Sprague-Dawley (SD) rats were obtained and reared from the Affiliated Hospital of Hebei University of Engineering. All experiments were performed in compliance with guidelines for the ethical use of animals of the Affiliated Hospital of Hebei University of Engineering. Transient focal cerebral ischemia was induced by intra-luminal MCAO as described previously (14,15). Briefly, male SD rats (280–320 g) were anesthetized and maintain at 37°C. A nylon filament with its cusp slightly rounded by heat was advanced from the right external carotid artery into the lumen of the internal carotid artery to occlude the right MCA for 2 h, and the suture was subsequently withdrawn to allow reperfusion. For sham group, surgery protocol ended after dissecting the internal carotid artery, thus no nylon filament was inserted. Rats were analgesia sacrificed to obtain the brains for biochemical assays.

HUVECs (Lonza, Walkersville, MD, USA) were grown in M199 medium supplemented with 20 mg/ml endothelial cell growth supplement (ECGS; Upstate Biotechnology, Inc., Lake Placid, NY, USA), 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) on 0.1% gelatin-coated culture flasks. The human embryonic kidney cell line 293T (HEK 293T) was preserved in our institute and cultured in DMEM supplemented with 10% heat-inactivated FBS. All cells were maintained in a humidified incubator with 5% CO₂ at 37°C. After the cells reached 80–90% confluence, they were digested by 0.25% trypsin (Beyotime Institute of Biotechnology, Haimen, China), passaged, and used for the following experiments.

For hypoxia, HUVEC cells were cultured in M199 medium without FBS in a hypoxia incubator (Sanyo, Japan) under hypoxic conditions (5% CO₂, 94% N₂, and 1% O₂,) for 12 h. Cells cultured under normoxic conditions were used as controls.

The miRNA microarray was performed as described previously (16,17). Briefly, total RNA of the rat brain was isolated by a miRNAeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), and followed by labeling and hybridization with the miRCURY^™ LNA Array (v.16.0, Exiqon). The feature extraction software (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to quantify the fluorescent intensity of each spot of microarray images, and signal intensities >10 were considered positive expression. The statistical significance of upregulated or downregulated miRNAs was analyzed by t-test. MEV software (v4.6, TIGR) was used to perform hierarchical clustering.

miR-195 mimic, miR-195 inhibitor and controls were purchased from Shanghai GenePharma (Shanghai, China). HUVEC cells were transfected with 20 nM miR-195 mimic, miR-195 inhibitor and miR negative control (NC) with Lipofectamine™ RNAiMAX (Life Technologies, Grand Island, NY, USA). Fourty-eight hours after transfection, cells were collected for further protein extraction.

The VEGFA sequence was subcloned into the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific). VEGFA ectopic expression was achieved through pcDNA3.1-VEGFA transfection using lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific). Plasmid vector (pcDNA3.1-VEGFA) for transfection was extracted using Midiprep kits (Qiagen GmbH, Hilden, Germany), and co-transfected into HUVEC cells with miR-195 mimic. After transfection for 48 h, cells were collected for cell invasion and tube formation.

Total RNA of the cultured cells and the tissues was extracted using a mirVana miRNA isolation kit (Ambion; Thermo Fisher Scientific). miR-195 was reverse transcribed using the PrimeScript RT reagent kit (Takara, Tokyo, Japan) and quantified by real-time PCR with the TaqMan MicroRNA assay kit (Applied Biosystems; Thermo Fisher Scientific). qRT-PCR analyses for VEGFA and the normalization control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were performed using SYBR Premix Ex Taq (Takara) on an ABI PRISM 7500 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific). The relative expression of each gene was calculated and normalized using the 2^−ΔΔCt method relative to RNU6B or GAPDH. All reactions were conducted in triplicate.

After transfection, cells were harvested and lysed in RIPA buffer. Equal amounts of protein extracts was subjected to 10% SDS-PAGE lysis and transferred to PVDF membrane, after blocking in 5% skimmed milk for 30 mins at room temperature, the member was incubated with antibodies against VEGFA (1:1,000; Cell Signaling Technology, Danvers, MA, USA), β-actin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h at room temperature, followed by incubating in with horseradish peroxidase-linked secondary antibody for 1 h at room temperature and visualized with ECL. And the results of western blots were analyzed using the ImageJ program.

HUVECs (2×10⁴ cells/well) were plated onto 96-well plate. The tubes formed in each well at 24 h were captured using an inverted microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA). The degree of tube formation under different treatments was quantified by counting the number of tubes in three random fields from each well. All experiments were repeated three times.

