Clinical data and specimens
We retrospectively harvested the HCC specimens and the adjacent normal tissues (ANTs) from ninety-two patients who received radical hepatectomy at the Department of Hepatic Surgery and Liver Transplantation Center of the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) between July 2014 and August 2018. All the enrolled patients were histologically diagnosed without any other pre-operative treatment, including chemotherapy and radiotherapy. After collected, the resected specimens were cut, snap-frozen in liquid nitrogen and subsequently stored at -80℃. This study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University, and the procedures were conducted abiding by the Declaration of Helsinki. All patients provided informed consents. The detailed information of patients was list in Supplemental Table 2.
Cell lines and culture
The normal human hepatocyte line LO2 and four human HCC lines, Huh7, HepG2, Hep3B, and SK-Hep1 were purchased from the Chinese Academy of Sciences (Shanghai, China) and maintained in high glucose (4.5 g/L)-Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, Life Technologies, Carlsbad, CA, USA) containing with 10% fetal bovine serum (FBS, PAN-Biotech, Germany). All the cells were cultured at 37℃ under an atmosphere of 5% CO2 confirming to be mycoplasma-free.
Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
The primer sequences used in this study were list in Supplemental Table 1. Total RNA extraction, reverse transcription and qRT-PCR were conducted following the previous described.[10] The tissues were cut into 0.5 cm3 and sequentially triturated in liquid nitrogen, while the cells were digested in 0.1% trypsin (Gibco, Life Technologies, Carlsbad, CA, USA). Then, total RNA extraction was performed using TRIzol (Invitrogen) according to the manufacturer’s protocol. After confirmed the amount and purity of total RNA using Biophotometer plus (Eppendorf, Germany), Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, USA) was used to reverse transcribe to cDNA from total RNA. cDNA was amplified using PCR Thermal Cycler (Bio-Rad, USA) by firstly heating at 65℃ for 10 min, incubating at 55℃ for 30 min, deactivating at 85℃ for 5 min and finally saving at 4℃ for 5 min. qRT-PCR was performed by SYBR Master Mix (Roche Applied Science) using reverse transcription system (LC-480, Roche, USA). GAPDH was identified as a housekeeping gene to normalization.
RNA fluorescence in situ hybridization
RNA fluorescence in situ hybridization (RNA-FISH) was conducted to visualize the location of circFOXK2 (RiboBio, Guangzhou, China) and miR-484 (BersinBio, Guangzhou, China) in cell using Fluorescent in Situ Hybridization Kits according to the manufacturer’s instructions. The cells were treated with the probes respectively specific to circFOXK2 (hsa_circ_0000817) and miR-484 at 4℃ overnight and, then, observed under a Zeiss 880 confocal microscope (Nikon Instruments, Melville, New York, United States) after stained with DAPI for 2 min.
Cell proliferation assay
The proliferation potential of HCC cells was detected using cell counting kit-8 (CCK8, KeyGEN BioTECH, Jiangsu, China) according to the manufacturer’s protocol. After treated following the group design, the cells were planted in a 96-well plate at a density of 4,000 cells per well and cultured for 24, 48, 72, 96 and 120 h. Cell viability of each group was determined by the optical density (OD) values at 450 nm using a microplate reader (Tecan Spark 10M, Austria).
Colony formation assay
Colony formation assay was used to evaluate the cloning ability of HCC cells. HCC cells in each group were seeded in 6-well plates at a density of 1,000 cells/well. Then, the cells were cultured in the incubator for 2 weeks. The colonies were visualized and counted after fixed with 4% paraformaldehyde (PFA) and stained with crystal violet.
Flow cytometry analysis
To measure cell apoptosis, a PI/Annexin V-FITC Apoptosis Detection Kit (BD, USA) was used according to the manufacturer’s instruction. Briefly, 1×106 HCC cells in each group were collected and rinsed twice with washing buffer. Subsequently, the cells were incubated with Annexin V and PI for 15 min in the dark at room temperature. The cell apoptosis was determined using flow cytometry (BD Biosciences, San Jose, CA, USA).
Transwell invasion assay
2×105 HCC cells were resuspended in serum-free DMEM medium and planted into the upper chamber of Transwell plates with 8.0-μm pores (Corning Costar, Corning, NY, USA). DMEM medium containing 10% FBS was added into lower chamber. The cells were maintained in the incubator at 37℃ for 24 h. Then, the non-migrated cells on the upper compartment were removed. After that, the cells were fixed with 4% PFA for 30 min and stained with crystal violet for 15 min. Image were captured under a light inverted microscope (Leica, German) and counted using ImageJ (National Institutes of Health, USA).
