RETRACTED ARTICLE: Long noncoding RNA ANRIL is activated by hypoxia-inducible factor-1α and promotes osteosarcoma cell invasion and suppresses cell apoptosis upon hypoxia

A total of 15 osteosarcoma samples were collected postoperatively from patients who underwent surgical resection between 2006 and 2011 at the Department of Orthopedics, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (Shanghai, China). Informed consent was obtained from the patients enrolled in this study. The study was approved by the Ethics Committee of Shanghai Jiao Tong University (2015-DY-126).

The MNNG and U2OS cell lines were purchased from the ATCC. The cell lines were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS, HyClone, Camarillo, CA, USA) as well as 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). Cells were maintained in a humidified incubator at 37 °C in the presence of 5 % CO₂. All cell lines have been passaged for fewer than 6 months. Hypoxia treatment (1 % O₂, 5 % CO₂, and 94 % N₂) was performed in hypoxia Workstation (InVIVO2). The cells were incubated under hypoxic conditions for 24 h. HIF-1α specific inhibitor 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA) and resolved in dimethyl sulfozide (DMSO). YC-1 treatment was used at a final concentration of 40 μM. YC-1 and equal volume of control (DMSO) were added into the culture medium 12 h before hypoxia treatment.

Total RNA was extracted from cancer cells utilizing the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and quantified with Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). First-strand cDNA was generated with the Primer-Script™ one step RT-PCR kit (TaKaRa, Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed with SYBR Green premix Ex Taq (TaKaRa) with β-actin as an internal control. MRNA values were normalized to that of β-actin. The relative expression of mRNA was calculated with the 2^−ΔΔCt method. Each sample was repeated in triplicate. QRT-PCR data were analyzed and converted to relative fold changes. The primer sequences used in the present study were as the followings: ANRIL, 5′-TTGTGAAGCCCAAGTACTGC-3′(forward), 5′-TTCACTGTGGAGACGTTGGT-3′(reverse); HIF-1α, 5′-CATCTCCATCTCCTACCCACA-3′(forward), 5′-CTTTTCTGCTCTGTTTGGTG-3′(reverse); GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′(forward), 5′-AGGGGCCATCCACAGTCTTC-3′(reverse); β-actin, 5′-CTGGGACGACATGGAGAAAA-3′(forward), 5′-AAGGAAGGCTGGAAGAGTGC-3′(reverse).

Cell lysates were extracted from cultured cells with RIPA buffer (Cell Signaling Technology) supplemented with the protease inhibitors. The lysates were subjected to ultrasonication and centrifugation. The protein concentrations were determined with the BCA Protein Assay Kit (Pierce). Equal amounts (30–40 μg) of proteins samples were separated by 8–12 % SDS–polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose filter membranes (Millipore). After blocking in phosphate-buffered saline/Tween-20 containing 5 % non-fat milk, the membrane was immunoprecipitated with indicated primary antibodies overnight at 4 °C: rabbit polyclonal anti-human HIF-1α antibody (1:1000, Abcam), rabbit polyclonal anti-human Bcl-2 antibody (1:500, Abcam), rabbit polyclonal anti-human Bax antibody (1:500, Abcam), and rabbit polyclonal anti-human β-actin antibody (1:1000, Abcam). The membranes were washed with TBST (3 × 5 min), and then immunoprecipitated with the secondary antibody [HRP-conjugated goat anti-rabbit IgG antibody (Abcam)] for 1 h at room temperature. Subsequent visualization was detected with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Intensities of the bands were normalized to the corresponding β-actin bands.

The ANRIL promoter constructs were generated using PCR-based amplification with human genomic DNA as the template and was subcloned in the pGL3 basic firefly luciferase reporter. The HRE mutations were created using a Quick-change Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) with ANRIL promoter plasmid as the template. The primers for Site-directed mutagenesis were: 5′-CAAATATGAGAAATGTATCGAGCTCCATCTGGGTAAGAC-3′(forward), 5′-GTCTTACCCAGATGGAGCTCGATACATTTCTCATATTTG-3′(reverse).

