Polymerase chain reaction (PCR) was used to obtain the VEGF gene expression sequence (NM_001171626), which was inserted into a LV5 vector (Shanghai GenePharma, Co., Ltd., Shanghai, China) using the Not I/Nsi I restriction enzymes to construct the LV-VEGF expression vector, as described previously (15). LV-NC served as the control vector. The VEGF-specific short hairpin (sh)RNA sequence, TTC TCC GAA CGT GTC ACGT, was obtained from Shanghai GenePharma, Co., Ltd. Following infection of the AML cell lines, K562, HL-60 and U937 (Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China) with the lentiviral vectors or VEGF-specific shRNA, infected cells were selected using purimycin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and untransfected cells were removed 48 h later, as described previously (16).

Total RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described previously (17). Total RNA was dissolved in diethylpyrocarbonate water and the optical density was measured at 260 and 280 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). The PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Biotechnology Co., Ltd., Dalian, China) was used for cDNA production from RNA, according to the manufacturer's protocol (18). The SYBR^® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Biotechnology Co., Ltd.) kit was used for qPCR on a StepOnePlus™ Real-Time PCR instrument (Thermo Fisher Scientific, Inc.). The cycling conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 34 sec. The primers were as follows: VEGF forward, 5′-CGCAGCTACTGCCATCCAAT-3′ and reverse, 5′-GTGAGGTTTGATCCGCATAATCT-3′; and GAPDH forward, 3′-ACCCAGAAGACTGTGGATGG-5′; and reverse, 3′-TCTAGACGGCAGGTCAGGTC-5′. The relative expression of the gene was calculated using the 2^−ΔΔCq method (19), with GAPDH as an internal control gene.

After reaching 80–90% confluence, the cells in culture plates were lysed using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1 mM phenylmethylsulfonyl fluoride protease inhibitor (Beyotime Institute of Biotechnology). The concentrations of proteins were determined using the bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). The proteins were boiled following addition of the sample loading buffer and the denatured proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology), as previously described (20). Subsequently, the membranes were incubated with primary antibodies against VEGF (cat. no. ab106041; 1:1,000 dilution; Abcam, Cambridge, MA, USA) and β-actin (cat. no. ab106045; 1:5,000 dilution; Abcam), followed by incubation with the secondary antibody (cat. no. ab6721; 1:5,000 dilution; Abcam). The bands of proteins were visualized using enhanced chemiluminesence (EMD Millipore, Billerica, MA, USA) and detected with the ChemiDoc MP imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total RNA was extracted from cells showing upregulation or downregulation of VEGF using the mirVana PARIS miRNA isolation kit (Ambion; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. After quantitation of the RNA using the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc.), microRNA expression profiling was performed using the Exiqon A/S Technology Platform (Exiqon A/S, Vedbaek, Denmark). MicroRNAs were labeled using the miRCURY™ Hy3™/Hy5™ Power kit and hybridized onto the miRCURY™ LNA (v.18.0) (Exiqon A/S). After washing with the washing buffer in the kit, the slides were scanned using the Axon GenePix 4000B Microarray Scanner, and the data were analyzed using GenePix Pro 6.0 software (Molecular Devices, LLC, Sunnyvale, CA, USA) (21,22).

The protocol of this study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. Written informed consent was obtained from all patients. In this study, 45 bone marrow samples from patients with AML were collected at our hospital from 2013–2014, in strict accordance with the approved protocol. The information of the patients in this experiment is summarized in Table I. Total RNA, including small RNAs, was isolated using the mirVana PARIS miRNA isolation kit from blood mononuclear cells isolated using Ficoll-Paque PLUS (GE Healthcare, Milwaukee, WI, USA), as described previously (23). RT-qPCR for the analysis of microRNAs was performed using the miScript SYBR Green PCR kit (Qiagen GmbH, Hilden, Germany). c-Abl served as the internal control gene. The relative expression levels of microRNAs were calculated using the 2^−ΔΔCq method (19). Furthermore, the mRNA expression levels of VEGF in the bone marrow samples were determined by RT-qPCR. The expression levels of VEGF were subsequently ranked from maximum to minimum and then divided into high and low groups.

SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. One-way analysis of variance was performed for the comparisons of three groups and Student's t-test for two groups. P<0.05 was considered to indicate a statistically significant difference.

To confirm that cells with stable knockdown and overexpression of VEGF were obtained, we extracted total protein for western blotting and total RNA for RT-qPCR. The RT-qPCR analysis showed that the mRNA expression levels of VEGF were significantly higher in K562-VEGFa-O, HL-60-VEGFa-O and U937-VEGFa-O cells compared with the control cells (K562-NC-LV, HL-60-NC-LV and U937-NC-LV) (P<0.01), and that those of K562-VEGF-shRNA, HL-60-VEGF-shRNA and U937-VEGF-shRNA cells were significantly lower compared with the control cells (P<0.01; Fig. 1A). In addition, the results of western blotting confirmed that the VEGF protein had a pattern of expression that resembled that of its mRNA (Fig. 1B).

For the detection of microRNA profiles, total RNA was extracted from the cells for microarray analysis. Genes that were screened using the microarray needed to meet the criteria of ≥2-fold increase or decrease in expression in the cells with overexpression or knockdown of VEGF compared with the controls. Overexpression of VEGF in leukemia cells (K562-VEGFa-O, HL-60-VEGFa-O and U937-VEGFa-O), despite being upregulated by 3-fold, had little effect on microRNA expression; only four microRNAs were identified as being altered in expression, including has-miR-1273f and has-miR-4679, which were upregulated, and eb-miR-BART10-3p and has-miR-3925-3p, which were downregulated (Fig. 2A).

In K562-VEGF-shRNA, HL-60-VEGF-shRNA and U937-VEGF-shRNA cells, microRNA profiling showed a significant change compared with control cells (K562-NC-LV, HL-60-NC-LV and U937-NC-LV). Among several microRNAs that showed a 5-fold change in expression, hsv2-miR-H12, has-miR-124-5p, has-miR-3924 and has-miR-4514 were upregulated, and 33 microRNAs, including has-miR-20a, were downregulated (Fig. 2B).

In the present study, several microRNAs showed changes in their levels of expression following overexpression or silencing of the expression of the VEGF gene. To confirm these results, bone marrow samples from 45 patients with AML were used to analyze the expression levels of microRNAs identified in the microarray and VEGF. Subsequently, the expression levels of VEGF were defined into low and high groups. The results indicated a positive association between VEGF expression and the expression of six microRNAs, including miR-20a, miR-93, miR-16-5p, miR-17-5p, miR-17b-3p and miR-124-5p. In addition, a negative association was observed between VEGF expression and the expression of miR-17b-3p (Fig. 3).