Patients and tissue microarray
Six pairs of HCC and adjacent (at least 5 cm from the tumor) normal tissues were collected from HCC patients at the First Affiliated Hospital of Third Military Medical University between 2011 and 2013. Informed consent was obtained from all patients. HCC tissue microarrays were purchased from Shanghai Biochip Company Ltd. (Shanghai, China), of which 145 cases with survival information and 85 with paired normal tissues were used for immunohistochemistry.
Cell culture
Human hepatoma cell lines Huh7 and LM3 were obtained from the American Type Culture Collection (ATCC) and authenticated by the Cell Bank of Type Culture Collection of Chinese Academy of Science. The cells were maintained under recommended conditions. For hypoxia treatment, the cells were either cultured in a sealed hypoxia chamber (Thermo fisher, Inc.) containing 1% O2, 5% CO2 and 94% N2, or treated with 100mM CoCl2 for 24 or 48 hours. Sorafenib-resistant Huh7 (Huh7-SR) and LM3 (LM3-SR) cells were enriched by steadily increasing the drug dose, while hypoxia-induced sorafenib resistant cell lines (Huh7-H-SR and LM3-H-SR) were established by culturing under 1% O2 with increasing drug doses.
Virus production
The pLVX-CMV-EGFP-3FLAG-PGK-Puro lentiviral vector expressing full-length human ATAD3A was purchased from SunBio (Shanghai. China). The pMAGic2.1-CMV-HygroR-U6 shRNA lentivirus vector purchased from SunBio (Shanghai. China), and the shRNA sequences are shown in additional file 1. Human HEK293T cells (American Type Culture Collection) were cultured in 6-well plates till ~70% confluent, and co-transfected with 2μg overexpression or knockdown virus vector, 1μg pMD2.G and 1μg psPAX2 lentivirus packaging vectors using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were maintained in high-glucose DMEM containing10% FBS, 2mM glutamine, and100 units/ml penicillin and streptomycin. The supernatants with virus were harvested twice at 48h and 72h, filtered through a 0.45μm syringe filter and frozen in liquid nitrogen.
Target cell transduction
HCC cells were cultured in complete DMEM, and 1.5x105 cells were mixed with 450μl virus-containing supernatant in the presence of 4μg/ml polybrene (Sigma). The cells were seeded into a 6-well plate, and the medium was changed 12h post-infection. After 48h, the infected cells were trypsinized and seeded into 10 cm culture dish with 4μg/ml puromycin (Thermo Fisher Scientific). The stably transduced cells were selected over 48h, and harvested 6 days post-infection to determine knockdown efficiency.
Western blotting
Total protein was extracted from cancer cells using Mammalian Protein Extraction Buffer (P0013, Beyotime, Beijing, China) supplemented with protease inhibitor cocktail (87786, ThermoFisher, USA). The mitochondrial fractions were separated using Mitochondria Isolation Kit (number: SM0020, Solarbio, Beijing, China) according to the manufacturer’s protocol, and the protein was extracted as above. Equal amounts of protein lysates were separated by SDS-PAGE gel and electro-transferred to PVDF membrane (Millipore, USA). After blocking in 5% milk-PBST for 2 hours at 37°C, the membranes were incubated overnight with primary antibodies (Additional file 2) at 4°C and with secondary antibodies at 37°C for 2 hours. The positive bands were visualized with Immobilon Western Chemiluminescent HRP Substrate detection reagent (Millipore, USA), and acquired using a ChemiDoc™ imaging System (Bio-Rad, USA).
Quantitative real-time PCR
Total RNA was extracted from cancer cells using Trizol Reagent (Invitrogen, USA), and qRT-PCR was performed using SYBR Prime Script RT-PCR kit (TaKaRa, Japan) on a Rotor-Gene 6000 real-time genetic analyzer (Corbett Life Science, USA). The primer sequences and the product sizes are listed in Additional file 3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The PCR conditions were as follows: denaturation at 95°C for 2 mins, followed by 40 cycles of amplification and quantification (95°C for 5 s, 55°C–57°C for 30 seconds), and melting curve (55°C–95°C, with 0.5°C increment each cycle). Each sample was tested in triplicates.
Apoptosis assay
For apoptosis assays, the suitably treated cells were washed twice with cold PBS, and resuspended in binding buffer at the density of 1×106 cells/ml, and distributed into 500μl aliquots (1×105) in 2ml tubes. 5μL of Annexin V-FITC were added to each tube. Cells were incubated at room temperature (25°C) for 15 min in a dark environment, and then analyzed on a FACS Calibur (BD Biosciences, USA), and results were calculated using Cell Quest software (BD Biosciences, USA).
Immunofluorescence assay
HCC cells were washed, fixed with 5% paraformaldehyde (PFA) and permeabilized in 0.1% Triton X-100. After incubating overnight with monoclonal mouse anti-HIF-1α antibody (cell signaling technology) or monoclonal rabbit anti-ATAD3A antibody (Invitrogen, PA5-03671) at 4℃, the cells were probed with fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG or Cy3-labeled anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, USA). The nuclei were counterstained with Hochest 33258 and observed under laser confocal scanning microscopy (Leica TCS-SP5, Germany).
