4.1 Bioinformatics analysis
COAD mRNA data were downloaded from the official website of TCGA. Unsupervised clustering was performed on the TCGA-COAD data to select a classification that includes glycolysis-related genes for hierarchical clustering40. ConsensusClusterPlus hierarchical clustering was used to classify colon cancer samples into low-glycolytic and high-glycolytic groups. The limma package of R was used to screen for differentially expressed genes in the two groups. Log2 |Fold change| ≥ 1 and a significance level of p < 0.05 were used as the cut-off value to identify genes that were up-regulated in the high-glycolysis group. The up-regulated genes were compared with the transcription factor subset (MsigDB: TF) of the Molecular Characterization Database for further analysis. The JASPAR database (http://jaspardev.genereg.net/), ALGGENdatabase (http://alggen.lsi.upc.es/home.html), and hTFtarget database (http://bioinfo.life.hust.edu.cn/hTFtarget) were used to predict the potential target genes regulated by ONECUT3.
4.2 Cell culture and reagents
We obtained human colon cancer cell lines CACO2, LOVO, SW480, SW620 from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), COLO205, HT29 and the colon immortalized cell line NCM460 from ATCC (American type culture collection). The cells were cultured in suggested standard medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 500 units/mL of penicillin, and 200 μg/mL of streptomycin. The cells were maintained at 37°C with 5% CO2. To create a hypoxic environment, the cells were cultured in a hypoxia incubator with an atmosphere containing 1% O2, 94% N2, and 5% CO2. The reagents used in this study included 2-DG (Sigma-Aldrich, D8375).
4.3 Clinical samples and IHC staining
We utilized paraffin-embedded tissue microarrays, which consisted of cancer tissues from 141 patients who underwent surgeries at Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, for immunohistochemical analysis. All patients included in this study were pathologically diagnosed with colorectal cancer and had complete clinicopathological characteristics. None of these patients received any preoperative antitumor therapy, such as chemotherapy or radiotherapy. The specimens were collected with informed consent from all patients and approved by the ethics committee of Renji Hospital (KY2021-120-B). Immunohistochemical (IHC) staining was conducted as the previously described 41. The primary antibodies used for IHC staining included ONECUT3 (1:200, abcam, ab181450), HDAC6 (1:200, Immunoway, YT2118), ALDOA (1:100, proteintech, 11217-1-AP), ENO1 (1:2000, proteintech, 11204-1-AP), TPI1 (1:100, proteintech, 10713-1-AP), GLUT1 (1:200, CST, D3J3A/12939S), and HIF-1α (1:100, proteintech, 20960-1-AP). The scoring was assessed by two investigators who were blinded to the clinical information, based on the staining intensity.
4.4 Quantitative real-time polymerase chain reaction (qRT-PCR)
TRIzol total RNA isolation reagent (share-bio, Shanghai, China) was used to extract total RNA from the indicated cells. The quality and quantity of RNA were determined using a Nanodrop™ spectrophotometer (NanoDrop products, Wilmington, CA). Then, 1 μg of total RNA was reverse transcribed into complementary DNA (cDNA) using All-in-One First-Strand Synthesis Master Mix (with dsDNase) (share-bio, Shanghai, China). Subsequently, the cDNA product was amplified by PCR on the ViiA 7 Real-Time PCR System (Thermo Scientific) to analyze mRNA expression. The 2*Universal SYBR Green qPCR Premix (share-bio, Shanghai, China) and specific primers were used for qPCR. 18sRNA was used as an internal control. The PCR primer sequences used in this study are listed in supplementary Table 1. Relative quantification was performed using the comparative 2–ΔΔCt method.
4.5 Western blot analysis and co-immunoprecipitation
Whole-cell protein lysates were obtained using IP lysis buffer (share-bio, Shanghai, China) supplemented with a protease and phosphatase inhibitor (share-bio, Shanghai, China). The cell lysates were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrophoretically transferred onto PVDF membranes. The membranes were blocked with 5% defatted milk for 1 h at room temperature (RT), hybridized with primary antibodies overnight at 4 °C, and then incubated with HRP-conjugated secondary antibodies at RT for 60 min. β-actin antibody was used as a loading control. Immunoblots were developed using the Basic Luminol Chemiluminescent Kit (share-bio, Shanghai, China) and the ChemiDoc Touch image system (Bio-Rad). The antibodies used were listed as follows: ONECUT3 (1:500, Abcam, ab181450), HIF1α (1:1000, Abcam, ab2185), GLUT1 (1:1000, Proteintech, 21829-1-AP), ALDOA (1:10000, Proteintech,11217-1-AP), HDAC6 (1: 10000, Abcam, ab133493), and β-actin (1: 5000, Abcam, ab6276). For co-immunoprecipitation, protein lysates obtained as described above were incubated with Pierce Anti-HA Magnetic Beads (Thermo Fisher Scientific, USA, #88836) or Pierce Protein-A/G Magnetic Beads (Thermo Fisher Scientific, USA, #88803), which were already incubated with anti-HIF1α (2 µg, Abcam, ab308433) or anti-IgG (as a negative control, 2 µg, Abcam, ab200699) for 15 min at RT, with rotation for 30 min at RT. The immuno-complexes were washed three times with TBS-T or PBS-T and then resuspended in 1 × SDS-PAGE sample buffer for western blotting analysis.
