HUVECs were purchased from Cell Systems Corporation (Kirkland, WA, USA) and were cultured in Dulbecco's modified Eagle's medium (GE Healthcare Life Sciences, Chalfont, UK) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified atmosphere containing 95% air and 5% CO₂, with subconfluent monolayers passaged 3–10 times prior to treatment.

Hypoxic exposure was performed using a tightly sealed molecular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA), which was tightly sealed and flushed with a gas mixture containing 1% O₂, 94% N₂ and 5% CO₂, as previously described (15,16), with the cell culture dishes containing 1×10⁵ cells/well placed in the chamber and incubated at 37°C for 24 h.

The HUVECs were divided into four groups: A normoxia group; a hypoxia group; a hypoxia-control group, which was transiently transfected with scramble small interfering (si)RNA; and a hypoxia-CCN1 siRNA group, which was transiently transfected with CCN1 siRNA. The HUVECs were transiently transfected with plasmids (500 ng/µl) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h.

The PI3K/Akt inhibitor, LY294002, was used in the present study to determine the effect of inhibiting the PI3K/Akt pathway on the normoxic and hypoxic HUVECs. The cells were cultured under normoxic or hypoxic conditions in six-well plates at a density of 1×10⁵ cells/well as described above, in the presence of LY294002 (Sigma-Aldrich, St. Louis, MO, USA). The solution comprised 40 µmol/l dissolved in dimethyl sulfoxide (DMSO), with a final concentration of DMSO in the cell culture of 0.1%. The cells were pretreated with LY294002 for 30 min prior to being placed in the incubator for hypoxic exposure. The mRNA and protein expression levels of CCN1 were then analyzed using RT-qPCR and western blotting, respectively, following 24 h normoxia or hypoxia.

Four pairs of CCN1 siRNA sequences were designed and synthesized (Shanghai GenePharma Co., Ltd., Shanghai, China), with one pair selected based on stability and effectiveness. The sequences were as follows: CCN1 (Cyr61-homo-553) forward, 5′-GGGAAAGUUUCCAGCCCAACUTT-3′ and reverse, 5′-AGUUGGGCUGGAAACUUUCCCTT-3′; CCN1 (Cyr61-homo-789) forward, 5′-GAGGUGGAGUUGACGAGAAACTT-3′ and reverse, 5′-GUUUCUCGUCAACUCCACCUCTT-3′; CCN1 (Cyr61-homo-1072) forward, 5′-GCAAGAAAUGCAGCAAGACCATT-3′ and reverse, 5′-UGGUCUUGCUGCAUUUCUUGCTT-3′; CCN1 (Cyr61-homo-1268) forward, 5′-GAUGAUCCAGUCCUGCAAAUGTT-3′ and reverse, 5′-CAUUUGCAGGACUGGAUCAUCTT-3′. In addition, a non-silencing siRNA sequence was selected for use as a negative control (forward 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′ reverse). The siRNAs were cloned using a pGPU6/green fluorescent protein (GFP)/Neomycin resistance screening marker (Neo) siRNA Expression Vector kit (cat. no. E-07/F-07; Shanghai GenePharma Co., Ltd.), according to the manufacturer's protocol, generating the pGPU6/GFP/Neo-CNN1 siRNA and the pGPU6/GFP/Neo-scramble siRNA plasmids, which contained Bbs1 and BamH1 restriction sites. The cells were transfected, according to the manufacturer's protocol, with the mRNA and protein levels assessed 48 h following transfection. siRNA was successfully transfected into HUVECs in six-well culture plates, with each well containing 240 pmol fluorescent labelled siRNA and 8 µl Lipofectamine^® 2000 for 6 h. Transfection efficiency was determined using fluorescence microscopy (FV1000; Olympus Corp., Tokyo, Japan).

A Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, Jiangsu, China) was used to measure cell proliferation, according to the manufacturer's protocol. Briefly, HUVECs were plated in 96-well plates at a density of 2,000 cells/well, and proliferation was measured each day for 4 days following transfection. A total of 10 µl CCK8 was added to each well and incubated for 2 h at 37°C. Following incubation, the samples were vortexed for 10 min and the absorbance of each was measured in a Sunrise™ microplate reader (Tecan Group, Ltd., Männedorf, Switzerland) at 450 nm.

Cellular apoptosis was investigated by flow cytometry using an Annexin V-Fluorescein Isothiocyanate (FITC) Apoptosis Detection kit (cat. no. KGA106; Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China), according to the manufacturer's protocol. The cells were washed twice in ice-cold phosphate-buffered saline at pH 7.5 (Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) and resuspended in 1X binding buffer (Zhongshan Jinqiao Biotechnology Co., Ltd.) at 1×10⁶ cells/ml. A total of 100 µl cells (1×10⁵ cells) were gently mixed with 5 µl annexin V-FITC and 5 µl propidium iodide (PI), and incubated for 15 min in the dark at room temperature. An additional 400 µl of 1X binding buffer was added, and cellular apoptosis was detected using a flow cytometer (FACSCalibur™; BD Biosciences, San Jose, CA, USA). The apoptotic rates of the cells were calculated as the ratio of early and late apoptotic cells to the total cells (17).

Total RNA was isolated from the HUVECs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse-transcribed into cDNA using a reverse transcription kit (DRR037S; PrimeScript™ RT Reagent kit-Perfect Real-Time; Takara Bio Inc., Dalian, China) as previously described (18). Primers were designed using Primer Express software version 2.0 (Life Technologies; Thermo Fisher Scientific, Inc.) and are presented in Table I. qPCR was performed using SYBR Green PCR Master mix (Premix Ex Taq™-Perfect Real Time; cat. no. DRR041S; Takara Bio, Inc.). The PCR mixture contained 10 µl 2X TaqMan PCR mix, 0.4 µl PCR forward and 0.4 µl PCR reverse primer, 1.0 µl cDNA and 8.2 µl double-distilled H₂O with a total volume of 20 µl and the reaction was performed in an Applied Biosystems 7300 Real-Time PCR system (Thermo Fisher Scientific, Inc.). The cycling conditions were as follows: 95°C for 30 sec, 50 cycles of 95°C for 5 sec and 60°C for 31 sec. β-actin was included in each reaction as an internal control, and the relative gene expression levels were calculated using the 2^−ΔΔCq method (19).

The cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate and phenylmethylsulfonyl fluoride (all from Sigma-Aldrich), and protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology, Haimen, China). The samples (60 µg) were separated by 8% or 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). Following blocking with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline-Tween-20 [20 mM Tris-HCl, 500 mM NaCl and 0.05% Tween-20 (Yesen Biotechnology Co., Ltd., Shanghai, China); TBST], membranes were washed four times for 5 min with TBST, and were then incubated with the following specific primary antibodies overnight at 4°C: Rabbit anti-CCN1 polyclonal antibody (1:2,000 dilution; cat. no. ab24448; Abcam, Cambridge, UK); rabbit anti-phosphorylated (p)AKT1/2/3 (Ser473) polyclonal antibody (1:2,000 dilution; cat. no. sc-101629); rabbit anti-VEGF polyclonal antibody (1:2,000 dilution; cat. no. sc-152) and rabbit anti-mouse β-actin polyclonal antibody (1:2,000 dilution; cat. no. sc-130656) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, membranes were incubated for 2 h at 37°C with horseradish peroxidase-conjugated anti-rabbit-immunoglobulin G secondary antibodies (1:2,000 dilution; cat. no. ZB-2010; Zhongshan Jinqiao Biotechnology Co., Ltd.). Protein bands were visualized using enhanced chemiluminescence reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) and an MF-ChemiBIS 3.2 (DNR Bio-Imaging Systems, Ltd., Jerusalem, Israel). Optical density (OD) was quantified using ImageQuant LAS 4000 software (GE Healthcare Life Sciences). Protein concentrations were established by calculating the ratio between the ODs of the protein of interest and β-actin.

