Cell culture and patient samples
HCC cell lines MHCC-97H, MHCC-97L, QGY-7701, Hep3B, Hep1, and Huh7 were purchased from Shanghai Institutes for Biological Sciences (China) and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C, 5% CO2. Cells were passed every 2–3 days to maintain logarithmic growth and cultured within 35 generations. The short tandem repeat (STR) analysis was used to verify the identity of cell lines.
The human HCC tissue samples and the benign counterparts used in IHC staining of NUPR1 were obtained from the Department of Pathology at Nanfang Hospital (Guangzhou, China) in 2018. A total of 13 specimens from HCC patients who underwent hepatectomy or ultrasonically guided liver biopsy before RT from 2011 to 2019 were also collected from the Department of Pathology at Nanfang Hospital. The therapeutic response of the tumor was evaluated according to the Modified Response Evaluation Criteria in Solid Tumor (mRECIST) as previously described . The collection of human specimens was approved by the Institute Research Medical Ethics Committee of Nanfang Hospital.
Plasmid constructs, lentivirus, siRNA, and drugs
Lentivirus containing pLent-NUPR1-RFP-Puro (LV-NUPR1) or empty vector (LV-NC) pLent-RFP-Puro were synthesized by Vigene (Vigene Biology, Shandong, China) and used to infect MHCC-97H and MHCC-97L cells with enhanced infection solution (EIS) (Vigene Biology). Similarly, pLent-GFP-sh-NUPR1-Puro (sh-NUPR1) or its negative control (sh-NC) pLent-GFP-Puro (Vigene Biology) was used to infect QGY-7701 and Hep3B cells. Seventy-two hours after the cells were infected with lentivirus, 5 μg/ml puromycin was added to kill the cells that had not been transfected.
The pcDNA3.1 vector (Vigene Biology) containing the full-length cDNA sequence of AhR and empty pcDNA3.1 vector as negative control were used for transient transfection by Lipofectamine 3000 reagents (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Small interfering RNA (siRNA) against NUPR1 (si-NUPR1) and its negative control (si-NC) were obtained from Genechem (Genechem, Shanghai, China) and transfected into HCC cells using Lipofectamine 3000 reagents. The RNA sequences used for transfection in this study are shown in Additional file 1: Table 1.
ROS inhibitor NAC, NUPR1 inhibitor ZZW-115, an agonist of AhR (6-formylindolo[3,2-b]carbazole, FICZ), and specific antagonist of AhR (CH223191) were obtained from MedChemExpress, LLC (Princeton, NJ). Chloroquine (CQ), bafilomycin A1 (BafA1), MG132, and alizarin were purchased from Selleck Chemicals (Houston, USA).
Colony formation assay
MHCC-97H/MHCC-97L/Hep3B cells (1000–6000/well) and QGY-7701 cells (250–1000/well) were seeded in 6-well plates and treated with different doses of IR (0, 2, 4, 6 Gy). Three thousand MHCC-97H/MHCC-97L/Hep3B cells and five hundred QGY-7701 cells were pretreated with different drugs following exposure to IR (6 Gy). After being cultured for approximately 2 weeks, cells were fixed in methanol and stained with 0.1% crystal violet. Plating efficiency was calculated as the eligible colonies (with > 50 cells)/the number of seeded cells. The survival fraction of cells was the ratio of the plating efficiency of treated cells to that of control cells. All related data were analyzed in GraphPad Prism 8 software, and survival curves of clone formation assays were plotted by using a single-hit multi-targeted model (y=1−(1−exp(−k*x))^N).
Western blot analysis
Protein was separated by 8–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore), and blocked in 5% BSA for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies. The sources of antibodies against the following proteins were as follows: NUPR1 (15056-1-AP), CYP1B1 (18505-1-AP), CYP3A4 (18227-1-AP), ARNT (14105-1-AP), HSP90 (60318-1-Ig), β-actin (20536-1-AP), LaminB1 (12987-1-AP), LC3 (14600-1-AP) and GAPDH (10494-1-AP) from Proteintech Group; AhR (A4000), CYP1A1 (A2159), caspase 3 (A19654), PARP (A19596), cleaved PARP (A19612) from ABclonal Technology. γH2AX (Ser139; #80312) and p62 (#8025S) was purchased from Cell Signaling Technology. The membranes were washed in PBST and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein-antibody complexed were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific).
