The human hepatoblastoma cell line, HepG2, was obtained from the American Type Culture Collection (Manassas, VA, USA). IGF-1R knockout HepG2 cells were constructed and conserved within the laboratory. HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. Curcumin and CoCl₂ were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Curcumin was dissolved in dimethylsulfoxide and CoCl₂ was dissolved in DMEM.

The candidate 20-base guide (g)RNA sequences were derived from the IGF-1R genome sequence (gene ID: 3480). The cDNA sequence was obtained from Han's lab (http://hanlab.xmu.edu.cn/cdna/Query.aspx, No. 25207). The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 design tool from Zhang's lab (crispr.mit.edu/) was used to search the functional gRNA sequence (5′-GAGAACTGCACGGTGATCGAGGG-3′), which contain the downstream 3′protospacer-adjacent motif with GG dinucleotide (N20-NGG). According to the functional gRNA sequence, a pair of oligo DNA were designed and synthesized (hIGF1R-QC-F, 5′-CACCGAGAACTGCACGGTGATCGA-3′; hIGF1R-QC-R, 5′-AAACTCGATCACCGTGCAGTTCTC-3′). The complementary oligo DNA were hybridized (50 ng) and then inserted to pGK1.1 (20 ng) (Genloci Biotechnologies, Inc., Jiangsu, China; http://www.genloci.com) and named pGK1.1-U6-IGF-1RgRNA. HepG2 cells were seeded in a 24-well plate 24 h prior to transfection and transfected with the pGK1.1-U6-IGF-1RgRNA plasmid via the electroporation method using a Neon^® Transfection system (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following transfection, the cells were diluted into 10 96-well plates and incubated at 37°C in an atmosphere containing 5% CO₂ for 2 weeks. The cells from each 96-well plate were then transferred to 48-well plates and incubated at 37°C in an atmosphere containing 5% CO₂ for further incubation. After cell cultures were 70% confluent, they were harvested and the genomic DNA was extracted using DNA Extraction kits (Genloci Biotechnologies, Inc.). PCR was conducted to amplify the target region with genomic DNA derived from the cells. DNA polymerase (cat. no. R060Q; Takara, Bio, Inc., Otsu, Japan) was use for PCR. The following primer sequences were used: hIGF1R-seq-F: 5′-GTTTACCCTCTTGTCTCCCT-3′ and hIGF1R-seq-R: 5′-CGGTAATGACCGTGAGCTTG-3′. PCR was performed using the following thermocycling conditions: 95°C for 10 sec, 60°C for 10 sec, 72°C for 20 sec for 30 cycles. Amplicons were sent to the Beijing Genomics Institute (Beijing, China) for deep sequencing.

HepG2 cells were plated in 96-well plates (5,000 cells/well) or 6-well plates (3×10⁵ cells/well) in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in an atmosphere containing 5% CO₂. Following 24 h, the medium was changed to fresh serum-free DMEM with different concentrations of CoCl₂ (0, 50, 100, 150, 200 and 400 µM) for different time periods (1, 2, 4, 6 and 8 h).

HepG2 cells were plated in a 96-well plate in 100 µl medium at a density of 5,000 cells/well and incubated using standard culture conditions overnight. The medium was removed carefully and the purple formazan was dissolved with 150 µl dimethyl sulfoxide. Cell viability was determined by an MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacturer's protocol. The optical density was determined at 570 nm by SpectraMax M2 (Molecular Devices, LLC, Sunnyvale, CA, USA).

Total protein was isolated from cells using a radioimmunoprecipitation buffer reagent (cat. no. P0013B, Beyotime Institute of Biotechnology, Shanghai, China). The lysates were ultrasonicated and centrifuged at 12,000 × g and at 4°C for 10 min. Supernatants were collected and stored at −70°C. Protein concentrations were determined using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Protein (50 µg per lane) was separated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane. The membrane was blocked with Tris-buffered saline (TBS)/5% nonfat dry milk at room temperature for 2 h and the membranes were subsequently incubated with primary antibodies overnight at 4°C. Following three washes with TBS+Tween-20 (TBST), the membranes were incubated with goat polyclonal immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (cat. no. sc-2004; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 45 min at room temperature. The membranes were washed with TBST three times prior to visualization using a Western Blotting Luminol Reagent (cat. no. sc-2048, Santa Cruz Biotechnology, Inc.). Image J 1.6 software (National Institutes of Health, Bethesda, MD, USA) was used to detect and analyze the relative densitometry. The primary antibodies used in the experiment were as follows: Rabbit anti-IGF-1R (cat. no. 9750), rabbit anti-HIF-1α (cat. no. 3716), rabbit anti-VEGF (cat. no. 2463), rabbit anti-Akt (cat. no. 9272), rabbit anti-phosphorylated (p)-Akt (cat. no. 5012), rabbit anti-extracellular signal-regulated kinases (Erk1/2; cat. no. 4695), rabbit anti-p-Erk1/2 (cat. no. 4377) (all 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-β-actin (cat. no. sc-7210; 1:4,000; Santa Cruz Biotechnology, Inc.).