Western blot assay
The cells were lysed in cold radioimmunoprecipitation assay (RIPA) Lysis Buffer containing Tris-HCL (pH 7.4, 50 mM), NaCl (150 mM), 0.1% Triton (100×), sodium dodecyl sulfate (SDS, 10%), sodium deoxycholate (10%), ethylene diamine tetraacetic acid (EDTA, 2 mM) and protease cocktail inhibitor (KeyGEN BioTECH, Jiangsu, China) for 20 min on ice. After equaled the total protein concentration using a bicinchoninic protein assay (KeyGEN BioTECH), 30 μg proteins were electrophoresed using 12% SDS polyacrylamide gel electrophoresis (PAGE) and subsequently transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). 5% non-fat milk was used to block non-specific antigen of the membranes for 1 h at room temperature, and, then, the membranes were respectively incubated with the primary antibodies at 4℃ overnight. After washed three times, the membranes were treated with secondary antibodies (anti-rabbit IgG, 1:5000, Sigma-Aldrich) for 1 h at room temperature. The blots were visualized by a ChemiDocTM MP Imaging System (Bio-Rad, CA, USA) after treated with an enhanced chemiluminescence (ECL) substrate. The intensities of the blots were evaluated using an image analyzer (ImageJ software).
Metabolism assay (including lactate, pyruvate, and ATP assays)
1) Lactate: Lactate concentration in the cell cultured supernatant was determined utilizing a Lactate Assay Kit (BioVision) following the manufacturer’s instruction. In brief, after treated according to the group design, the supernatant was harvested and centrifuged for 10 min to remove insoluble portion. The samples were incubated with a Lactate Assay Kit for 30 min in the dark and detected by a microplate reader (Tecan Spark 10M, Austria) at a wavelength of 570 nm.
2) Pyruvate activity: For the determination of pyruvate activity, the cells in each group were collected and extracted in the Pyruvate Assay Buffer (4 volume, BioVision). After centrifugation for 10 min to remove insoluble material, the supernatant was detected using a Pyruvate Colorimetric Assay Kit (BioVision). The reaction mixture was assayed at a wavelength of 570 nm using a microplate reader after incubation at room temperature in the dark for 30 min.
3) Intracellular ATP: Intracellular ATP was measured by an ENLITEN ATP Assay System Bioluminescence Detection Kit (Promega, USA) following the manufacturer’s instruction. After treated according to the group design and cultured in high-glucose DMEM containing 10% FBS for 48 h, the cells were washed with PBS three times and subsequently extracted in the ATP Assay Buffer (Promega). The ENLITEN ATP Assay System Bioluminescence Detection Kit was used to mix with the supernatant followed by the incubation in the dark for 30 min at room temperature. The 570 nm OD was detected using a microplate reader.
The NADH/NAD+ assays
The NADH/NAD+ Quantification Colorimetric Kit (BioVision) was used to determine the NADH/NAD+ ratio of HCC cells. All the procedures of this assay were conducted following the manufacturers’ protocol.
Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) measurements
Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were respectively detected using the Seahorse XFe 96 Extracellular Flux Analyzer (Seahorse Bioscience) according to the manufacturer’s instructions. The results were analyzed by a Seahorse XF-96 Wave software. The procedures of these two assays are briefly described as follows:
ECAR: The cells from each group were seeded in a 24-well cell culture XF microplate (Seahorse Bioscience) at a density of 25,000 cells/well. After cultured overnight to allow cells adherence, the cells were washed twice with assay medium and subsequently incubated with assay medium [the DMEM containing L-glutamine (2 mM), pH 7.4] culturing in a CO2-free incubator at 37℃ for 1 h. In addition, 80 mM glucose, 9 μM oligomycin and 1 M 2-DG were respectively loaded to the cartridge port A, B, and C. All values were normalized according to the cell number through the crystal violet assay. ECAR was identified as glycolysis rate after treated with glucose and glycolysis capacity after oligomycin treatment.
OCR: ATP synthesis was inhibited by oligomycin (1 μM, Sigma), then maximal OCR was measured by adding with trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP, 500 nM, Sigma), and, finally, mitochondrial respiration was closed using antimycin A (the mitochondrial complex I inhibitor rotenone + the mitochondrial complex III inhibitor, 1 μM, Sigma). The values were also normalized according to the cell number.
Detection of intracellular mitochondrial morphology
The mitochondrial morphology in the HCC cells was observed by staining with MitoTracker Green FM probe (Invitrogen) in the dark for 30 min at 37℃. Images were obtained under a Zeiss 880 confocal microscope to assess the number and morphology of mitochondria. And the percentage of cells with small and round mitochondria was respectively calculated.
Co-immunoprecipitation
The cells were lysed in 4℃ cold co-IP buffer [HEPES (10 mM, pH 8.0), NaCl (300 mM), EDTA (0.1 mM), NP-40 (0.2%), glycerol (20%), protease and phosphatase inhibitors] for 30 min. The lysates were centrifuged at 10,000 g for 5 min and cleared after treated with protein A/G agarose (Gibco BRL, Crand Island, NY, USA) for 15 min at 4℃. The pre-cleared supernatant was subjected to a horizontal shaker and incubated with the indicated primary antibodies at 4℃ overnight. After that, the protein complexes were harvested and incubated with the protein G beads for 120 min at 4℃. Finally, the samples were separated via SDS-PAGE.
