EGM-2 medium, fetal bovine serum (FBS), Lipofectamine^® 2000, MTT, dimethyl sulfoxide (DMSO), TRIzol^® reagent and a miRNA Reverse Transcription kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The All-in-One™ miRNA qRT-PCR Detection kit was purchased from GeneCopoeia, Inc. (Rockville, MD, USA). miR-138 mimics, miR-138 inhibitor and scramble miR mimics were purchased from Genechem, Co., Ltd. (Shanghai, China). Rabbit anti-HIF-1α polyclonal antibody (ab69836), rabbit anti-VEGF polyclonal antibody (ab69479), rabbit anti-phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK) polyclonal antibody (ab60999), rabbit anti-p38 MAPK polyclonal antibody (ab31828), rabbit anti-p-AKT monoclonal antibody (ab81283), rabbit anti-AKT monoclonal antibody (ab32505), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (ab9485) and mouse monoclonal 2A9 Anti-Rabbit IgG heavy chain (HRP) (ab99702) were all purchased from Abcam (Cambridge, MA, USA). The Pierce ECL Western Blotting KIT and polyvinylidene difluoride membrane were purchased from Pierce; Thermo Fisher Scientific, Inc. pRL-TK plasmid, lysis buffer and the Dual-Luciferase^® Reporter assay system were purchased from Promega Corp. (Madison, WI, USA).

An umbilical cord (UBC) was donated by a patient that had just given birth in the Second Affiliated Hospital of Nangchang University (Nanchang, China) and informed consent was obtained. The UBC was homogenized and immersed in Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Inc.) at a ratio of 1:1 and overlaid onto 1.077 g/ml Ficoll (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), followed by centrifugation at 4°C for 30 min at 400 × g. UBC-monocytes were collected and seeded into tissue culture plates coated with fibronectin (EMD Millipore, Billerica, MA, USA) and cultured in EGM-2 (Lonza Group, Basel, Switzerland) at 37°C, in a humidified incubator with 5% CO₂. The culture medium was changed every 2 days until EPC colonies appeared. Typically, colonies appeared between day 5 and 10, and were passaged at sub-confluence.

EPCs (5×10⁷) were cultured in serum-free EGM-2 at 37°C and 5% CO₂ for 12 h prior to hypoxia treatment. Hypoxia treatment consisted of EPCs undergoing culture in EGM-2 containing 20% fetal bovine serum at 37°C, in a humidified incubator containing 1% O₂, 94% N₂ and 5% CO₂. Hypoxia treatment lasted for 3 h prior to subsequent analyses.

An MTT assay was performed to determine cell proliferation. In brief, 10 mg/ml MTT was added to the medium. Following 4 h of incubation, the reaction was terminated by removal of the supernatant and addition of 100 µl DMSO to dissolve the formazan product. After 30 min, the optical density of each well was measured at 570 nm using an ELx808 absorbance microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

EPCs were resuspended in 70% ethanol. Following fixation overnight at −20°C, cells were pelleted, washed twice in 1X PBS with 3% bovine serum albumin (BSA; Thermo Fisher Scientific, Inc.) and pelleted at 1,000 × g for 5 min at 4°C. Subsequently, EPCs were resuspended and incubated for 30 min at room temperature in propidium iodide (PI) staining buffer containing 3% BSA, 40 µg/ml PI and 0.2 mg/ml RNase in 1X PBS. DNA content analyses were performed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).

TRIzol reagent was used to extract total RNA from cells following the manufacturer's instructions. Total RNA was transcribed into complementary (c)DNA using the miRNA Reverse Transcription kit following the manufacturer's protocols. Reverse transcription was performed at 16°C for 30 min, followed by an incubation step at 42°C for 30 min and enzyme inactivation at 85°C for 5 min. miRNA levels were determined using the All-in-One™ miRNA qRT-PCR Detection kit on an ABI 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's instructions. For qPCR, 0.33 µl cDNA solution, 10 µl 1X TaqMan universal PCR master mix, 2 µl 1X gene-specific primer (Thermo Fisher Scientific, Inc.) and 7.67 µl H₂O were mixed to obtain a final reaction volume of 20 µl. The reaction conditions were 95°C for 5 min, and 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation step at 60°C for 60 sec. The U6 gene was used as an internal reference. The relative expression was analyzed by the 2^−ΔΔCq method (14).

