Study populations
A total of 165 formalin-fixed, paraffin-embedded samples were excised from fresh LIHC surgical samples. The clinicopathological features included sex, age at diagnosis, differentiation, vascular invasion, TNM stage, tumour size and cirrhosis. None of the patients received radiotherapy, chemotherapy, or immunotherapy prior to surgery. The overall survival duration was defined as the interval from the date of first biopsy to the date of death from disease.
Immunohistochemistry (IHC)
LIHC tissue microarray (TMA) slides from patients were used for NRF1 staining with a Tissue Microarray System (Quick-Ray, UT06, UNITMA, Korea). Core tissue biopsies (2 mm in diameter), which were taken from individual paraffin-embedded sample sections, were arranged in new recipient paraffin blocks. IHC analysis was performed as previously described [21]. The slides were incubated with the primary antibody against NRF1 (Abcam, Cambridge, MA, USA) at 4 °C overnight. Three trained pathologists were blinded to evaluate NRF1 immunostaining. There were two estimated variables: intensity (0 to 3 as negative, weak, moderate or strong) and percentage (0% to 100%). The degree of NRF1 expression was quantified using a two-level grading system defined as follows: score ≤ 60 defined as low, otherwise defined as high.
Tumour Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA) Database Analysis
The level of NRF1 mRNA expression in different tumour types was obtained from TIMER2.0 (http://timer.cistrome.org/) [22, 23]. GEPIA2 (http://gepia2.cancer-pku.cn/#index) was employed to profile the expression of NRF1 in different cancer stages and generate disease-free survival curves based on the expression status of NRF1 [24].
Cell culture, cell transfection and lentivirus infection
HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, UT, USA) containing 10% foetal bovine serum (HyClone, UT, USA) and were cultured at 37 °C with 5% CO2 in an incubator. Cells were transiently transfected with plasmids or siRNA duplexes using Lipofectamine 2000 Transfection Reagent (Invitrogen, CA, USA) following the manufacturer's protocol. NRF1 overexpression constructs were generated into the Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin lentiviral vector (GeneChem, Shanghai, CHN). The lentivirus infection was manipulated according to the instructions.
Chromatin immunoprecipitation sequencing (ChIP-Seq) dataset of NRF1 binding sites and molecular pathway analysis
ChIP was performed using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, MA, USA) as described in the manufacturer’s protocol. Briefly, cells were washed and fixed in 1% formaldehyde at room temperature. Then, the cells were collected and lysed to release the nuclei. Nuclei were then isolated before being subjected to micrococcal nuclease. The lysate was then immunoprecipitated with NRF1 antibodies (Abcam, MA, USA) or a negative control IgG. The pulled-down chromatin was washed, reverse-crosslinked, purified and detected by deep sequencing (Vazyme Biotech, Nanjing, China). To identify the pathways relevant to ChIP-Seq-based NRF1 target genes, we used Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 (https://david.abcc.ncifcrf.gov/) to analyse the sequencing data.
Gene Silencing
Human NRF1-specific siRNA (siNRF1) duplexes were designed and synthesized by GenePharma Co., Ltd. (GenePharma, Shanghai, CHN). The siNRF1 sequences were as follows: siNRF1, 5’-CACAUUGGCUGAUGCUUCAUU-3’.
RNA Isolation and Quantitative Real-time PCR
RNA was isolated using TRIzol reagent (Invitrogen, CA, USA) and treated with DNase I (Promega, WI, USA) before cDNA synthesis. cDNA was synthesized by a Transcript First-Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). Quantitative real-time PCR was performed using AceQ qPCR SYBR Green Master Mix (High ROX Premixed) (Vazyme, Nanjing, CHN) in a StepOne Plus Real-time PCR System (Applied Biosystems, Singapore city, Singapore). The primer sequences were as follows: E2F1, F: 5’-CATCCCAGGAGGTCACTTCTG-3’ and R: 5’-GACAACAGCGGTTCTTGCTC-3’; ACTB, F: 5’-CATGTACGTTGCTATCCAGGC-3’ and R: 5’- CTCCTTAATGTCACGCACGAT-3’; CCNE1: F: 5’-ACTCAACGTGCAAGCCTCG-3’ and R: 5’-GCTCAAGAAAGTGCTGATCCC-3’; CDK2, F: 5’-CCAGGAGTTACTTCTATGCCTGA-3’ and R: 5’-TTCATCCAGGGGAGGTACAAC-3’. Melting curves were generated to confirm primer specificity.