Migration assays were performed using Costar Transwell plates with 6.5 mm-diameter polycarbonate filters (8 µm pore size). The lower surface of the filter was coated with 10 µg of gelatin. Fresh M199 containing 5% FBS were placed in the lower wells. HUVECs with a final concentration of 1×10⁶ cells/ml were seeded onto the upper surface of the filter. Then, the chamber was incubated at normoxia or hypoxia. After 24 h, non-migrating cells were removed from the upper surface of the filter and migration was observed using an inverted microscope. Four randomly chosen fields were counted per well.

A whole fragment of 3′UTR VEGFA mRNA and a mutant form were cloned into pGL-3-Luc. The HEK 293T cells were seeded in 12-well plates and co-transfected with pGL-3-VEGFA wild-type or mutant portion and TK100 Renilla combined with miR-195 mimic, miR-195 inhibitor or NC control using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific). After 48 h of incubation, cells were collected for application in the Dual-Luciferase Reporter system (Promega, Madison, WI, USA) following the manufacturer's recommendations. All of the dual-luciferase reporter assays were done in triplicate within each experiment, and three independent experiments were conducted.

All values were expressed as mean ± SD and processed by GraphPad Prism 5.0 software. Differences among the groups were assessed by Student's t-test, and they were considered statistical significance if P<0.05.

As hypoxia is an important factor to induce angiogenesis, we established the cell model as described previously (22,23). Then, we performed qRT-PCR to detect the expression of miR-195 in HUVECs under normoxic and hypoxic conditions. We found that hypoxia treatment caused a significant decrease in the expression of miR-195 in HUVECs, which declined to the lowest levels at 6 h and followed by returning to normoxia at 24 h (Fig. 2A). Results were consistent with in vivo findings. Therefore, the data suggests that hypoxia could decrease the expression of miR-195.

To further investigate the role of miR-195 in the regulation of angiogenesis, HUVECs were transfected with miR-195 mimics or their inhibitor. The miR-195 level was significantly increased in HUVECs that were treated with miR-195 mimics, and miR-195 was downregulated by miR-195 inhibitor (Fig. 2B). Because endogenous vascular endothelial cell proliferation, migration, and tube formation play important roles in angiogenesis (24), we determined the effects of miR-195 on tube formation and cell migration by cell migration assay and tube formation assay. As shown in Fig. 2C and D, miR-195 mimic could obviously attenuated hypoxia-induced tube formation and cell migration, while miR-195 inhibitor showed a significant improvement in tube formation and cell migration (Fig. 2E and F). These findings demonstrate that miR-195 plays an important role in angiogenesis.

To elucidate the underlying mechanism by which miR-195 regulates angiogenesis of HUVECs, we explored miR-195 targets using the microRNA.org bioinformatics algorithm. According to bioinformatics analysis, we focused on VEGFA because of its positive roles in angiogenesis (25). The binding sites between miR-195 and VEFGFA were illustrated in Fig. 3A. To confirm the direct binding relationship between miR-195 and VEFGFA, a luciferase activity assay was conducted. As shown in Fig. 3B, co-transfection of miR-195 mimic and pGL-3-VEGFA-wt significantly decreased the luciferase activity, whereas co-transfection of miR-195 inhibitor and pGL-3-VEGFA-wt increased the luciferase activity. Likewise, cells co-transfected with miR-195 mimic, miR-195 inhibitor and pGL-3-VEGFA-mut showed no obvious change in luciferase activity. Then, we explore whether miR-195 can modulate the expression of miR-195. As shown in Fig. 3C and D, miR-195 overexpression down-regulated VEGFA expression at both the protein and mRNA levels, whereas increased after inhibition of miR-195.

In addition, we measured miR-195 and VEGFA expression levels in 10 rat brains samples after MCAO. As shown in Fig. 1C, an inverse correlation was observed between VEGFA expression and the miR-195 expression level (R²=0.9274; P<0.01). All these data indicate that miR-195 may inhibit post-transcriptional VEGFA expression by targeting its 3′UTR.

Given that VEGFA was the target of miR-195, we hypothesized that miR-195 might play a role in angiogenesis by regulating the expression of VEGFA. To test this hypothesis, we performed a rescue experiment. HUVECs were co-transfected with miR-195 mimic and pcDNA-VEGFA, then incubated at normoxia or hypoxia. Subsequently, we determined the tube formation ability and cell migration of HUVECs. We found that the overexpression of VEGFA reversed the inhibitory effects of miR-195 overexpression on tube formation (Fig. 4A). Similarly, overexpression of VEGFA also reversed the inhibitory effect of miR-195 overexpression on cell migration of HUVECs (Fig. 4B). These results indicate that miR-195 regulates angiogenesis of HUVECs under hypoxia by targeting VEGFA.