Downregulation of microRNA-195 promotes angiogenesis induced by cerebral infarction via targeting VEGFA.

Angiogenesis, the formation of new blood vessels from preexisting endothelium, is a process that involves a series of interassociated and mutually interactive pathophysiological processes. It is accepted that microRNAs (miRNAs) regulate endothelial cell behavior, including their involvement in angiogenesis. However, it remains unclear whether miRNAs are involved in the regulation of angiogenesis following cerebral ischemia. Therefore, the present study aimed to investigate the role of miRNAs in angiogenesis and the underlying mechanism following cerebral ischemia. Expression profiles of miRNAs in rat brain samples following middle cerebral artery occlusion (MCAO) were investigated using a miRNA microarray. The expression of candidate miRNA, miR‑195 was further validated using reverse transcription‑quantitative polymerase chain reaction. Then, the effects of miR‑195 on cell migration and tube formation of human umbilical vein vascular endothelial cells (HUVECs) were investigated following miR‑195 silencing, and overexpression. The specific target genes of miR‑195 were predicted using microRNA prediction bioinformatics software (http://www.microrna.org/microrna/home.do), and then confirmed using a dual‑luciferase reporter assay and rescue experiment. It was demonstrated that miR‑195 was significantly downregulated in the brains of rats following MCAO and in hypoxia‑induced HUVECs. Furthermore, it was revealed that miR‑195 overexpression inhibited the invasion ability and tube formation of HUVECs in vitro, while miR‑195 silencing enhanced these functions. In addition, vascular endothelial growth factor A (VEGFA) was identified as a direct target of miR‑195 and was negatively correlated with miR‑195 expression. In addition, the rescue experiment revealed that overexpression of VEGFA reversed the inhibitory effects of miR‑195 overexpression on the invasion ability and tube formation of HUVECs. The present study has provided a novel insight into the promoting roles of miR‑195 downregulation on angiogenesis following cerebral infarction and suggests that the miR‑195/VEGFA signaling pathway is a putative therapeutic target in cerebral ischemia.

Introduction

Cerebral infarction is a kind of acute ischemic cerebrovascular disease with high morbidity, high disability rate and high recurrence rate, accounting for 60–80% of the total number of stroke (1). Ischemia irreversibly resulted in substantial neuronal degeneration and necrosis and caused severe neurological deficits. It has been proved that promoting angiogenesis in ischemic brain to increase the number of new collateral circulation will increase the blood supply and improve the ischemic brain function (2). However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood.

MicroRNAs (miRNAs) are endogenous, evolutionarily conserved, and single chain noncoding small RNAs consisting of about 22 nucleotides, which inhibit mRNA transcription or induce mRNA degradation (3,4). Emerging evidence has demonstrated an important role of miRNAs in angiogenesis and other aspects of endothelial cell behavior (5). For example, Guduric-Fuchs et al found that miR-21 promoted the tube-forming capacity of primary bovine retinal microvascular endothelial cells (6), and its inhibition restricted the antiangiogenic effect of cardamonin on human umbilical vein vascular endothelial cells (HUVECs) (7). Furthermore, a study performed by van Mil et al showed that upregulation of miR-1 expression enhanced the tubes formation of human-derived cardiomyocyte progenitor cells (hCMPCs) (8). However, limited studies have pay attention on the role and underlying mechanisms of miRNAs in cerebral infarction. miR-195 is a miRNA that is dysregulated in several cancers and is correlated with angiogenesis (9–11). A recent study demonstrated that suppression of miR-195 promoted angiogenesis of hepatocellular carcinoma (HCC) by inhibiting the expression of VEGF (12). Vascular endothelial growth factor A (VEGFA) signaling is involved in vasodilation, proliferation, permeability, migration and survival of endothelial cells and available evidence has suggested that activation of this pathway in endothelial cells could promote angiogenesis (13). However, whether suppression of miR-195 promotes angiogenesis induced by cerebral infarction remains unclear.

In this study, the miRNA expression profiles in rat brain after middle cerebral artery occlusion (MCAO) are analyzed using microarray assay. Further results demonstrate that miR-195 is downregulated, and that miR-195/VEGFA axis is a key factor in the regulation of angiogenesis.