Mass spectrometry analysis by LC-MS/MS
To identify the protein, mass spectrometry analysis was conducted according to previously described.[10] In brief, total proteins were firstly separated by SDS-PAGE (12%). Then, the protein-bands near 15 kDa were manually cut according to the size and digested by sequencing-grade trypsin (Promega, Madison, WI, USA). Peptide mixtures were extracted, dried, and finally loaded to LC-MS/MS (Thermo Fisher Scientific, Waltham, MA, USA) for detecting protein sequen—
ce. The National Center for Biotechnology Information nonredundant protein database with Mascot (Matrix Science, Boston, MA, USA) was used to analyze the fragment spectra.
RNA immunoprecipitation (RIP)
After cross-linked with 1% formaldehyde in ice-cold PBS for 10 min, the cells were collected and lysed in RIP lysis buffer followed by treating with Dynabeads protein G conjugated with anti-AGO2 or anti-IgG and rotating overnight at 4℃. The immunoprecipitated RNAs were obtained using TRIzol reagent and measured by RT-qPCR with specific primers.
Dual-luciferase reporter assay
The sequences of circFOXK2 with muted miR-484 binding sites or wild-type were established and inserted into luciferase vectors. Then, these vectors and miR-484 mimics were co-transfected into HEK-293T cells for 48 h. The luciferase activity was detected by a dual-luciferase reporter assay system (Promega, USA) following the manufacturer’s instruction. The Renilla luciferase internal control was used to normalize the results.
Biotin-labeled miRNA capture
The RNA pull-down assay was carried out according to the previous studies.[25] The biotin-labeled miR-484 mimic (GenePharma, China) was transfected into the stably overexpressing circFOXK2 HCC cells for 48 h. After washed and blocked by yeast tRNA for 2 h at 4℃, the streptavidin-Dyna beads M-280 were incubated with the cell lysates overnight at 4℃ to pull down the biotin-coupled RNA complex. The abundance of circFOXK2 in bound sections was detected by qRT-PCR.
Animals
5-week-old, male BALB/c nude mice were purchased from Biomedical Research Institute of Nanjing University (Jiangsu, China) and maintained abiding by the Guideline of Sun Yat-sen University for Animal Experimentation. All mice were taken care in laminar flow cabinets under specific pathogen free (SPF) conditions of the Laboratory Animal Center of Sun Yat-sen University with 12 h dark/light cycle, 50% humidity, 20℃ temperature and freely received standard laboratory diet and water.
Xenograft model establishment
All in vivo experiments were approved by the Ethics Committee of Sun Yat-sen University (Guangzhou, China) and performed complying with the institutional guideline. The establishment of the xenograft model was conducted according to the previous study.[26, 27] 5×106 HCC cells (Huh7) were suspended in Matrigel and injected subcutaneously into the left-backs of the mice. The length and width of the tumors were measured every three days when the tumors grew to palpable. Four weeks of implantation later, the tumors were harvested for further analysis by sacrificing all mice. The weight and volume of samples were measured. The formula for calculating the tumor volume was listed as follow: tumor volume= (tumor length × tumor width2)/2.
Immunohistochemistry staining
The protocol of immunohistochemistry staining was conducted according to the previous study.[28] In brief, 4 μm thick paraffin-embedded sections were dewaxed and dehydrated followed by the incubation with H2O2 (3%) for 10 min at 37℃. To repair antigen, the sections were treated with EDTA (pH 8.0) at 95℃ for 20 min and, then, cooled down to 25℃. After blocked the non-specific antigens by normal goat serum, the sections were respectively incubated with primary antibodies overnight at 4℃ and subsequently the secondary antibody for 30 min at 37℃. Finally, the sections were treated with diaminobenzidine and detected under a light microscope (Leica, Germany).
Oligonucleotide transfection
The control plasmid (vector control), circFOXK2 overexpressing vector, circFOXK2-flag vector, circFOXK2-mut vector, circFOXK2-flag-mut vector and FOXK2-142aa vector were obtained from General Biosystems (Anhui, China). ShRNA knocking down circFOXK2, siRNA targeting LDHA and miR-484 mimics or inhibitors were purchased from GenePharma (GenePharma Corporation, Shanghai, China). After reached approximately 60% confluence, the vectors were respectively transfected to the cells by a Lipofectamine® 3000 transfection kit (Invitrogen, Carlsbad, CA, USA), while the shRNA, siRNA and miRNA mimics or inhibitor were respectively transfected using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol. The efficiency of transfection was determined by qRT-PCR.
Statistical analysis
Statistical analyses were performed by GraphPad Prism (version 5.0, USA) and SPSS 23.0 (IBM, SPSS, Chicago, IL., USA). The relationship between circFOXK2 expression and the clinicopathological characterizations of HCC was analyzed by the Chi-square test. In addition, univariate and multivariate analyses were performed using a Cox proportional hazards regression model to detect the clinical value of circFOXK2 and FOXK2-142aa. OS was carried out by the Kaplan-Meier method and analyzed using a log-rank test. All in vitro experiments were independently repeated three times. As appropriate, the significant difference was assessed by Mann-Whitney U test, Wilcoxon rank-sum test or unpaired two-tailed Student’s t-test. A probability (p) value less than 0.05 were considered significant difference. *p <0.05, **p <0.01, ***p <0.001.