Data are presented as the mean ± standard deviation and were analyzed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). Differences between the groups at each time point were examined for statistical significance using one-way analysis of variance and a least significant difference post-hoc comparison. The correlation between HIF-1α and VEGF protein levels was evaluated statistically using Pearson's correlation coefficient analysis if the quantitative data had a normal distribution; if the data was not normally distributed, the Spearman rank correlation coefficient analysis was used. P<0.05 was considered to indicate a statistically significant difference.

An IGF-1R knockout HepG2 cell line was successfully constructed using a CRISPR/Cas9 genome-editing system. To investigate the effect of hypoxia on the IGF-1R knockout HepG2 cell line, cells were treated with different concentrations of CoCl₂ for 1–8 h and the viability was subsequently analyzed using an MTT assay. No significant differences were observed between the control cells and those treated with low CoCl₂ doses (50–100 µM; Fig. 1). However, the viability of IGF-1R knockout HepG₂ cells was significantly reduced in the 150, 200 and 400 µM CoCl₂ treated groups compared with the control (no CoCl₂ treatment) at the same time points (P<0.05; Fig. 1).

Western blot analysis was performed to examine the effects of CoCl₂-induced hypoxia on HIF-1α, IGF-1R and VEGF protein expression in IGF-1R knockout HepG2 cells. The cells were treated with 0, 50, 100, 150, 200 or 400 µM CoCl₂ for 6 h and the expression of HIF-1α, IGF-1R and VEGF was determined by western blotting. The protein expression of HIF-1α and VEGF was significantly higher in cells treated with 150 or 200 µM CoCl₂ compared with the control group (P<0.05; Fig. 2A and B). Correlation analysis revealed that the expression of the HIF-1α was positively correlated with the expression of VEGF (r=0.85, P<0.05) in a dose-dependent manner (data not shown).

The IGF-1R knockout HepG₂ cells were treated with 150 µM CoCl₂ for the indicated time periods and the protein expression of HIF-1α, IGF-1R and VEGF was determined by western blotting (Fig. 2C). The relative expression of each protein was calculated and it was observed that the protein expression of HIF-1α and VEGF was significantly increased at 2–8 h compared with the control group (P<0.05; Fig. 2D). In addition, HIF-1α expression was positively correlated with the expression of VEGF (r=0.71, P<0.05) in a time-dependent manner (data not shown).

IGF-1R knockout HepG2 cells were incubated with increasing concentrations of curcumin (5, 10, 20, 30 and 40 µM) in the presence of 150 µM CoCl₂. It was observed that the cell survival rate decreased as the incubation time or dose increased, and all doses of curcumin caused a reduction in cell survival (Fig. 3). The incubation of cell cultures with different concentrations of curcumin at each time point (8, 12, 24 and 48 h) had significant inhibitory effects on the survival of IGF-1R knockout HepG2 cells compared with the control group (P<0.05; Fig. 3).

HepG2 cells and IGF-1R knockout HepG2 cells were treated with or without curcumin (20 µM) under CoCl₂-induced hypoxic conditions. Following 24 h of treatment, total cell protein was extracted and analyzed using western blotting (Fig. 4A). The expression of IGF-1R, p-Akt, p-Erk1/2, HIF-1α and VEGF proteins was significantly reduced in HepG2 cells treated with curcumin compared with untreated cells (P<0.05; Fig. 4B-F). IGF-1R knock out cells treated with curcumin also exhibited markedly reduced p-Akt, p-Erk1/2, HIF-1α and VEGF protein expression compared with the IGF-1R knock out cells not treated with curcumin (P<0.05). Knockdown IGF-1R also inhibited the expression of HIF-1α, VEGF, p-Akt and p-Erk proteins in CoCl₂-induced HepG2 cells (P<0.05, Fig. 4C-F). Additionally, the difference between control group (lane A) and HepG2 cells with CoCl₂ plus curcumin (lane B) seems greater than the difference between IGF-1R knockout HepG2 cells with CoCl₂ (lane C) and IGF-1R knockout HepG2 cells with CoCl₂ plus curcumin (lane D).