THP-1 and nephroblastoma cell line WiT49 was bought from National Collection of Authenticated Cell Cultures (Shanghai, China), and cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 ℃, 5% CO2. THP-1 cells were induced to differentiate to macrophages by Phorbol ester-12-myristate-13-acetate (PMA). WiT49 cells were treated in Normoxia or Hypoxia (95% N2 and 5% CO2) conditions, and then they were co-cultured with macrophages using Transwell inserts (Millipore) to induce inflammation, in which THP-1-induced macrophages in the upper and WiT49 in the lower chamber.
Vector construction and transfection
The siRNAs of hypoxia-inducible factor 1α (HIF-1α) and CCAAT/enhancer-binding protein β (C/EBPβ), the pcDNA of HOXA11-AS, and their negative control were synthesized by GenePharma (Shanghai, China). According to the manufacturer's manual, the transfection treatment was performed using Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Cell viability, migration, and invasion analyses
The cell viability was evaluated by Cell counting kit-8 (CCK-8) assay. The cells were seeded into 96-well plates, incubated at 37℃ with 5% CO2, and it’s OD450 values were calculated every 6h of 48h.
For cell migration and invasion experiments, the Transwell assays were conducted. The 24-well Transwell chamber and 8.0-μm pore membranes (Corning USA) coating with Matrigel (BD Biosciences) were employed for the cell invasion experiment. Approximately 1.0×105 cells/well in 100 mL serum-free DMEM were seeded in the top chamber for 6 h at 37℃, and 500mL medium containing 10% FBS was placed into the lower chamber. Subsequently, the Transwell was washed, fixed with glutaraldehyde, and stained with 0.1% crystal violet. Finally, five visuals were randomly selected, and the cell numbers were counted under a 400× microscope. The cell migration analysis was similar with that of invasion, except the supplementation of DMEM and culture.
Cell migration was further confirmed by wound-healing assay. The stable transfected cells were seeded into 6-well plates, cultured overnight, scratched by a sterile plastic pipette tip, and washed with culture medium. At the end of the experiment, the cells were further cultured for 48h with medium containing 1% FBS.
Quantitative real-time PCR (qRT-PCR) analysis
The expression levels of HOXA11-AS, HIF-1α, and C/EBPβ were evaluated in WiT49 cells. The total RNA in cells were extracted by TRIzol (Invitrogen, Carlsbad, CA, USA). The primers were designed and synthesized by Sangon (Shanghai, China) and listed in Table 1. Then, the qRT-PCR analysis was conducted by applying HiScript II One Step qRT-PCR SYBR Green Kit (#Q221-01), tested on an ABI 7900 system (Foster City, CA, USA), and calculated using 2–ΔΔCt method, using β-actin as the internal gene.
Enzyme-linked immunosorbent assay (ELISA)
The levels of pro-inflammatory cytokines of interleukin-1β (IL-1β), Interferon γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were assessed using the ELISA kits (Abcam), according to the manufacturer’s instructions.
Fluorescence in situ hybridization (FISH) and immunofluorescence (IF) analyses
The transfected cells were fixed with 4% formaldehyde, washed by PBS, and supplemented with 0.5% Triton X-100, and culture at 4°C for 5min. Then, the cells were treated with HOXA11-AS FISH probe (RiboBio; Guangzhou, China) mix in 100 μL hybridization buffer at 37°C overnight. After hybridization, the slide was washed and mounted with 4'6-diamidine 2-phenylindole (DAPI; Shanghai Beyotime Biotechnology Co., Ltd., China).
The IF analysis was conducted similarly with FISH analysis. Briefly, the cells were fixed with 4% formaldehyde, blocked with 5% BSA, and incubated with anti-HOXA11 antibody (#ab72591, Abcam; 1:800) and goat anti-mouse fluorescent secondary antibody (#ab150115, Abcam; 1:500) for 48 h. The cells were stained with DAPI and observed under a fluorescence microscope (Leica, Germany). Five randomly selected visuals in each group were observed, photographed, and calculated.