Co-immunoprecipitation (Co-IP) assays were performed using MHCC-97H/MHCC-97L cells with NUPR1 overexpression and QGY-7701/Hep3B cells. The cells were harvested in RIPA lysis buffer (P0013D, Beyotime) with a protease inhibitor cocktail for 30 min on ice. The supernatant was collected by centrifugation at 12,000 × g for 15 min. The protein A/G agarose beads were incubated with antibodies overnight at 4°C while rotating. After washing, the complexes were subjected to western blotting analysis. Antibodies used in the study were anti-Flag (F1804, Sigma-Aldrich), NUPR1 (15056-1-AP, Proteintech), AhR (GTX22770, GeneTex), and AhR (A4000, ABclonal). Mouse IgG (B900620) and secondary antibody HRP-goat anti-rabbit IgG (SA00001-2) were from Proteintech.
Immunofluorescence staining analysis
Cells were seeded on culture dishes and allowed to grow to 70–80% confluency. Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and washed again with PBS before being blocked with goat serum for 30 min. The fixed cells were incubated overnight at 4°C with primary antibodies against NUPR1 (ab234696, 1:100, Abcam), AhR (GTX22770, 1:100, GeneTex), ARNT (14105-1-AP, 1:200, Proteintech), and LAMP1 (ab208943, 1:100, Abcam). After that, cells were incubated with Alexa Fluor 488-conjugated or Alexa Fluor 555-conjugated secondary antibodies (1:100, Bioss, Beijing) and then mounted with DAPI.
RNA isolation and real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (RR037A, TaKaRa Bio). Quantitative real-time PCR assays were performed by using TB Green Premix Ex Taq II (RR820A, TaKaRa Bio) through an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). Relative expressions were normalized to the geometric mean of housekeeping gene β-actin and were analyzed by using the 2-ΔΔCt method. The primer sequences were listed in Additional file 1: Table 2.
MHCC-97H cells transfected with NUPR1 overexpression vector or control vector were seeded in 6-well plates and exposed to 8 Gy irradiation. After 24 h, total RNA was isolated and subjected to the construction of RNA-seq libraries. The quality of the RNA libraries was evaluated using the Agilent 2200 TapeStation (Agilent Technologies, USA). Library sequencing was performed on a HiSeq 3000 sequencing platform (Illumina Company, USA) by Guangzhou RiboBio Corp., China.
ROS and lipid peroxidation assay
Cells were seeded in triplicate in 6-well plates and allowed to grow to 70-80% confluency. The cells were pretreated with or without drugs for 24 h and then irradiated. After irradiation for 24 h, MHCC-97H/MHCC-97L cells transfected with RFP protein were replaced with fresh medium containing 5 μM CM-H2DCFDA (C6827, Thermo Fisher) for ROS measurements. QGY-7701/Hep3B cell lines carrying GFP protein were treated with 5 μM CellROX Deep Red Reagent (C10422, Thermo Fisher) to determine ROS levels. A fresh medium with 5 μM BODIPY 581/591 C11 dye (D3861, Invitrogen) was added to each well for lipid peroxidation measurements. After incubation for 30 min in a humidified incubator (37°C, 5% CO2), the cells were washed with PBS, digested with trypsin, and measured by flow cytometry using FACS Canto II cytometer (BD Biosciences). The results were analyzed by Flow Jo 7.6.1 software (Treestar).
Cell death analysis
Cells were seeded in triplicate in 6-well plates and exposed to 8 Gy of irradiation. After IR, cells were replaced with a fresh medium and cultured for three days. Next, cells were washed with PBS, digested by trypsin solution without EDTA, and resuspended in 500 μL assay buffer containing 5 μM of 7-aminoactinomycin D (7-AAD) (C1053S, Beyotime). After incubation for 15 min at room temperature, cell samples were detected and analyzed by flow cytometry (BD Biosciences).
For apoptosis analysis, cells were pretreated with or without drugs for 24 h and exposed to a single dose of radiation (8 Gy) for 48 h. The cells were washed by PBS, trypsinized, and resuspended in 500 μL of FITC-Annexin V or APC-Annexin V solution (KeyGen BioTech, Nanjing). After incubation on ice for 15 min, DAPI was added to a final concentration of 10 μg/mL. The samples were then analyzed by flow cytometry (BD Biosciences).
