b binding site were made as UGCACUG to UGCCGGG (Figure 2). To test the interaction between miR-148b and 3′ UTR of Wnt1 gene, 293T cells (ATCC) were co-transfected with miR-148b mimics (100 ng/10⁶ cells) or miR-148b mimic negative control (miR-148b mimic-con, 100 ng/10⁶ cells) and Wnt1-3′UTR (100 ng/10⁶ cells) or Wnt1-3′UTR-m (100 ng/10⁶ cells) by using lipofectamine-2000 transfection reagent (Life technologies, USA). Twenty-four hours later, cells were lysed and treated with a Dual-luciferase assay kit (Genecopoeia, USA). Luciferase activity was detected using a multimode microplate reader (PerkinElmer/Fusion, USA) and normalized to its corresponding Renilla luciferase activity.

Seven days after MCAo, NSCs were isolated from rats SVZ and cultured as previously published papers (Reynolds and Weiss, 1992; Morshead et al., 1994; Liu et al., 2016). NSCs were dissociated and expanded at a density of 2 × 10⁴ cells/ml in proliferation medium [DMEM/F-12 medium (Life Technologies), 20 ng/mL of EGF (R&D System), bFGF (R&D System)] in a cell culture incubator at 37°C, 5% CO₂. NSCs differentiation were induced in differentiation medium [DMEM/F-12 medium contains L-glutamine (2 mmol/L, Life Technologies), glucose (0.6%, Sigma-Aldrich), NH₂(CH₂)₄NH₂ (9.6 mg/Ml, Sigma-Aldrich), insulin (0.025 mg/mL, Sigma-Aldrich), progesterone (6.3 ng/mL, Sigma-Aldrich), apo-transferrin (0.1 mg/mL, Sigma-Aldrich), and Na₂SeO₃ (5.2 ng/mL, Sigma-Aldrich)] for 5 days, then followed the miRNA transfection.

miR-148b mimic, MiRNA-148b mimic negative control (miR-148b mimic-con), MiRNA-148b inhibitor, and MiRNA-148b inhibitor negative control (miR-148b inhibitor-con) were synthesized by RiboBio Co., China. Wnt-1 siRNA was purchased from Qiagen, Germany. Transfection was performed using Lipofectamine RNAiMAX Transfection reagent (Thermo-Fisher, USA) following the manufacturer's instructions.

For proliferation assay, NSCs were cultured on polyornithine/laminin-coated 24-well plates at a density of 5 × 10⁴ cells per well in growth medium for 24 h. To label the dividing cells, 5 μM 5-Bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich) was added into medium during the last 8 h of culture. Cells were fixed in 4% paraformaldehyde for 20 min, incubated with 50% Formamide for 30 min at 65°C, and washed with 1 M HCl for 10 min at 37°C, and 0.1 M borate buffer for 3 min. Non-specific binding sites were blocked with 1% bovine serum albumin (BSA) and 0.3% Triton PBS for 1 h at room temperature. Then, NSCs were incubated with rabbit anti-BrdU (1: 2,000, Abcam) overnight at 4°C. After washing with PBS, cells were incubated with FITC-conjugated secondary antibodies, and followed by 4-6-diamidino-2-phenylindole (DAPI, 1: 10,000, Abcam) incubation for 1 h at room temperature. For differentiation assay, cells were fixed and incubated with blocking buffer and incubated with mouse anti-Neuron-specific class III beta-tubulin (Tuj1, 1:200, Abcam) or rabbit anti-glial fibrillary acidic protein (GFAP, 1:200, Abcam) overnight at 4°C. The next day, cells were washed and incubated with the secondary antibodies and DAPI as the same procedures described above. Cells were counted from three wells/group. Ten view fields per well were imaged under a 40X objective lens. Data is showed as the number of marker-positive cells divided by the total number of DAPI.

To determine neurogenesis in SVZ of ischemia-affected hemisphere, paraffin sections of rat brains were used for staining the following markers, mouse anti-DCX (1:100, Santa Cruz Biotechnology), mouse anti-NeuN (1:100, Abcam), and moue anti-GFAP (1:100, Abcam). All primary antibodies were incubated at overnight at 4°C, and followed by FITC-or Cy3-conjugated secondary antibodies incubation at a concentration of 1:300. To quantify the regional differences in the number of NPCs, neurons and NSCs, we used 40X objective lens to count positive cell in five view fields per section and 1-in-5 series of sections were stained for each animal.

Total RNA was isolated from cultured cells or tissue using the miRNeasy Mini kit (Qiagen, Germany), for formalin-fixed, paraffin-embedded tissue we used miRNeasy FFPE (Qiagen, Germany). Total 10 ng of RNA from each sample was reverse transcribed using TaqMan® MicroRNA Reverse Transcription kit (Applied Biosystems). Quantification of miRNA was performed using standard TaqMan® PCR kit protocol (Applied Biosystems). The method of 2^−ΔΔCT was used to calculate relative miRNAs expression levels. Each sample was tested in triplicate and at least three samples obtained from independent experiments were examined. U6 snRNA served as the internal control (Thermo-Fisher; Livak and Schmittgen, 2001).