Materials and methods

Animals and the MCAO model

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.

Cell culture and treatment

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.

MiRNA microarray

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.

Cell transfection

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.

Plasmid construction

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.

Quantitative real-time polymerase chain reaction (qRT-PCR)

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.

Western blotting

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.

In vitro tube formation assay

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.

Cell migration assay

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.

Luciferase reporter assays

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.

Statistical analysis

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.

Results

miR-195 is downregulated in rat brains after MCAO. In recent developments, miRNA expression profiling has been examined in stroke, and these studies indicate that miRNAs have emerged as key mediators in ischemic stroke biology (18,19). To explore the role of miRNAs in cerebral ischemia, we profiled miRNA expression patterns through miRNA microarray analysis using rat brain isolated from rat subjected to MCAO, which is a classic model to induce cerebral infarction. A summary of these differentially expressed genes is presented in Fig. 1A. Among the aberrantly expressed miRNAs, miR-195 was one of the most robustly reduced of these miRNAs. In addition, previous studies have been reported that miR-195 play important roles in the angiogenesis of endothelial cells, mesenchymal stromal/stem cells and cancer cells (12,20,21). Therefore, miR-195 was chosen for further study.

Figure 1.

The expression of miR-195 is significantly downregulated after MCAO in rats. (A) The hierarchical clustering of miRNAs differentially expressed in rat brain after MCAO compared with the sham group. (B) miR-195 mRNA level was decreased in rat (n=10) undergoing MCAO. **P<0.01 vs. sham group. (C) Pearson analysis for the correlation of VEGFA and miR-195 expression levels in rat brain (n=10; r=0.9247; P<0.01). MCAO, middle cerebral artery occlusion; VEGFA, vascular endothelial growth factor A.

To validate the microarray results, miR-195 expression in rat brain tissues after ischemic injury was determined using qRT-PCR, and miR-195 expression was markedly lower in the ischemic group than in the sham control group (Fig. 1B). These finding suggests that downregulation of miR-195 may be involved in cerebral ischemia.

miR-195 regulates cell migration and tube formation in vitro

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.

Figure 2.

miR-195 regulates migration and tube formation of HUVECs. (A) HUVECs were treated with hypoxia for the indicated time-points. The relative expression level of miR-195 was detected by qPT-PCR. (B) miR-195 mimics and miR-195 inhibitor were transfected into HUVECs. After 24 h, cells were treated with hypoxia for 12 h. The relative expression level of miR-195 was detected by qRT-PCR. (C and E) HUVECs were transfected with miR-195 mimic, miR-195 inhibitor or miR mimic scramble. After 24 h, cells were treated with hypoxia for 12 h. The tube formation assay was performed as described in Materials and methods. The length of the tubes was quantified. (D and F) HUVECs were transfected as in C and E, and then cells were treated with hypoxia for 24 h. The cell migration assay was performed as described in Materials and methods. The histogram represents the quantification of cells that migrated. All data are expressed as the mean ± SD. *P<0.05; **P<0.01 vs. normoxia, ##P<0.05 vs. inhibitor NC or mimic NC. HUVECs, human umbilical vein vascular endothelial cells.

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.

VEGFA is a direct target of miR-195

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.

Figure 3.

VEGFA is a direct target of miR-195. (A) Schematic of the VEGFA 3′UTR containing the miR-195 binding sites. (B) Relative luciferase activity in HEK-293T cells co-transfection with wild-type or mutant-type 3′UTR VEGFA reporter plasmids and miR-195 or miR-NC. wt, wild-type; Mut, mutant-type. (C and D) The expression levels of VEGFA protein and mRNA after treatment with miR-195 mimics, miR-195 inhibitor or miR NC. All data are expressed as the mean ± SD. **P<0.01 vs. mimics NC, ##P<0.01 vs. inhibitor NC. VEGFA, vascular endothelial growth factor A.

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.

miR-195 promoted angiogenesis after cerebral ischemic by targeting VEGFA

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.

