Down-regulation of miR-361-5p promotes the viability, migration and tube formation of endothelial progenitor cells via targeting FGF1

Abstract Transplantion of bone marrow-derived endothelial progenitor cells (EPCs) may be a novel treatment for deep venous thrombosis (DVT). The present study probed into the role of microRNA (miR)-361-5p in EPCs and DVT recanalization. EPCs were isolated from male Sprague–Dawley (SD) rats and identified using confocal microscopy and flow cytometry. The viability, migration and tube formation of EPCs were examined using MTT assay, wound-healing assay and tube formation assay, respectively. Target gene and potential binding sites between miR-361-5p and fibroblast growth factor 1 (FGF1) were predicted by StarBase and confirmed by dual-luciferase reporter assay. Relative expressions of miR-361-5p and FGF1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. A DVT model in SD rats was established to investigate the role of EPC with miR-361-5p antagomir in DVT by Hematoxylin–Eosin (H&E) staining. EPC was identified as 87.1% positive for cluster of difference (CD)31, 2.17% positive for CD133, 85.6% positive for von Willebrand factor (vWF) and 94.8% positive for vascular endothelial growth factor receptor-2 (VEGFR2). MiR-361-5p antagomir promoted proliferation, migration and tube formation of EPCs and up-regulated FGF1 expression, thereby dissolving thrombus in the vein of DVT rats. FGF1 was the target of miR-361-5p, and overexpressed FGF1 reversed the effects of up-regulating miR-361-5p on suppressing EPCs. Down-regulation of miR-361-5p enhanced thrombus resolution in vivo and promoted EPC viability, migration and angiogenesis in vitro through targeting FGF1. Therefore, miR-361-5p may be a potential therapeutic target for DVT recanalization.


Introduction
Deep venous thrombosis (DVT) refers to the formation of a blood clot within a deep vein in contrast with venous thromboembolism, which includes superficial thrombophlebitis and pulmonary embolism [1]. DVT will lead to morbidity and mortality in several conditions [2]. Anti-coagulation is a major therapeutic strategy for DVT, but it cannot dissolve thrombus and restore the function of valves, at the same time, thrombosis may occur with post-thrombotic syndrome (PTS) [3]. Thus, it is of great significance to explore a new therapeutic method for DVT treatment.
Recent discoveries showed that successful DVT-related thrombi resolution plays a key role in DVT treatment [4], however, detailed mechanisms remained obscure. Endothelial progenitor cells (EPCs), derived from bone marrow and resident to tissues, act as precursor for endothelial cells. EPCs have the ability to differentiate into mature endothelial cells and play a major role in vascular integrity maintenance and endothelial damage as well as the resolution of thrombus in vivo [5][6][7]. EPCs can be homed and integrated into the injured blood vessel and thrombus to secrete angiogenesis factors, thus increasing the formation of new blood vessels and improving the resolution of vascular thrombosis when DVT occurs [2]. Therefore, the effective recruitment of EPCs into thrombus may help treat DVT.
As previous study demonstrated, microRNAs (miRNAs, miRs) are involved in the biological functions of EPCs [8]. MiRNAs are a highly conserved family of small non-coding RNAs (ncRNAs) with 19-25 nucleotides in length and regulate the gene expression through the combination with 3 -untranslated region (3 -UTR) of target messenger RNAs (mRNAs) [9]. MiR-361-5p, in particular, has been reported to suppress many human cancers progression, such as hepatocellular carcinoma [10], hemangioma [11] and papillary carcinoma [12]. According to Wang et al.'s study, miR-361-5p suppresses vascular endothelial growth factor (VEGF) expression and EPC activity [13]. We predicted that the target gene of miR-361-5p was fibroblast growth factor 1 (FGF1), which aroused our research interest. FGF1, which plays a part in cell proliferation, migration, invasion and angiogenesis, can promote the viability of EPCs and neovascularization [14,15]. Herein, in our study, the roles of miR-361-5p in the development and progression of DVT were explored through examining their regulatory functions in EPCs based on an animal model, hoping to find a potential therapeutic method for DVT.