Ten weeks old male Sprague-Dawley rats weighing 180–220 g were performed MCAo. The next day, animals were randomly divided into the following three groups (n = 10/group): Lentivirus miR-148b inhibitor (LV-148b-inhibitor, CMV-ZsGREEN1-148b-miRNAinhibitor-PGK-puromycin, 3.0 × 10⁶ IU in 3 μL saline, Genomeditech, China), LV-GFP or saline vehicle control. Rats were anesthetized with 10% chloral hydrate, and fixed on a stereotactic frame (RWD, China). The liquid was injected into the right striatum at a rate of 0.2 μL/min via a mini-pump (WPI, USA). Behavioral tests, including modified neurological severity score (mNSS) test, foot-fault test, and adhesive patch removal test were performed on day 1, day 4, day 7, and day 14 after MCAo (Chen et al., 2001; Shen et al., 2007). On day 14, all the animals were perfused using saline, followed by 4% paraformaldehyde solution. Brains were removed and fixed in 4% formalin, dehydrated, and embedded in paraffin separately. To measure lesion volume, brain slices were stained with hematoxylin and eosin (HE), and the infarct lesion volume (mm³) was measured as appropriate area (mm²) multiply section interval thickness (2 mm).

mNSS was measured according to the published record (Chen et al., 2001). All animals were tested the abilities of motor (score 0–6), sensory (score 0–2) balance (score 0–6), reflex and abnormal movements (score 0–4). Total maximum score is 18, higher score represents severer injury. For foot-fault test, animals were placed on horizontal ladder and trained to go cross it. When testing, total steps and the foot-faults steps for left forelimb were record separately, and the results were presented as the percentage of foot-fault steps to the total number of steps (Hernandez and Schallert, 1988). For adhesive patch removal test, adhesive paper was placed at distal-radial region on the wrist of left forelimb as tactile stimuli, and recorded the time required to remove the stickers. For each animal, three trials per testing day were performed (Shen et al., 2007).

Cells were lysed in lysis buffer containing 1% protease and 1% phosphatase inhibitor (Cell Signaling Technology) followed by centrifuging for 15 min at 15,000 rpm, 4°C to remove cell debris. Protein concentrations were determined using BCA assay (Thermo-Fisher). For each sample, 30 μg of total protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. Membrane was blocked and incubated with an appropriate primary antibody and a secondary antibody. The following antibodies were used: mouse anti-β-actin (1: 5,000, Abcam), rabbit anti-Wnt1 (1: 1,000; Abcam), rabbit anti-β-catenin (1: 1,000; Abcam), rabbit anti-Cyclin D1 (1: 1,000, Abcam), rabbit anti-PROX1 (1: 1,000, Abcam), rabbit anti-NeuroD1 (1: 1,000, Abcam), rabbit anti-DCX (1: 1,000, Abcam), and mouse anti-glial antigen 2 (NG2, 1: 1,000, Abcam). Proteins were visualized by enhanced chemiluminescence (Thermo-Fisher).

All data are presented as mean ± SE. Significant differences between groups were analyzed using One-way ANOVA. Statistical significance was set at p < 0.05.

To explore the potential involvement of miR-148 in ischemic SVZ, sub-ventricular zone was isolated from sham-operation (Sham) and MCAo applied rat brains (Figure 1A). Quantitate RT-PCR analysis indicated the expression levels of both miR-148a and miR-148b in ischemic SVZ were obviously increased compared with sham group (Figure 1B). However, the enrichment of miR-148a in sham and ischemic brains were significantly lower than miR-148b, and the following study was focused on miR-148b.

Using targetscan.org, we searched predicted target genes of miR-148b. A potential miR-148b broadly conserved sequence was found to be seed matched with 3′UTR of Wnt1 mRNA (Figure 2A). To confirm the directly targeting relationship of miR-148b and Wnt1, we constructed luciferase expression vectors harboring wild type or mutant Wnt1 3′UTR binding site (WNT1-3UTR and WNT1-3UTR-m; Figure 2). Luciferase assay revealed that WNT1-3UTR had significantly lower activity than WNT1-3UTR-m in HEK-293T cells when miR-148a mimic co-transfected. In contrast, miR-148b mimics did not reduce luminescence activity when miR-148b seed sequences at 3′ UTR of Wnt1 gene was mutated (Figure 2B). These data indicated miR-148b could target Wnt1 mRNA in HEK 293T cells.

