Liver tumor specimens and adjacent non-tumorous specimens were collected from 20 patients who underwent surgical resection at the Second Affiliated Hospital of Chongqing Medical University (Chongqing, China). Patients had not received chemotherapy or radiation therapy before surgery. The samples were frozen immediately after surgery and stored in liquid nitrogen. All patients provided informed consent and the study was conducted with the approval of the Institutional Ethical Review Board of Chongqing Medical University (project license number: 2017012).
Cell cultures and drug treatment
The human hepatoma cell lines HepG2, SK-Hep1, and PLC/PRF/5 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Other hepatoma cell lines like Huh7 and normal human liver cells MiHA were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Apart from HepG2, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD, USA), 100 IU penicillin, and 100 mg/mL streptomycin. HepG2 cells were cultured in minimum essential medium (MEM; Hyclone). Cell lines involved in this study were recently authenticated by short tandem repeat (STR) profiling (Beijing Microread Gene Technology Co., Beijing, China). All cells were incubated in a humidified atmosphere at 37 °C containing 5% CO2.
For cell cycle analysis, cells were collected at different time points post-synchronization. For low-glucose treatment, cells were incubated in glucose-free DMEM (Hyclone) supplemented with 10% FBS, 100 IU penicillin, 100 mg/mL streptomycin, and the indicated concentrations of glucose (Hyclone) for 12 h. For immunoblotting and cell cycle analysis under glucose-deprived conditions, cells were incubated in DMEM containing 1 mM glucose and metformin (5 mM; Cat# S1950; Selleck, Houston, TX, USA) for 12 h and then collected for analysis. For proliferation and colony formation assays, cells were cultured in normal DMEM supplemented with 0.5 mM metformin.
Plasmid constructs and adenovirus production
The full-length cDNA of PCK1 (coding sequence of NM_002591) was amplified from plasmid pOTB7-PCK1 (Cat# FL07339; GeneCopoeia, Rockville, MD, USA). Primers are listed in Additional file 1: Table S1. The amplified PCK1 fragment was inserted into the shuttle vector pAdTrack-TO4 (kindly provided by Dr. Tong-Chuan He, University of Chicago, USA). Adenoviral recombinant pAd-PCK1 was generated using the AdEasy system as described previously . The analogous adenovirus expressing GFP only (AdGFP) was used as control.
Two pairs of oligonucleotides encoding short hairpin RNA (shRNA) targeting isoform α1 of AMPK (Additional file 1: Table S1) were designed and subcloned into the lentiviral vector pLL3.7 (kindly gifted by Prof. Bing Sun from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China) to generate shAMPK lentivirus. A control virus (shControl), with a scrambled shRNA insert, was also generated. For lentiviral supernatant production, HEK-293 T cells were transfected with 2 μg VSVG, 3 μg Δ8.9, and 4 μg of the shRNA lentiviral construct for lentiviral package using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s instructions. SK-Hep1 cells were transduced with the packaged lentiviruses in the presence of 5 μg/mL polybrene.
RNA-sequencing (RNA-seq) and expression analysis
Hepatoma cells were infected with AdGFP or AdPCK1 for 36 h and then RNA was extracted using TRIzol reagent (Invitrogen, Rockville, MD, USA) according to manufacturer’s instructions. RNA-seq and bioinformatic data analysis were conducted at Shanghai Novel Bio Ltd. (Shanghai, China). Strand-specific RNA-seq libraries were prepared using the Total RNA-seq (H/M/R) Library Prep Kit (Vazyme Biotech, Nanjing, China) and were sequenced on an Ion Torrent Proton Sequencer (Life Technologies, Carlsbad, CA, USA) according to the Ion PI Sequencing 200 Kit v2.0 (Life Technologies). Raw reads in FASTQ format were quality-controlled using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). RNA-seq reads were aligned to the reference genome using Bowtie and uniquely mapped reads were used for further analysis. Gene expression levels were expressed as reads per kilobase per million reads (RPKM) and differences in gene expression were calculated with rSeq (http://www-personal.umich.edu/~jianghui/rseq/).
RNA extraction and real-time reverse transcription (qRT)-PCR
Total cellular RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) and then reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) and random hexamers (Promega). The synthesized cDNA was then used as template for qPCR of the respective genes using SYBR Green and a CFX Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The actin beta gene (ACTB) was used as a reference gene for normalization. Relative mRNA levels were calculated using the 2–ΔΔCt method. All primers are listed in Additional file 1: Table S1.
Western blot analysis
Protein was extracted from cells or tissue samples using lysis buffer (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime), after which the protein concentration was determined using a BCA protein assay kit (Beyotime). Proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with primary antibodies against PCK1 (1:1000; Cat# BS6870; Bioworld, Atlanta, GA, USA), phospho-AMPKα1 (T172; 1:1000; Cat# 2535; Cell Signaling Technology, Danvers, MA, USA), AMPKα (1:1000; Cat# 2795; Cell Signaling Technology), p27Kip1(1:1000; Cat# 3686; Cell Signaling Technology), phospho-Rb (S780; 1:1000; Cat# BS4164; Bioworld), Rb (T774; 1:1000; Cat# BS1310; Bioworld), cyclin E1 (L389;1:1000; Cat# BS1086; Bioworld), CDK2 (78B2; 1:1000; Cat# 2546; Cell Signaling Technology), phospho-CDK2 (Thr160; 1:1000; Cat# 2561; Cell Signaling Technology), E2F2 (K236; 1:1000; Cat# BS2057; Bioworld), LKB1 (1:1000; Cat# GTX130697; GeneTex, Irvine, CA, USA), and CAMKK2 (1:1000; Cat# GTX115461; GeneTex). Then, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Abcam, Cambridge, UK). Proteins were detected using the Super Signal West Pico Chemiluminescent Substrate Kit (Millipore) and quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MA, USA; http://imagej.nih.gov/). GAPDH (Cat# AF0006; Beyotime) or β-actin (Cat# BL005B; Biosharp) were used as internal control. All experiments were independently repeated three times.