Materials and methods
EPCs isolation and culture EPC isolation was conducted following a previous description [16]. Male Sprague-Dawley (SD) rats (3 weeks old, 80-100 g) were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China) and kept in microisolator cages under a 12-hour (h) day/night cycle at 23 • C with free access to standard laboratory diet and tap water for 2 weeks before our experiment. Then, after 2 weeks of stabilization, five male SD rats were anesthetized and sacrificed with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Subsequently, bone marrow of the rats was harvested via femurs and tibias. Mononuclear cells were available from density-gradient centrifugation with Ficoll-Paque (GE Healthcare, Piscataway, NJ, U.S.A.). EPCs at the density of 0.8-1.0 × 10 6 cells/cm 2 were inoculated to the culture flask and cultured with microvascular endothelial cell growth-2 (EGM-2) medium; catalog number: CC-3125; Lonza, Greenwood, SC, U.S.A.), which contained penicillin-streptomycin (P4333, Sigma-Aldrich, St Louis, MO, U.S.A) at 37 • C with 5% CO 2 . Non-adherent cells were washed after the cell culture for 4 days, and the medium was refreshed every 2 days. EPCs in passage 3 were selected for subsequent experiments.

MTT assay
MTT assay was performed to measure the EPC viability. In brief, the EPCs (1 × 10 3 cells/well) were cultured in 96-well plates and 10 μl MTT assay kit (#30006, Biotium, Inc., Fremont, CA, U.S.A.) was added into the wells at 12, 24 and 48 h of the culture. The supernatant was discarded 4 h after incubation at 37 • C, and 100 μl dimethyl sulfoxide (DMSO; 472301, Sigma-Aldrich, U.S.A.) was added into the wells to dissolve formazan crystals. The OD values at an absorbance of 490 nm were measured and recorded by a microplate reader (Model 680, Bio-Rad, U.S.A.).

Wound healing assay
After 48-h cell transfection, the EPCs (1 × 10 4 cell/ml) were seeded in a 24-well tissue culture plate. The straight wound in the middle of the culture was then created by a sterile pipette tip after the cells reached 100% confluence. After washing the cells by PBS twice to smoothen the edge of scratch and the removal of the floating cells, the EPCs were incubated in an incubator at 37 • C with 5% CO 2 . Cell images at 0 and 48 h were captured under an inverted optical microscope (SW380T, Swift Optical Instruments, Schertz, TX, U.S.A.). Cell migration was measured by Image-Pro Plus Analysis software (Version 6.0, Media Cybernetics Company, U.S.A.).

Tube formation assay
The vascular formation of EPCs was evaluated using tube formation assay and Matrigel plug assay. Pre-heated matrigel (R&D Systems, Minneapolis, MN, U.S.A.) at 4 • C overnight was diluted with non-serum medium, layered in 96-well plates and incubated at 37 • C for 30 min to allow polymerization. Subsequently, the EPCs (2 × 10 4 cell/ml) were plated on to the Matrigel layer in EGM™-2 MV medium, and later the capillary-like structures formation was captured using an inverted microscope (IRB20, Microscope World, Carlsbad, CA, U.S.A.) with Tube formation ACAS Image Analysis Software (v.1.0, ibidi GmbH, Gräfelfing, Germany).

Target gene and dual-luciferase reporter assay
StarBase (http://www.starbase.sysu.edu.cn) predicted that the target gene of miR-361-5p was FGF1, which aroused our research interest. Previous study found that miR-361-5p plays an important role in cell proliferation and invasion [18]. FGF1 has also been found to promote the activity, proliferation, angiogenesis and anti-apoptosis of EPCs [15]. The predicted targeted relationship was subsequently confirmed by dual-luciferase reporter assay. PMIR-REPORT Luciferase vector (catalog number: AM5795; Thermo Fisher Scientific, U.S.A.) containing the sequences of wildtype or mutated FGF1 3 -UTR was cloned into the pMirGLO reporter vector (Promega, Madison, WI, U.S.A.) to form FGF1-WT and FGF1-MUT. A total of 1 × 10 4 cells/ml (EPCs) were then co-transfected with FGF1-WT and FGF1-MUT, miR-361-5p agomir and miR-NC-agomir by Lipofectamine 2000 Transfection reagent (Thermo Fisher Scientific, U.S.A.) at 37 • C. Renilla reporter gene in the luciferase reporter vector was used as an internal control. Then cells were harvested 48 h after the transfection for luciferase detection in dual-luciferase reporter assay system (E1910; Promega, Madison, WI, U.S.A.) following the producer's protocols. The firefly luciferase activity was normalized to that of Renilla luciferase activity.