Figure 4.

pcDNA-VEGFA reversed the inhibitory effect of miR-195 overexpression on migration and tube formation of HUVECs. (A) HUVECs were transfected with miR-195 mimic and pcDNA-VEGFA. After 24 h, cells were treated with hypoxia for 12 h. The tube formation assay was performed as described in Materials and methods. The length of the tubes was quantified. (B) HUVECs were transfected as in A, and then cells were treated with hypoxia for 12 h. The cell migration assay was performed as described in Material and Methods. All data are expressed as the mean ± SD. *P<0.05, **P<0.01 vs. normoxia, ##P<0.05 vs. mimic NC. VEGFA, vascular endothelial growth factor A; HUVECs, human umbilical vein vascular endothelial cells.

Discussion

In the present study, we found that miR-195 was downregulated and inversely correlated with VEGFA in rat brain after MACO. Moreover, we demonstrated that miR-195 promoted cell migration and tube formation by targeting VEGFA in vitro. Taken together, these results suggest that miR-195 is involved in the regulation of angiogenesis after cerebral ischemia and maybe as a potential therapeutic target for cerebral infarction.

Emerging evidence has demonstrated that a number of miRNAs had been involved in the regulation of angiogenesis (26,27). For example, miR-21 has been found to induce tumor angiogenesis by enhancing VEGF expression (28). A study performed by Wang et al showed that the endothelial cell-restricted miR-126 promoted angiogenesis via direct suppression of Sproutyrelated protein 1 (Spred-1, encoded by SPRED1 gene), a negative regulator of VEGF signaling (29). However, evidence is limited about the role of miRNAs in regulation of angiogenesis after cerebral ischemia. A recent study from Lou et al showed that miR-210 promoted angiogenesis in HUVEC Matrigel cultures by enhancing the expression of VEGF and VEGFR-2 (30). In this study, we identified the miRNA expression profile in rat brain in comparison with the sham group by miRNA microarray, and miR-195 was found significantly down-regulated. Our data imply its potential role in cerebral ischemia.

We focused on miR-195, as this miR has been previously reported as a miR with potential angiogenic activity. In a previous study, miR-195 down-regulation resulted in enhanced VEGF levels in the tumor microenvironment, which subsequently activated VEGF receptor 2 signaling in endothelial cells and thereby promoted angiogenesis (12). In another study, miR-195 inhibition promoted cell proliferation, migration and angiogenesis of human endothelial progenitor cells (hEPCs) under hypoxia (31). In line with these studies, our data demonstrated that downregulation of miR-195 promoted angiogenesis of HUVECs under hypoxia, indicating that miR-195 is a potential promising factor for regulating angiogenesis after cerebral ischemia.

It is well known that VEGF are involved in the signaling pathway that regulates angiogenesis (25,32). Among VEGF family members, VEGFA is the primary and important member in angiogenesis (33). Yan et al found that miR-210 is involved in isoprenaline-mediated angiogenesis through upregulation of VEGFA expression (34). miR-1 and miR-206 negatively regulates developmental angiogenesis in zebrafish by inhibiting VEGF-A (35). In addition, VEGFA protein is a chemical signaling molecule that plays a central role in tumor-induced angiogenesis and is also a target of antiangiogenic therapies (36). For instance, miR-1 regulated osteosarcoma angiogenesis by targeting VEGFA (37). Therefore, we speculate that regulation of VEGFA might be involved in miR-195-induced angiogenesis. Interestingly, according to the bioinformatics analysis, VEGFA was predicated as a direct target of miR-195 and validated by a dual luciferase experiment. Meanwhile, an inverse relationship was observed between miR-195 and VEGFA in rat brain samples, suggesting that miR-195 negatively regulated the expression of VEGFA. Moreover, VEGFA overexpression reversed the inhibitory effects of miR-195 mimic on cell migration and tube formation of HUVECs. Therefore, VEGFA may be required for the function of miR-195 in regulating angiogenesis.

In conclusion, we found a downregulation of miR-195 in rat brain after MACO and highlighted the function of miR-195 in the angiogenesis. The identification of the miR-195-regulated VEGFA pathway may provide a new insight into the potential molecular mechanisms of cerebral infarction.