ANRIL specific siRNA, HIF-1α siRNA and Allstars Negative Control siRNA were purchased from Biotend (Shanghai, China). HIF-1α siRNA, 5′-CCGCTGGAGACACAATCATAT-3′; ANRIL siRNA-1, 5′-GGUCAUCUCAUUGCUCUAU-3′; ANRIL siRNA-2, 5′-AAAUCCAGAACCCUCUGACAUUUGC-3′; siRNA control, 5′-UUCUCCGAACGUGUCACGUTT-3′. Vector were transfected into cells by Lipofectamine-mediated gene transfer according to the manufacturer’s protocol. For RNA extraction and western blotting assays, cells were used 48 h after transfection.

HIF-1α siRNA and the ANRIL promoter/luciferase reporter construct were cotransfected into osteosarcoma cells by Lipofectamine 2000-mediated gene transfer. Each sample was cotransfected with the pRL-TK plasmid in order to monitor transfection efficiency (Promega, Madison, WI, USA). Luciferase assays were carried out utilizing the dual-luciferase reporter assay system kit (Promega). The luciferase expression was measured with Modulus single-tube multimode reader (Promega). The relative luciferase activity was normalized to the renilla luciferase activity.

OSTEOSARCOMA cells were incubated under hypoxic conditions for 24 h. EMSA was carried out utilizing a light shift chemiluminescent EMSA kit (Pierce). Nuclear proteins from OSTEOSARCOMA cells were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA). Nuclear proteins (4 μg) were incubated with binding buffer [2.5 % glycerol, 5 mM MgCl₂, 0.05 % NP-40, 1 μg poly(dI–dC), 10 mM Tris, 50 mM KCl, 1 mM DTT, pH 7.5] with or without of 200-fold (3 pmol) unlabeled probes (cold probe or mutant probe) for 20 min, followed by the incubation with the biotin labeled probes (10 fmol) for 20 min. Samples were electrophoresed on a 6 % polyacrylamide gel. The probes used were as the followings: HRE probe, 5′-AATGTATCACACGCCATCTGGG-3′; HRE mutant probe, 5′-CGAGCGCAGTGGCATGGGGCTGTAATCCCA-3′.

Chromatin immunoprecipitation (ChIP) assays were carried out using the EZ ChIP Chromatin Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. Briefly, Chromatin was sonicated on ice to an average length of 200–300 bp in an ultrasound bath. Human IgG was employed as the negative control. Chromatin was immunoprecipitated with rabbit polyclonal anti-human HIF-1α antibody (Abcam, Cambridge, MA, USA). ChIP-derived DNA was quantified using qRT-PCR. Quantitative PCR reactions were then performed on real-time PCR machine (Realplex2, Eppendorf) with ChIP primers: 5′-AAGATCTCGGAACGGCTCT-3′(forward), 5′-TCAGGTGACGGATGTAGCTA-3′(reverse).

Cell viability assays were performed utilizing the CCK-8 assay kits following by the manufacturer’s protocol. Cells transfected with desired vector were seeded into the 96-well plates at an initial density of 5000 cells/well and cultured under hypoxic conditions. At the indicated time points, CCK-8 solution 10 µl was added into each well and incubated for 2 h. The absorbance was determined by scanning at 450 nm with a microplate reader. Data were presented as the percentage of viable cells as the followings: relative viability (%) = (A450_treated − A450_blank)/(A450_control − A450_blank) × 100 %.

Cells transfected with desired vector were seeded into the 6-well plates and cultured under hypoxic conditions for 48 h. The cultures were then stained with annexin V-FITC and propidium iodide (Beyotime, Haimen, China) following the manufacture’s instructions and examined using a flow cytometer (Becton–Dickinson, USA).

Cells transfected with desired vector (2 × 10⁵ in 100 µl serum-free medium) were seeded in the upper chamber of an insert (8.0 µm, Millipore, MA). The insert was pre-coated with Matrigel (Sigma, USA). The lower chamber was added with 400 µl of culture medium supplemented with 10 % FBS for 24 h hypoxia treatment before final examination. The cells on the upper surface that didn’t invade were wiped off with a cotton swab, whereas cells on the lower membrane surface were fixed with methanol for 15 min and stained with 0.01 % crystal violet in PBS for 10 min. Cells from three random fields were counted under a phase contrast microscope (original magnification, ×200) and the relative number was determined. Experiments were repeated in triplicate.