Construction of animal model
SD rats (10 weeks old, 280-300 g) regardless of gender were obtained from Guangdong Medical Laboratory Animal Center (Foshan, Guangdong, China), and were then kept in specifically made pathogen-free animal rooms. For this, rat model construction was well described in previous studies [2,16]. In brief, the rats were anesthetized by intraperitoneal 7% pentobarbital injection and underwent midline laparotomy to dissect inferior vena cava (IVC) from aorta. IVC was subsequently ligated just below the upper renal vein using 7-0 Prolene sutures, meanwhile, the posterior venous branches were tightened. Then, confluence in iliac vein was discontinued using a pair of vascular clip for 15 min. After that, the incision was closed and the rats were allowed to recover after the surgery. The rats in Sham group received a dissection of IVC but without ligation.
Three days after the construction of animal model, the SD rats were divided into four groups at random (total number = 40; n=10 for each group): (A) Sham group received 2 ml EGM™-2 MV medium (Lonza, U.S.A.) injection with IVC exposure, (B) Model group received 2 ml EGM™-2 MV medium injection after model construction, (C) EPCs group received the injection of 1 × 10 6 EPCs containing miR-NC carriers via tail vein injection, (D) EPC+miR-361-5p antagomir group received injection of 1 × 10 6 EPCs via tail vein injection after transfection with miR-361-5p antagomir.

Histopathologic examination with Hematoxylin-Eosin (H&E) staining
The rats were anesthetized and sacrificed with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) 7 days after the injection. Segments of IVC containing the thrombus were harvested with caution and fixed in 4% paraformaldehyde, subsequently embedded in dissolved paraffin. Excess blood on thrombi was removed using filter paper. Specimens were finally stained with Hematoxylin-Eosin (H&E) and analyzed with a light inverted microscope (CKX53; Olympus, Tokyo, Japan) in the dark.

Statistical analysis
In our study, all the experiments were independently performed more than three times. The experimental data were expressed as mean + − standard deviation. Statistical analysis was performed with SPSS 21.0 software (IBM Corporation, Armonk, NY, U.S.A.). Normal distribution and variance homogeneity were tested for all the data. Comparison of differences between multiple groups was determined by one-way ANOVA. Comparison of differences between two groups was determined by Student's t test. P-value <0.05 was considered as statistically significant.

Culture and identification of EPCs
In line with previous studies, changes in the morphology and numbers of EPCs were observed under an inverted optical microscope. Shortly after the isolation, colony of Peripheral blood mononuclear cells (PBMCs) exhibited a round morphology and suspended in the medium ( Figure 1A). Then, 7 days after the culture, an elongated spindle-shaped morphology and formed central cluster was observed ( Figure 1A). Also, PBMCs began to merge in passage 3 ( Figure  1A). The isolated PBMCs were identified by confocal microscopy, and the double staining with functional marker FITC-UEA-I and Dil-Ac-LDL suggested that the isolated PBMCs were EPCs ( Figure 1B). EPCs were further characterized by flow cytometry. CD31, CD133, vWF and VEGFR2 were markers of EPCs [20], and therefore their expressions were measured using flow cytometry in order to confirm the identity of EPCs. In Figure 1C, the results from flow cytometry showed that P10 cells were 87.1% positive for CD31, 2.17% positive for CD133, 85.6% positive for vWF and 94.8% positive for VEGFR2, suggesting that the isolated mononuclear cells were EPCs.
To further uncover the effects of miR-361-5p on EPC functions, we measured the viability, migration and tube formation of EPCs after the transfection with miR-361-5p agomir or antagomir. MTT assay showed that the viability of EPCs at 24 and 48 h was reduced in miR-361-5p agomir group ( Figure 2B, P<0.01), while that in miR-361-5p antagomir group showed an opposite result at 24 and 48 h ( Figure 2B, P<0.05), indicating that down-regulating miR-361-5p could promote the cell viability. Then the migration of EPCs was determined using wound-healing assay. The data from the experiments revealed that relative migration rate of EPCs in miR-361-5p agomir group was decreased ( Figure 2C, P<0.001), while that in miR-361-5p antagomir group was increased ( Figure 2C, P<0.05), suggesting that down-regulating miR-361-5p expression promoted the migration of EPCs. Finally, the EPC tube formation was detected with tube formation assay, and it has been observed that both branch points and relative tube length in miR-361-5p agomir group were reduced ( Figure 2D, P<0.01), while that in miR-361-5p antagomir group was increased ( Figure 2D, P<0.01), suggesting that the EPC tube formation could be enhanced by down-regulating miR-361-5p.