4.6 Lentivirus production and transfection
To achieve overexpression, plasmids expressing HA-tagged ONECUT3 were constructed by Shanghai Generay Biotech Co., Ltd. The cDNAs encoding full-length human ONECUT3 (NM_001080488.2) with HA tag were synthesized and inserted into the pCDH-CMV-MCS-EF1-Puro vector (Generay, Shanghai, China). For knockdown, lentiviral siRNA negative control and siRNA oligonucleotides targeting human ONECUT3 were designed and synthesized by Genepharma (Shanghai, China). The sequences for the siRNA were as follows: siONECUT3-1: 5’-CGCTGATCGCCATCTTCAAGGAGAA-3’; HDAC6 siRNA sequence: 5’-GGACAACATGGAGGAGGACAATGTA-3’. 293T packaging cells were used to produce lentivirus, which was subsequently transfected into target cell lines with 6 µg/ml polybrene for 24 h. Transfected cells used for overexpression or knockdown, as well as their control cells, were selected with 5 µg/ml puromycin for 2 weeks. The overexpression or knockdown efficiency of ONECUT3 was assessed using qRT-PCR and western blotting.
4.7 siRNA targeting ONECUT3 or HDAC6
ONECUT3 or HDAC6 specific siRNA and non-targeting control (NTC) siRNA were purchased from Genepharma (Shanghai, China). According to the instructions of jetPRIME® in vitro DNA & siRNA transfection reagent (Polyplus, Shanghai, China), dilute ONECUT3 or HDAC6 specific siRNA (25 nM) or NTC siRNA (25 nM) into 200 µL of jetPRIME® buffer, then add 4 µL jetPRIME® reagent, incubate for 10 to 15 min at RT, add the transfection mix to the cells in serum containing medium dropwise, and incubate the plate at 37 °C. As mentioned earlier, transfection efficiency was determined by quantitative PCR (qPCR) and immunoblot analysis. Relevant experiments were conducted between 24 and 36 hours after siRNA introduction.
4.8 Colony formation assay
Different types of cells (1 × 103 cells per plate) were seeded in 6-well plates and incubated for approximately 14 days. After the experiments, the formed colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.2% crystal violet for 30 min. Colonies larger than 100 μm in diameter were counted for each plate.
4.9 Extracellular acidification rate (ECAR) and oxygen consumption ratio (OCR) assays
The Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Billerica, Massachusetts, USA) was used to measure the real-time extracellular acidification rate (ECAR) of CRC cells in vitro, following the manufacturer’s instructions. Briefly, CRC cells were seeded at a density of 2-3 × 104 cells per well in an XF96-well plate and allowed to attach overnight. Cells were incubated in non-buffered media under basal conditions for 1 hour. Subsequently, they were sequentially injected with 10 mM glucose, 1 mM mitochondrial poison (oligomycin, Sigma-Aldrich, Saint Louis, Missouri, USA), and 80 mM glycolysis inhibitor (2-deoxyglucose, 2-DG, Sigma-Aldrich). ECAR measurement was normalized by total protein content, as demonstrated by the BCA assay. The experimental data was processed using the Seahorse XF96 Wave software.
4.10 Subcutaneous xenograft model
2 × 106 cells (ONECUT3 knockdown colon cancer cells, and their control cells), resuspended in 100 μl of PBS, were subcutaneously injected into the backs of male Balb/c nude mice (5-6 weeks old, five mice per group). After 5 weeks of subcutaneous inoculation, the experiment was terminated, and the mice were euthanized. The tumors were then collected, weighed, and fixed in formalin. All animal studies were approved by the Animal Care and Use Committee of Shanghai East Hospital, Tongji University School of Medicine. The mice received humane care in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health.
4.11 HIF-1α activity measurement
We quantified HIF-1α activity by using the HIF-1 alpha Transcription Factor Assay Kit (Abcam, ab133104) following the manufacturer’s instructions, after extracting the nuclear fraction. Briefly, processed colorectal cancer cells was collected and the nuclear and cytoplasmic protein were extracted by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027) . The samples were added to the wells of the HIF-1α transcription factor plate and then incubated overnight at 4°C. Diluted HIF-1α primary antibody was added to each well and incubated at room temperature for 1 hour. Subsequently, diluted goat anti-rabbit HRP conjugate was added to each well and incubated at room temperature for 1 hour. After adding the stop solution, the HIF-α DNA binding activity level was measured at 450 nm using a microplate reader.
4.12 Chromatin immunoprecipitation (ChIP) assays
ChIP assay was were carried out using the EZ-Magna ChIP Assay Kit according to the manufacturer’s protocols (Millipore, 17-10086). Briefly, cells were cross-linked with 1% PFA/PBS at room temperature for 10 minutes. Then, unreacted PFA was eliminated using a 10-fold concentration of glycine. Subsequently, samples were sonicated in lysis buffer to obtain DNA fragments ranging from 200 to 1,000 bp. Immunoprecipitation was performed using 5 μg of ONECUT3 or IgG antibodies. Primers targeting the promoter region of the HDAC6 gene were used for quantitative RT-PCR. The results were presented as the relative mRNA expression, calculated by comparing the delta CT values of ONECUT3-specific antibodies with IgG antibodies.
4.13 Luciferase reporter assay
To evaluate the activity of the HDAC6 gene promoter, processed cells and control cells were seeded into 96-well plates and co-transfected with a PTRF luciferase reporter plasmid. The plasmid contained a tandem repeat of the PTRF transcriptional response element, while the Renilla control reporter served as an internal control. After 48 hours, the cells were lysed, and the enzymatic activity of luciferase and Renilla was measured using the Dual-Luciferase Assay kit (Promega, E1910) following the manufacturer’s protocol.
4.14 Statistical analysis
We conducted statistical analyses using SPSS 19.0 for Windows (IBM Corporation) and GraphPad Prism 7 software (San Diego, CA). The results were reported as mean ± standard deviation (SD) and compared using a two-tailed, unpaired Student’s t-test or one-way ANOVA. p-value of <0.05 was considered statistically significant.