Transfection was performed using Lipofectamine^® 2000. In brief, EPCs were cultured to 70% confluence and re-suspended in EGM-2 medium without FBS. miR-138 mimics, miR-138 inhibitor and Lipofectamine^® 2000 were all diluted with serum-free medium. The diluted Lipofectamine^® 2000 was added to the diluted miR-138 mimics or miR-138 inhibitor and incubated for 20 min at room temperature, prior to addition to the cell suspension. Following 6 h of incubation at 37°C in 5% CO₂, the medium was replaced by EGM-2 medium containing 20% FBS and EPCs were cultured for 24 h prior to further assays.

EPCs were solubilized in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.). Proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with Tris-buffered saline containing Tween-20 and 5% skimmed milk at 4°C overnight. Subsequently, the membrane was incubated for 3 h at room temperature with rabbit anti-HIF-1α monoclonal antibody (1:50), rabbit anti-VEGF monoclonal antibody (1:100), rabbit anti-p-p38 MAPK monoclonal antibody (1:50), rabbit anti-p38 MAPK monoclonal antibody (1:50), rabbit anti-p-AKT monoclonal antibody (1:50), rabbit anti-AKT monoclonal antibody (1:50) and mouse anti-GAPDH monoclonal antibody (1:100). Following washing with PBS containing Tween-20 for three times, the membrane was incubated with the mouse anti-rabbit secondary antibody (1:5,000) at room temperature for 1 h. The ECL kit was used to detect chemiluminescence. Relative protein expression was determined using Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.

Targetscan 3.1 online software (http://www.targetscan.org) was used to predict the putative target genes of miR-138. The species was ‘Human’ and ‘miR-138’ was entered as the miR name.

The wild-type (WT) and mutant (Mut) 3′UTR sequences of HIF-1α were designed by GeneCopoeia, Inc. and were inserted into a dual luciferase reporter vector (Promega Corp.). EPCs (5×10⁷) were seeded into 24-well plates and co-transfected with 200 ng pMIR-HIF-1α or pMIR-HIF-1α-Mut vector and 100 ng miR-138 mimic or scramble miR mimic, and the pRL-TK plasmid for internal normalization. Cells were harvested after 36 h and lysed using the lysis buffer. A luciferase reporter gene assay was perfored using the Dual-Luciferase Reporter assay system, following the manufacturer's instructions.

Values are expressed as the mean ± standard deviation of three independent experiments. Statistical analysis of differences was performed using Student's t-test with SPSS version 17 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of hypoxia treatment on the proliferation capacity of EPCs was examined. An MTT assay was performed to determine EPC proliferation under hypoxia or normoxia. The proliferation of EPCs was significantly upregulated following culture under hypoxia for 12, 24, 48, 72 and 96 h, compared with the proliferation of cells cultured under normoxia (P<0.01; Fig. 1A), demonstrating that hypoxia enhances EPC proliferation. It has been suggested that HIF-1α is associated with EPC proliferation (11); therefore, the levels of HIF-1α expression in EPCs cultured under hypoxic or normoxic conditions were determined. Hypoxia was found to significantly enhance the levels of HIF-1α mRNA and protein in EPCs compared with the control group (P<0.01; Fig. 1B and C).

The role of miR-138 in regulating hypoxia-induced EPC proliferation was investigated. EPCs were transfected with miR-138 mimics or scramble miR mimics as a negative control (miR-NC). Following transfection, RT-qPCR was performed to determine the expression of miR-138 in each group. The results demonstrated that miR-138 expression was significantly increased in EPCs transfected with miR-138 mimics, compared with those transfected with the miR-NC and the control group, indicating that the transfection was successful (P<0.01; Fig. 2A). EPCs in each group were cultured under hypoxic conditions for 12, 24, 48, 72 and 96 h. The results indicated that proliferation was significantly decreased in EPCs overexpressing miR-138 compared with that of the control group (Fig. 2B). These findings indicated that upregulation of miR-138 inhibits hypoxia-induced EPC proliferation.