FGF1 was the target of miR-361-5p and overexpressed FGF1 reversed the effects of miR-361-5p agomir on FGF1 expression
MiRNAs combine with 3 -UTR of target mRNAs to regulate gene expressions [9]. By applying StarBase, we successfully found that FGF1 might be a possible target of miR-361-5p, because it contained miR-361-5p binding sites at 3 -UTR ( Figure 3A). To further confirm that miR-361-5p could bind with FGF1, we built a luciferase reporter vector containing 3 -UTR. For the assay, the results demonstrated that the relative luciferase activity in FGF1-WT group was reduced in the presence of miR-361-5p agomir ( Figure 3B, P<0.001). However, no significant difference was detected in luciferase activity of miR-361-5p agomir in FGF1-MUT group ( Figure 3B). These results suggested that FGF1 was the target of miR-361-5p.

Overexpression of FGF1 reversed the inhibitory effects of miR-361-5p agomir on the viability, migration and tube formation of EPCs
To uncover the effects of miR-361-5p and FGF1 on the EPC viability, migration and tube formation, EPCs were transfected with miR-361-5p agomir or antagomir. MTT assay showed that the EPC viability was reduced after miR-361-5p agomir was transfected into the cells, while overexpressed FGF1 showed an opposite effect ( Figure 4C, P<0.001). In addition, overexpressed FGF1 reversed the effects of miR-361-5p agomir on the EPC viability ( Figure 4C, P<0.01). In wound-healing assay, relative migration of EPCs in miR-361-5p agomir+NC group was reduced, whereas overexpression of FGF1 led to an opposite result ( Figure 4D, P<0.001). Furthermore, overexpressing FGF1 in EPCs reversed the effects of miR-361-5p agomir on the cell migration ( Figure 4D, P<0.001). Moreover, from tube formation assay, it could be observed that the relative branch points and tube length of the EPCs were reduced following upregulating miR-361-5p ( Figure 4E, P<0.01), while overexpressed FGF1 resulted in an opposite effect ( Figure 4E, P<0.001), and overexpression of FGF1 in the EPCs reversed the effects of miR-361-5p agomir ( Figure 4E, P<0.01).

EPCs with miR-361-5p antagomir showed promotion on thrombus resolution in the vein
As shown in Figure 5, H&E staining showed a normal vein in Sham group, while in Model group, nucleated cells  (monocytes, endothelial cells and neutrophil granulocytes) were found entering the thrombus perimeter on day 7. Moreover, the red blood cells, platelets and fibrin were dried red in the center of thrombus in Model group. Besides, in EPC/miR-NC antagomir group, more nucleated cells and channels with reduced thrombus were detected on day 7 compared with Model group. In addition, in EPC/miR-361-5p antagomir group, more nucleated cells were found entering the thrombus. For EPC/miR-361-5p antagomir group, we observed small fracture in the perimeter of thrombus and the formation of tube structure and red blood cells. Collectively, the experimental results suggested that EPCs with miR-361-5p antagomir promoted thrombus resolution in the vein.