In vivo xenograft mouse models
For the establishment of HCC xenografts, 2 × 106 MHCC-97H cells transfected with LV-NC/LV-NUPR1 were suspended in 150 μL of serum-free DMEM containing 50 μL Matrigel and subcutaneously injected into nude mice (male, 4–6 weeks). Tumor volumes were measured using digital vernier calipers and calculated by a standard formula: length × width2/2. When tumor volume reached 100 mm3, irradiation (8 Gy/day × 2 days) was administered to each xenograft. Mice were divided into 4 groups (n = 5/group): control, NUPR1 overexpressing, control plus IR, or NUPR1 overexpression plus IR. 1 × 107 Hepa1-6 cells were subcutaneously injected into C57/BL6 mice (male, 4–6 weeks). When xenografts reached about 200 mm3, mice were randomly divided into 4 groups (n = 5/group): control, ZZW115, IR, or ZZW115 plus IR. A single dose of IR (10 Gy) was given on the first day, and ZZW115 (1 mg/kg) was concurrently given by intraperitoneal injection for 7 consecutive days. The tumors were measured every 4 days and collected when the biggest reached about 1000 mm3.
Immunohistochemistry (IHC) analysis
In brief, paraffin-embedded tissues were cut into 3 μm sections. Sections were deparaffinized, rehydrated, subjected to antigen retrieval, and treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Antibodies against NUPR1 (15056-1-AP, 1:500, Proteintech), AhR (GTX22770, 1:200, GeneTex), CYP1A1 (13241-1-AP, 1: 200, Proteintech), Ki67 (#9449, 1:800, Cell Signaling), PCNA (A12427, 1:200, ABclonal), γH2AX (#9718, 1:400, Cell Signaling), MDA (ab24066, 1:100, Abcam) were incubated with the sections overnight at 4°C, respectively. After incubation with a secondary antibody, the visualization signal was stained with 3, 3′-diaminobenzidine (DAB) and then counterstained with hematoxylin. We regarded the multiplication of staining intensity and the extent of staining as the final score (0–12). Staining intensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The extent of staining scored was as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (>75%). The stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters.
Comet assay was analyzed using DNA Damage Detection Kit (KeyGen BioTech, Nanjing) according to the manufacturer’s instructions. Briefly, transfected cells were irradiated at a dose of 8 Gy. The following day, cells were collected and suspended in PBS containing 1% low-melting agarose and layered onto adhesive microscope slides previously covered with 0.5% normal-melting agarose. The cells were dipped in a specific lysed buffer at 4°C for 2 h. Then, the DNA was uncoiled and unwound in an alkalescent electrophoresis buffer for 30 min. Electrophoresis was carried out and the cells were stained with DAPI solution for 10 min in a dark room. The slides were examined with an Olympus BX63 fluorescence microscope. Tail moment was calculated by using CASP software.
Cells were seeded at 6-well plates, allowed to attach, and exposed to 8 Gy irradiation. After 24 h, cells were washed with cold PBS and extracted with NADP+/NADPH extraction buffer, followed by centrifugation at 10,000 × g for 10min to remove insoluble material. Samples were deproteinized by filtering through a 10-kDa cut-off spin filter. To detect NADPH, NADP+ was decomposed by centrifuging tubes and heating to 60°C for 30 min in a water bath followed by cooling on ice. Samples were quickly spun to remove any precipitates, leaving only NADPH. NADP+ and NADPH samples were incubated with a Master Reaction mixture for an appropriate time before the absorbance was measured at 450 nm according to the manufacturer’s protocol (MAK038, Sigma-Aldrich).
Statistical analysis was performed using SPSS 20.0 software, GraphPad Prizm 8, ImageJ. Two-tailed and unpaired Student’s t-tests and two-way ANOVA tests were performed to compare differences. The differences in NUPR1 expression levels between the paired HCC tissues and adjacent nontumorous liver tissues in the TCGA database were compared by paired t-tests. Pearson correlation analysis was performed to analyze the correlation between two molecules. Survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Data were presented as the means ± standard deviation (SD). Statistical significance was defined as a P-value less than 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ns, no significance.