Luciferase reporter assay
The 3′-UTR region of human ATAD3A was amplified by PCR from genomic DNA and cloned downstream to the firefly luciferase coding region in the pMIR-REPORTTM plasmid. The 293 T cells were seeded in 96-well plates, and co-transfected 24h later with 100ng/ml reporter plasmid and 50nM miR-210-5P or NC mimics using Lipofectamine 2000. Luciferase activity were measured after 72h using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). All experiments were performed in triplicates.
Cell proliferation assay
Cells were seeded in 96-well plates at the density of 5000 cells/well. After treating with 5.13µM (Huh7) or 7.92µM (LM3) sorafenib for varying durations (0h,12h,24h,48h and 72h), the cells were incubated for 2 hours with 0.5 mg/ml 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich) in serum free medium. To measure the IC50 of different cell lines, 10-3,10-2,10-1,100,101 and 102µM sorafenib were added in the 96-well plates for 72h, then treated same as above. The optical densities were measured at 450nM spectral wavelength using the microplate reader (ELx800; Bio-Tek Instruments, Inc.).
Colony formation assay
For foci formation assay, 5x102 cells were seeded in 6-well plates with sorafenib 5.13µM (Huh7) or 7.92µM (LM3) as appropriate. The conditioned medium was removed 3 days later and replaced by medium containing 10% serum for 2 weeks. The ensuing colonies were stained by crystal violet and counted. The experiment was performed thrice.
Mitophagy assay
Western blotting was used to test LC3 mobility shift by analyzing the expression levels of LC3 I/II, and mitochondrial marker protein TOMM20 and TOMM70. Subcellular localization of mitophagy bodies was tracked by transmission-electron-microscopy (TEM, JEM-1230, Japan). The cells were fixed in cooling 2% glutaraldehyde and 0.1M cacodylate buffer at 4 °C overnight, then exposed to phosphate buffer containing 1% osmium tetroxide for 1 h. Following dehydrated in different concentrations of acetone, these cells were infiltrated and embedded into Epon. The embedded cells were sectioned and stained with 3% uranyl acetate and lead citrate. Mitochondrial content was analyzed by Mito-Tracker staining (number: C1049, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol and then analysed by FACS (BD Biosciences, USA), and results were calculated using Flow Jowo software (BD Biosciences, USA).
Lactate measurement
The suitably treated cells were seeded in 6-well plates in triplicate at the density of 5*104 cells/well. After culturing for 2 days, the number of viable cells was counted, and the culture medium was collected. Lactate levels were detected in the latter using a specific analytical kit (Nanjing Jian cheng Bioengineering Institute, China), and normalized to cell number and calculated as relative units per cell.
Immunohistochemistry (IHC)
IHC was performed on tissue array slides as previously described. The tissues were probed with rabbit anti-human ATAD3A (Invitrogen, PA5-03671 1:200) and rabbit anti-human HIF-1α (abcam, ab51608, 1:200) antibodies, and counterstained with hematoxylin (Sigma). The staining intensity (negative = 0, weak = 1, moderate = 2, or strong = 3) and the percentage of positively stained cells (<25% = 1, 25 to 50% = 2, >50% to <75% = 3, ≥75% = 4) were scored independently by two pathologists. The staining index was calculated by multiplying the intensity score with percentage score, and the samples were classified as negative/low expression (0-2) or positive/high expression (3-12) accordingly.
RNA sequencing and bioinformatic data analysis
LM3-shControl and LM3-shATAD3A cells were generated as before and total RNA was extracted. RNA libraries were constructed using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions, and sequenced on an Illumina platform (HiSeqTM 2500 or Illumina HiSeq X Ten) into 125bp/150bp paired-end reads by OE Biotech (Shanghai, China). The differentially expressed genes (DEGs) were identified using Cuff-diff with q value <0.05 and fold change >2 as the criteria. Gene set enrichment analysis was performed using software.broadinstitute.org/gsea. Heatmaps were generated with the heatmap package of R program.
Tumorigenicity analysis in vivo
Female BALB/c (nu/nu) athymic nude mice, 5 weeks of age, were purchased from hfkbio Inc. Mice were maintained in specific pathogen-free conditions: 20–24°C, 12/12h of dark/light cycle, 60±5% of humidity, and plastic cage (3–4 mice/cage). Bedding materials were changed every week, and environmental enrichment was done with sterile materials. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Medicine University of Electronic Science and Technology of China’s. Control and miR-210-5P antagomir-transfected Huh7-shControl and Huh7-shATAD3A cells were inoculated subcutaneously (n=5 each, 2×106 cells per mouse) in the right flank of 4-6 weeks old BALB/c athymic female nude mice. Once the tumors grew to approximately 100mm3 (around 5 days), the mice were intraperitoneally injected with sorafenib tosylate (10mg/kg) daily for a week, and monitored every 4 days for the appearance of subcutaneous tumors. We conducted the sacrifice of mice at 28 days, we placed the mice in the chamber and introduced 100% carbon dioxide. After we removed each tumor, we maintained the carbon dioxide flow for a minimum of 1min after respiration ceases. The tumor weight and tumor volume (TV; mm3) were calculated. Tumor volume was calculated as d2×D/2 (d and D represent the shortest and the longest diameters respectively). All animal experiments were performed in accordance with the guidelines of the ARRIVE reporting guidelines.
Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad, La Jolla, CA, USA). Two tailed unpaired t-test or one-way ANOVA were used to compare two or multiple groups. Survival curves were plotted using the Kaplan–Meier method and compared by the log-rank test. All data was presented as mean ± SD or mean ± SEM. P<0.05 was considered statistically significant.