SPSS software, version 15.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Data are presented as the mean ± standard deviation of three independent experiments. Statistical significance was evaluated using one-way analysis of variance, with a least significant difference test for post-hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

At 6 h post-transfection with CCN1 siRNA, the percentage of GFP-positive HUVECs was >80% (Fig. 1A and B). RT-qPCR was performed to measure the mRNA expression of CCN1. Compared with the hypoxia-control, mRNA expression of CCN1 in the hypoxia-CCN1 siRNA group was downregulated by 78.21% (P<0.05; Fig. 2). Western blotting indicated that, compared with the hypoxia-control group, the protein expression of CCN1 in the hypoxia-CCN1 siRNA group was downregulated by 32.43% (P<0.05; Fig. 3).

The major hallmark of angiogenesis is endothelial cell proliferation (20); therefore, HUVEC proliferation was measured using a CCK8 assay. The proliferation rate was reduced in the hypoxia-CCN1 siRNA group, compared with the proliferation rates in the hypoxia and normoxia groups (P<0.05; Fig. 4). These results indicated that CCN1 siRNA has an anti-proliferative effect on HUVECs (21), possibly due to an anti-angiogenic effect.

To investigate whether hypoxia can induce apoptosis in HUVECs, cellular apoptotic ratios were measured using flow cytometry, in which apoptotic cells determined as annexin V-FITC positive and PI negative. The results of the flow cytometric analysis indicated a moderate increase in apoptosis in the HUVECs transfected with CCN1 siRNA (69.24±0.85%; P<0.05), compared with the HUVECs transfected with scramble siRNA (40.14±0.78%), under hypoxic (32.28±0.23%) or normoxic (18.68±0.43%) conditions (Fig. 5). These results indicated that CCN1 siRNA had a pro-apoptotic effect on HUVECs (21), possibly due to an anti-angiogenic effect.

The results of the RT-qPCR (Fig. 2) and western blot analysis (Fig. 3) indicated that the mRNA and protein levels of CCN1 and VEGF were increased in the hypoxia and hypoxia-control groups, compared with the normoxia group (P<0.05), however, no significant differences were observed between the hypoxia and hypoxia-control groups (P>0.05). The mRNA levels of Akt were increased, and western blotting indicated an increase in the expression levels of p-Akt in the hypoxia and the hypoxia-control groups, compared with the normoxia group. Additionally, the mRNA and protein expression levels were reduced in the hypoxia-CCN1 siRNA group, compared with the hypoxia and hypoxia-control groups (P<0.05; Figs. 2 and 3). Compared with the hypoxia-control, the hypoxia-CCN1 siRNA group demonstrated reduced mRNA expression levels of CCN1, Akt and VEGF, which were reduced by 78.21, 67.19 and 77.65%, respectively (Fig. 2). The protein levels of CCN1, Akt and VEGF were reduced by 32.43, 48.48 and 57.76%, respectively, in this group (Fig. 3). These results demonstrated that the hypoxia-induced expression of CCN1 was mediated through the PI3K/Akt-VEGF signaling pathway.

In the present study, RT-qPCR and western blotting were performed to measure the mRNA and protein expression levels of CCN1 following exposure of the cells to LY294002 under normoxic or hypoxic conditions. Compared with the hypoxia group, the mRNA expression of CCN1 in the LY294002 hypoxia group was downregulated by 82.38% (P<0.05; Fig. 6A), and LY294002 treatment reduced the protein expression of CCN1 by 37.32% in the hypoxic cells (P<0.05; Fig. 6B and C). Compared with the normoxia group, the mRNA expression level of CCN1 in the normoxia-LY294002 group was downregulated by 32.57% (P<0.05; Fig. 6A), as were the protein levels of CCN2, which were reduced by 21.87% in the normoxic group (P<0.05; Fig. 6B and C). These results suggested that the PI3K/Akt inhibitor, LY294002, reduced the expression levels of CCN1, and that this process involved an autocrine loop.