To further investigate the effect of miR-148b on Wnt/β-catenin signaling pathway, primary ischemic SVZ NSCs were transfected with miR-148b mimic and miR-148b mimic negative control (miR-148b mimic-con), respectively. The expression of miR-148b was increased by 5.92 ± 0.44 and 5.58 ± 0.38 times higher than blank (vehicle control) and miR-148b mimic-con, respectively (Figure 3A). Western blot data showed the expression of Wnt1 and β-catenin were significantly decreased (p < 0.05, n = 3) in miR-148b mimic group compared with blank and miR-148b mimic-con group. Three downstream proteins of Wnt/β-catenin pathway, Cyclin D1, NeuroD1 and Prox1 were also detected and showed significantly downregulated in miR-148b mimic transfected NSCs (p < 0.05, n = 3; Figures 3C,D). On the contrary, cells transfected with miR-148b inhibitor substantially decreased the expression of miR-148b by 0.31 ± 0.06 and 0.29 ± 0.04 times compared with blank and miR-148b inhibitor control transfected cells, respectively (Figure 3B). And the increasing proteins expression of Wnt/β-catenin signaling and downstream was also confirmed by the means of western blot (Figures 3C,E). These results further demonstrated that Wnt1/ β-catenin signaling was regulated by miR-148b in SVZ NSCs.

The proliferation and differentiation activities of NSCs are curial for neurogenesis process. First, we tested the effect of miR-148b on SVZ NSCs proliferation. Ischemic SVZ cells cultured in growth medium were transfected with miR-148b inhibitor and miR-148b inhibitor negative control (miR-148b inhibitor-con), respectively. Immunofluorescent staining showed a robustly up-regulated BrdU-positive cell percentage as 56.5 ± 9.1% compared with miR-148b inhibitor-con group (41.6 ± 8.5%, p < 0.01; Figure 4A).

Then, we tested the effect of miR-148b on NSCs differentiation. Primary ischemic SVZ cells were transfected with miR-148b inhibitor/inhibitor negative control and cultured in differentiation medium for another 5 days. Immunofluorescent staining analysis revealed that suppress of miR-148b resulted in a significant increase in the number of Tuj1-positive cells (12.2 ± 1.9% in miR-148b inhibitor vs. 8.4 ± 1.2% in miR-148b inhibitor-con group, p < 0.05) and GFAP-positive cells (37.8 ± 3.1% in miR-148b inhibitor group vs. 26.5 ± 6.4% in miR-148b inhibitor negative control group, p < 0.05; Figures 4A,B). Western blot further demonstrated the expression of NG2 (neural/glial antigen 2) and DCX was also increased significantly in miR-148b inhibitor transfected NSCs than inhibitor negative control transfected cells (Figures 4C,D).

Additionally, we silenced the expression of Wnt1 in ischemic SVZ cells by Wnt1 siRNA. As shown in Figure 4C, the expression of Wnt/β-catenin signaling and the downstream proteins were obviously down-regulated when Wnt1 siRNA was applied, and miR-148b inhibitor could not increase the expression of these proteins (p < 0.01, vs. miR-148b inhibitor). These results indicating Wnt/β-catenin signaling mediated the neurogenesis inhibitory effect of miR-148b on SVZ NSCs.

To detect the effect of miR-148b in neurological functional recovery after ischemic stroke in vivo, we inhibited the miR-148b expression level in rats by Lentivirus miR-148b inhibitor injection. Saline, LV-GFP, and LV-148b inhibitor was transferred to rats 1 day after MCAo was performed. All animals were scarified 14 days after MCAo. Quantitate RT-PCR demonstrated the expression level of miR-148b was significantly reduced in LV-148b inhibitor injection group (Figure 6C), which indicating the miR-148b inhibitor transduction succeed. HE staining revealed that the brain infarct volume of rats treated with LV-148b inhibitor (28.8 ± 5.7 mm³) was significantly reduced than saline and LV-GFP administrated group (40.3 ± 4.5 mm³ and 39.2 ± 4.8 mm³, Figures 5A,B). Functional behavior test results demonstrated LV-148b inhibitor group showed a better neurological functional recovery outcome compared with saline and LV-GFP groups at day 4, day 7, and day 14 (Figures 5C–E). These data strongly indicated LV-148b inhibitor reduced the infarct volume and improved neurological function recovery of ischemic stroke rats.

To further explore the effects of miR-148b on neurogenesis in vivo, Paraffin coronal sections were used for DCX, NeuN, and GFAP staining. Compared with LV-GFP injection group, quantity of DCX⁺, NeuN,+ and GFAP⁺ cells in SVZ of LV-148b inhibitor treated rats were significantly higher at day 14 after MCAo (Figures 6A,B). Adult NSCs are radial, astrocytes-like GFAP⁺ cells. LV-148b inhibitor injection obviously increased the expression of GFAP, and induced the differentiation of NSCs into neuroblast which express DCX as a marker. Mature neurons in SVZ were marked with NeuN staining, according our results, LV-148b inhibitor up-regulated the number of mature neurons at day 14, indicating neurogenesis was improved in SVZ and this process could at least partially explain the effects of miR-148b inhibitor on neurological function recovery after stoke.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.