Discussion
Thrombosis, which referred to the blood clot formation inside blood vessels, could obstruct blood flow in the circulatory system [16]. Endothelial cells at normal state express the molecules with anticoagulant effect and inhibit the formation of fibrin [21]. Moreover, as endothelial cells may induce tissue repair and tube formation [22], it now plays an important role in thrombosis prevention and treatment. EPCs function as precursor cells for mature endothelial cells, and its potential to differentiate into all capillary niches allows it to contribute to vascularizing engineered tissues [23]. Many researches uncovered the relation between proliferation, migration and tube formation of EPCs and DVT recanalization. Mo et al. demonstrated that down-regulation on miR-195 could regulate the proliferation, migration, angiogenesis and autography of EPCs by targeting GABA type A receptor-associated protein-like 1 (GABARAPL1), and Li et al. discovered that miR-3120 was implicated in the mechanisms via which long non-coding RNA Wilms Tumor 1 Associated Protein Pseudogene 1 (LncRNA WTAPP1) promoted EPCs migration and angiogenesis [24,25]. In our study, consistent with previous discoveries, we found that EPCs transfected with miR-361-5p antagomir could partially promote thrombus resolution in vein.
Recently, the functions of miRNAs in the regulation of vascular development, homeostasis and differentiation have been widely explored both at home and abroad [26,27]. MiRNAs also affect EPC function in angiogenesis [28]. MiR-361-5p, in particular, has been found overexpressed in vascular cells, including EPCs, to inhibit their activities [13]. Wang et al. demonstrated that miR-361-5p could suppress EPCs activities via targeting VEGF in patients with coronary artery diseases [29]. In our present study, we conducted a series of studies on the biological behaviors of EPCs at the cellular level, and found that after miR-361-5p antagomir was transfected into EPCs, the viability, migration and tube formation of EPCs were promoted, suggesting that down-regulating miR-361-5p may have promoting effects on EPCs, which was consistent with previous studies [29]. However, upregulating miR-361-5p significantly reduced the viability, migration and angiogenesis of EPCs. Such a result encouraged us to further study its mechanism of action. Using the starBase site, we predicted the target genes and potential binding sites of miR-361-5p. FGF1, which was predicted as a target for miR-361-5p in the present study, has been found to promote activity, proliferation and angiogenesis of EPCs [15]. Moreover, we also confirmed the targeting relationship between miR-361-5p and FGF1 by dual-luciferase reporter gene assay.
FGF1 is a member of FGF family and a growth factor involved in cell proliferation, migration, invasion and angiogenesis [14]. Up-regulating FGF1 expression modulates to ameliorate atherosclerosis [30]. FGF1 induced by ERK1/2 signaling could reciprocally regulate proliferation and smooth muscle cell differentiation of ligament-derived EPC-like cells [31]. In addition, adipose-derived mesenchymal stem cells (AD-MSCs) transfected with FGF1  has been found to promote angiogenic proliferation [32]. However, the relation of FGF1 with miR-361-5p was hardly discussed. In our studies, according to the results of bioinformatics analysis, a binding site between FGF1 and miR-361-5p was identified, suggesting that FGF1 was the target of miR-361-5p. Then we discovered that down-regulation of miR-361-5p expression could promote FGF1 expression and FGF1 overexpression reversed the effects of up-regulating miR-361-5p on inhibiting the viability, migration and tube formation of EPCs. MiRNAs also participate in promoting DVT recanalization and resolution [28], but the effects of miR-361-5p in promoting DVT recanalization has not been examined yet. The results of in vitro experiments showed that miR-361-5p regulated the proliferation, migration and angiogenesis of EPCs through FGF1. Next, we carried out in vivo experiments to further explore the effect of miR-361-5p on venous thrombosis. In our studies, we found that miR-361-5p played an important role in DVT recanalization. Moreover, histopathological observation showed that EPCs transfected with miR-361-5p antagomir promoted DVT recanalization, indicating that down-regulating miR-361-5p expression in EPCs promoted thrombus resolution in vein, and that miR-361-5p could be a potential biomarker for DVT treatment.
Our study has some limitations that should be noted, with limited in vivo studies, the mechanisms of action of miR-361-5p on EPCs and DVT recanalization have not been fully elucidated, and this will be addressed in our future studies. The present study did not identify the aging of cells, which is also one of the limitations.
In conclusion, our studies revealed a novel role of miR-361-5p in DVT recanalization based on a rat model in vivo, and we also discovered that down-regulation of miR-361-5p expression played a vital role in promoting the viability, migration and tube formation of EPCs by targeting FGF1. Therefore, down-regulation of miR-361-5p could serve as a potential therapeutic strategy for DVT diagnosis and prognosis in clinical practice.