To start with, we examined the expression pattern of ANRIL in osteosarcoma tissues. According to the results of qRT-PCR, the expression levels of ANRIL were significantly higher in osteosarcoma tissues compared with adjacent normal tissues (n = 15, P < 0.001; Fig. 1a). As ANRIL was frequently upregulated in osteosarcoma, we would like to determine the transcriptional factors that contributed to its upregulation. In the first place, we examined the effect of hypoxia on the expression level of ANRILA. Hypoxia obviously increased the transcript levels of ANRIL in MNNG and U2OS cells (Fig. 1b). Meanwhile, hypoxia markedly enhanced the promoter activities of ANRIL (Fig. 1c). Together, it suggests that hypoxia promoted the transcriptional activity and expression level of ANRIL in osteosarcoma cells.

We sought to investigate whether HIF-1α took a part in upregulating the expression level of ANRIL under hypoxic conditions. We identified one putative binding site for HIF-1 (HRE, −714 to −705 bp) in the upstream region of ANRIL with online bioinformatical software programs MatInspector (http://​www.​genomatix.​de/​online_​help/​help_​matinspector/​matinspector_​help.​html) (Fig. 2a). We detected the expression of HIF-1α under normoxic and hypoxia conditions. Western blotting analysis demonstrated that hypoxia obviously upregulated the protein levels of HIF-1α, whereas hypoxia had no effects on the mRNA levels of HIF-1α (Fig. 2b, c). The direct interaction between HIF-1α and putative HRE was confirmed with the EMSA assay (Fig. 2d). Furthermore, HIF-1α immunoprecipitates were highly enriched in the DNA fragments compared with negative control IgG immunoprecipitates in the ChIP assay (Fig. 2e). Together, it suggests that HIF-1α directly binds to hypoxia responsive elements (HREs) of the ANRIL promoter region upon hypoxia in both MNNG and U2OS cells.

To explore whether HIF-1α transcriptionally activates ANRIL expression in osteosarcoma cells under hypoxic conditions, we constructed the wild and mutant type of HRE in the promoter region of ANRIL. A significant reduction in the ANRIL promoter activity was observed when we mutated the putative HREs in both MNNG and U2OS cells upon hypoxia (Fig. 3a). A HIF-1α specific siRNA was transfected in MNNG and U2OS cells to suppress HIF-1α expression under hypoxic conditions. HIF-1α specific siRNA potently decreased the mRNA and protein levels of HIF-1α (Fig. 3b, c). Meanwhile, HIF-1α specific siRNA significantly reduced the promoter activities (Fig. 3d) and transcript levels (Fig. 3e) of ANRIL in both MNNG and U2OS cells. In the meantime, HIF-1α-specific inhibitor YC-1 significantly reduced the mRNA (Fig. 3f) and protein levels (Fig. 3g) of HIF-1α under hypoxic conditions. YC-1 also ameliorated the upregulation of ANRIL in hypoxic osteosarcoma cells (Fig. 3h). Collectively, these data showed that HIF-1α could transcriptionally activate ANRIL in hypoxic osteosarcoma cells.

To investigate the biological effects of ANRIL under hypoxic conditions, we transfected U2OS cells with the most efficient ANRIL specific siRNA that have been confirmed in other studies [19]. The hypoxia-induced ANRIL expression was knocked down by ANRIL specific siRNAs (Fig. 4a). ANRIL silencing reduced the hypoxic cell viability compared to the negative control cells (Fig. 4b). Furthermore, ANRIL silencing increased the apoptosis rate of U2OS cells compared to the negative control cells under hypoxic conditions (Fig. 4c). ANRIL-knocked down cells demonstrated increased Bax/Bcl-2 ratio under hypoxic conditions (Fig. 4d). Thus, it indicates that ANRIL increases cell viability and inhibits apoptosis of osteosarcoma cells under hypoxic conditions.

In the functional aspect, we investigated the effects of ANRIL on invasive capacity of osteosarcoma cells. Hypoxia significantly increased the invasive potencies of U2OS cells (Fig. 5a, b). However, such as effect was attenuate by ANRIL silencing (Fig. 5a, b). These data suggest that ANRIL is involved in hypoxia-mediated invasion of osteosarcoma cells.