Western blot and analyses
The protein expression levels of HOXA11, β-catenin, receptor–related protein 6 (LRP6), Phosphorylation-LRP6 (p-LRP6), E-cadherin (E-cad), N-cadherin (N-cad), Vimentin, HIF-1α, C/EBPβ, and β-actin were assessed in WiT49 cells. The proteins were isolated by RIPA lysis buffer (#R0278, Sigma). Standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method was used to separate the proteins . Then, the gels were transferred, blocked, and cultured with primary antibodies against HOXA11 (#ab54365; 1:1000), β-catenin (#ab32572; 1:800), LRP6 (#ab134146; 1:1000), p-LRP6 (#ab76417; 1:800), E-cad (#ab6528; 1:800), N-cad (#ab6258; 1:800), Vimentin (#ab92547; 1:800), HIF-1α (#ab51608; 1:1000), C/EBPβ (#ab32358; 1:1000), and β-actin (#ab32572; 1:5000) bought from Abcam (Cambridge, MA, USA). The membranes were washed and incubated with the HRP conjugated Goat Anti-Rabbit IgG H&L (1: 5000, #ab6721, Abcam). Finally, the bands were observed under a Tanon 6600 Luminescence imaging workstation (Tanon, China).
PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and JASPAR(http://jaspar.genereg.net/) databases were used for the transcription factor prediction of HOXA11-AS with the threshold.
Chromatin immunoprecipitation (ChIP)-qPCR
The ChIP-qPCR assay was performed to verify the effect of HIF-1α and C/EBPβ on the HOXA11-AS promoter region. The experiment was carried out following a method described previously . In brief, DNA-protein complexes were cross-linked with 1% formaldehyde for 15 min. Immune complexes were formed with control (IgG) or antibodies against HIF-1α or C/EBPβ. DNA was eluted and purified from complexes, followed by PCR amplification, and qPCR detection. The fold-enrichment has calculated the ratio of amplification efficiency of HIF-1α or C/EBPβ to IgG.
Dual-luciferase reporter assay
Dual-luciferase reporter assay was firstly used to detect the transcription factors binding region of HOXA11-AS promoter. The truncated fragments of HOXA11-AS promoter were amplified and inserted into the pGL3-Basic luciferase reporter (Promega, USA) using restriction enzymes Mlu1 I and Xho I (TaKaRa, Japan), and then ligated by T4 DNA ligase (TaKaRa, Japan) for the transfection of WiT49 cells. The vector was transfected into WiT49 cells by Lipofectamine 2000 kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 80%–90% confluent. After 48 h transfection, firefly and renilla luciferase activities were measured using Dual Luciferase Reporter Assay System (Promega). Relative luciferase activity was determined with renilla luciferase activity as an internal control.
Next, Dual-luciferase reporter assay was used to confirm the binding of HIF-1α and HOXA11-AS, C/EBPβ and HOXA11-AS. Firstly, the binding sequences of HIF-1α or C/EBPβ on HOXA11-AS were synthesized (WT) and mutated (Mut), then the pGL3-promoter vectors (Thermo Fisher Scientific) containing the binding sites were constructed by GenePharma (Shanghai, China). Next, the vectors were transfected into WiT49 cells treated with Control and Hypoxia. Finally, one Dual-Luciferase Reporter Assay System bought from Promega Corporation (Madison, WI, USA) was employed to detect luciferase activity.
Electrophoretic mobility shift assay (EMSA) and shift western blotting
The oligonucleotide probes specific for the HOXA11-AS-binding site of the HIF-1α (5′-ACGTGG-3′) and C/EBPβ gene (5′-GCTCAAT-3′) were synthesized (Generay Biotech; Shanghai, China) and detected by employing DIG Gel Shift Kit (Roche, Mannheim, Germany). The HOXA11-AS binding specificity was confirmed by mutant biotin-labeled oligonucleotide probes (Biotin-Mut-probe). Then, the complexes were separated by 6% non-denaturing polyacrylamide gels. After electrophoresis, the gels were transferred to nylon membranes, detected chemiluminescent, and analyzed using an ImageQuant LAS 4000 Scanner of GE Healthcare.
GraphPad Prism 8 (San Diego, CA, USA) was used for the statistical analysis and chart drawing. Student’s t-test and one-way ANONA were performed for comparison analyses depends on the groups. Data are exhibited as mean ± standard deviation (SD). P<0.05 was considered a significant difference.