Western Blot
Cells were collected and lysed with cell lysis buffer (Beyotime, Shanghai, China). Whole-cell extracts were resolved by 10% SDS–PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Roche Diagnostics, Mannheim, Germany). The membranes were blocked and then incubated with anti-NRF1, anti-β-actin or anti-E2F1 antibodies (Abcam, Cambridge, MA, USA) at 4 °C overnight, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA). The chemiluminescence reaction was performed using ECL reagent (Thermo Scientific, IL, USA).
Clone-forming assay
The cells were seeded (103 cells/well) onto 12-well plates and cultured for 3 days. The cells were fixed with 4% paraformaldehyde for 30 min and stained with crystal violet (Sigma–Aldrich, MO, USA). The cell clones were photographed and counted. Each experimental group was performed in triplicate.
Cell proliferation assay
The cells were seeded onto 96-well plates at a density of 2 × 103 cells/well and cultured for 96 h. Then, 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT; Sigma–Aldrich, MO, USA; 5 mg/mL) in PBS was added to each 96-well plate, and the cells were incubated for an additional 4 h. Then, the supernatants were removed and replaced with 100 μL dimethyl sulfoxide to dissolve the formazan crystals. Optical density (OD) was measured at 570 nm wavelength by an ELX-800 Microplate assay reader (Bio-tek, USA). The OD570 values indicated changes in cell proliferation.
Cell cycle analysis
Cells were treated with serum-free medium for synchronization. To assess the cell cycle distribution, all the above cells were collected and fixed in 70% ethanol overnight. After removal of the ethanol, samples were washed three times with PBS and then incubated with RNase A at 4 °C for 30 min. Next, samples were stained with propidium iodide (50 μg/ml) and evaluated by a Gallios flow cytometer (Beckman). The subsequent analysis was conducted by MultiCycle software.
Chromatin immunoprecipitation
Cells were fixed with formaldehyde, and sonicated nuclear lysates were processed for immunoprecipitation with NRF1 antibody or normal IgG (Abcam, Cambridge, MA, USA). ChIP DNA fragments were processed for quantitative real-time PCR. The amount of amplified DNA was roughly comparable to that obtained using approximately 2% of the total input chromatin as templates. Primers were designed with E2F1 promoter binding sites: primer 1 (-333/-17), F: 5’-AGAAAGGTCAGTGGGATGCG-3’ and R: 5’-CCAAATCCTTTTTGCCGCGA-3’, which was amplified region of 317-bp; primer 2 (-1291/-869), F: 5’-AGCCTCTGTTTCTTTCATAACCT-3’ and R: 5’-TCGAGACCAGCCTGATCAACA-3’, which was amplified region of 422-bp.
Plasmid Constructs
Genomic DNA was used as the template to construct E2F1 promoter reporter plasmids. Different truncations of the human E2F1 promoter were cloned into the pGL3-Basic vector (Promega, WI, USA). Primer sequences for E2F1 (-333/-17) are F: 5’-GCTAGCAGAAAGGTCAGTGGGATGCG-3’ (NheI site is underlined) and R: 5’-AAGCTTCCAAATCCTTTTTGCCGCGA -3’ (HindIII site is underlined); E2F1 (-1291/-869) primer sequences are F: 5’-GCTAGCAGCCTCTGTTTCTTTCATAACCT-3’ (NheI site is underlined) and R: 5’-AAGCTTAGCCTCTGTTTCTTTCATAACCT-3’ (HindIII site is underlined) The NRF1 binding sites in E2F1 promoter were mutated, respectively. Site-directed mutagenesis of putative NRF1 binding sites was generated using a QuikChange site-directed mutagenesis kit (Stratagene, CA, USA). The expression plasmids for wild-type NRF1 and DN NRF1 (a dominant-negative form) were constructed according to a method described previously [25, 26]. All constructs were verified by sequencing.
Dual-luciferase Reporter Assays
Each well of cells was transiently cotransfected with E2F1 promoter luciferase constructs and pRL-TK (Promega, WI, USA) as an internal control. Cells were lysed and collected to detect luciferase activity by the Dual-Luciferase Reporter Assay System (Promega, WI, USA). The firefly/Renilla luciferase activity measurements were recorded according to the manufacturer’s protocol.
Statistical Analysis
The differences in NRF1 expression in tumour and adjacent tissue were assessed using paired t tests. Correlations between clinicopathologic features and NRF1 expression were evaluated by the chi-square test. Multivariate survival analysis was performed with Cox regression. Statistical significance was determined by one-way ANOVA, followed by the post hoc Tukey multiple comparison test or two-way ANOVA, followed by Bonferroni's multiple comparisons test. All P values reported are from two-sided tests, and the threshold for significance was set at P = 0.05. The statistical analyses were performed using STATA version 13.0 (StataCorp, TX, USA).