Subsequently, the cell cycle distribution of EPCs in each group was examined. The results showed that the percentage of cells in the G1 stage was significantly higher in the miR-138 group, compared with that in the miR-NC and control groups (P<0.01; Fig. 3). This suggested that overexpression of miR-138 induces cell cycle arrest at G1, resulting in the decreased proliferation of EPCs.

It has been demonstrated that the expression of HIF-1α is negatively regulated by miR-138 in ovarian cancer cells and clear cell renal cell carcinoma cells (12,13). However, it remains elusive whether miR-138 affects the expression of HIF-1α. Therefore, in the present study, EPCs were transfected with miR-138 mimics or miR-138 inhibitors. Following transfection, RT-qPCR was performed to determine the level of miR-138 expression in each group. Compared with the control group, the expression of miR-138 was significantly increased in EPCs transfected with miR-138 mimics but reduced in EPCs transfected with miR-138 inhibitor, compared with the control (P<0.01; Fig. 4A). Subsequently, RT-qPCR and western blotting analysis were performed to examine HIF-1α mRNA and protein expression, respectively, in each group. Neither miR-138 overexpression or miR-138 knockdown affected HIF-1α mRNA expression in EPCs (Fig. 4B). However, compared with the control group, overexpression of miR-138 led to a significant decrease in HIF-1α protein levels, while knockdown of miR-138 significantly increased HIF-1α protein levels (P<0.01; Fig. 4C). These findings indicated that miR-138 downregulated the expression of HIF-1α in EPCs at the post-transcriptional level.

Bioinformatical analysis using Targetscan indicated that HIF-1α was a potential target gene of miR-138. To confirm this prediction, WT or Mut 3′UTR sequences of HIF-1α (Fig. 5A) were respectively inserted into the dual luciferase reporter vector (Fig. 5B). Subsequently, miR-138 mimic/miR-NC and the reporter plasmids driven by the WT/MUT sequence from the 3′UTR of HIF-1α were co-transfected into EPCs. The results of the dual luciferase reporter assay demonstrated that luciferase activity was significantly repressed in the cells co-transfected with miR-138 mimic and plasmid containing the WT 3′UTR sequence of HIF-1α, compared with that in the control group (P<0.01; Fig. 5C). However, the miR-138-mediated repression of luciferase activity was abolished in the group transfected with the reporter plasmid driven by the MUT 3′UTR sequence of HIF-1α (Fig. 5C). These results demonstrated that miR-138 directly targets the HIF-1α 3′UTR in EPCs.

The effect of miR-138 upregulation on hypoxia-induced HIF-1α expression in EPCs was assessed. The expression of HIF-1α mRNA and protein was lower in miR-138-overexpressing EPCs cultured under hypoxia, when compared with non-transfected EPCs and EPCs transfected with miR-NC cultured under hypoxia (P<0.01; Fig. 6). These findings indicated that miR-138 suppresses hypoxia-induced HIF-1α upregulation in EPCs.

It has been demonstrated that VEGF, MAPK and AKT signaling are associated with EPC proliferation (15). Therefore, the effect of miR-138 overexpression on the expression of VEGF, as well as the activity of p38 MAPK and AKT signaling in EPCs cultured under hypoxia, was investigated. Hypoxia treatment significantly enhanced the expression of VEGF protein in EPCs compared with EPCs cultured under normoxic conditions; however, transfection with miR-138 mimics significantly reduced the expression of VEGF protein in EPCs cultured under hypoxia compared with untransfected cells cultured under hypoxia (P<0.01; Fig. 7A). Hypoxia treatment significantly enhanced the levels of phosphorylated p38 MAPK and AKT protein in EPCs, when compared with those in EPCs cultured under normoxia (P<0.01; Fig. 7B and C), indicating that the activity of p38 MAPK and AKT was upregulated. However, overexpression of miR-138 inhibited the hypoxia-induced upregulation of phosphorylated protein levels of p38 MAPK and AKT in EPCs (P<0.01; Fig. 7B and C). Taken together, these results suggested that miR-138 inhibits the hypoxia-induced upregulation of VEGF expression as well as MAPK and AKT signaling.