Histological and immunohistochemistry (IHC) analysis
Liver samples were fixed in fresh 4% paraformaldehyde and subjected to routine histological procedures for embedding in paraffin. Then, the samples were cut in 4.5 μm-thick sections that were processed for hematoxylin and eosin (HE) or IHC staining with antibodies targeting PCK1 (1:500), pAMPK (1:200), or p27Kip1 (1:500). For IHC assays, the sections were incubated with secondary anti-rabbit IgG (ZSGB-BIO, Beijing, China) and stained with 3,3′-diaminobenzidine (ZSGB-BIO). Stained slides were scanned with a Pannoramic Scan 250 Flash or MIDI system and images were acquired using Pannoramic Viewer 1.15.2 (3DHistech, Budapest, Hungary).
CRISPR/Cas9-mediated knockout of PCK1
The CRISPR/Cas9 plasmids lentiCRISPR v2, pMD2.G, and psPAX2 were kindly provided by Prof. Ding Xue from the School of Life Sciences, Tsinghua University (Beijing, China). Single-guide RNAs targeting human PCK1 were designed using the E-CRISP online tool (http://www.e-crisp.org/E-CRISP/designcrispr.html). The PCK1 targeting sequences (listed in Additional file 1: Table S1) were synthesized by TsingKe Biological Technology (Chongqing, China) and cloned into lentiCRISPR v2 vectors. Lentiviruses were generated by co-transfecting HEK293T cells with lentiCRISPR v2, envelop plasmid pMD2.G, and packaging plasmid psPAX2 using Lipofectamine 2000 according to manufacturer’s instructions. After 48 h, PLC/PRF/5 cells were incubated with medium containing virus along with 5 μg/mL polybrene. Two days post-infection, cells were selected in the medium containing 2 μg/mL puromycin and then single-cell colonies were selected after seeding in 96-well plates. For genotyping, clonal cell genomic DNA was extracted with a Genomic DNA Purification Kit (Genloci Biotechnologies Inc., Jiangsu, China) and then cloned into the pMD19-T TA cloning vector (Takara, Kyoto, Japan) for sequencing. PCK1-knockout efficiency was confirmed by western blotting. PCK1-knockout and control cells are hereafter referred to as PCK1-KO and parental cells, respectively. All primers are listed in Additional file 1: Table S1.
Cells were seeded in 96-well plates at a density of 1 × 103 cells/well and cultured for 5 days. The absorbance at 490 nm was measured in real time every 24 h after incubation with 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) labeling reagent solution (Promega) for 2 h.
Colony formation assay
Cells (1 × 103 cells/well) were seeded in 6-well plates and cultured at 37 °C and 5% CO2 for 14 days. The medium was replaced every three days. Cell colonies were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.1% crystal violet for 20 min. The experiment was repeated at least three times.
Cell cycle analysis
Cells were harvested at a density of 1.0 × 106 cells/mL, washed with PBS, and fixed with 70% ethanol at 4 °C overnight. Then, the cells were washed with PBS, stained with propidium iodide, and subjected to cell cycle analysis using a FACSCalibur instrument (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest software.
Determination of intracellular ATP levels
Cellular ATP production was detected using an ATP Assay Kit (Beyotime) according to manufacturer’s instructions . Briefly, cells were collected in a 1.5-mL tube and centrifuged. Then, 200 μL of lysis buffer from the ATP Assay Kit was added to each tube, after which the lysates were centrifuged at 12,000×g for 5 min. ATP production was measured by a luciferase assay of cell lysates and normalized to cellular protein concentrations (nM ATP/mg protein). Protein levels of the supernatant were measured at 562 nm with a BCA assay kit (Beyotime).
For the subcutaneous xenograft tumor model, 18 male BALB/c nude mice (5–6 weeks of age) were randomly divided into three groups. MHCC-97H cells were mock-infected or infected with AdGFP or AdPCK1 for 24 h, then collected for subcutaneous injection (1 × 105 cells/injection) into the flanks of athymic BALB/c nude mice. Tumor volume was monitored by measuring the length (L) and width (W) at 3-day intervals for 5 weeks. Tumor volume [cm3] was calculated as L [cm] × (square of W [cm2])/2. After 5 weeks, the mice were sacrificed and tumor tissues were collected for histological analysis.
For the orthotopic implantation model, 15 BALB/c nude mice were randomly divided into parental, PCK1-KO, and metformin-treated PCK1-KO groups (five mice per group). The PLC/PRF/5 parental and PCK1-KO cells (1 × 105 cells/injection) were collected and implanted into the left lobes of nude mice livers. On day 7 after implantation, the mice were treated with metformin (250 mg/kg per day, intraperitoneally) or PBS (equal volume, intraperitoneally) for 6 weeks. One mouse in a treatment group died due to postoperative infection during the experiment. Seven weeks after implantation, the mice were sacrificed and liver tissues were collected for histological examination.
All animal experiments were carried out according to the guidelines of the Institutional Animal Care and Use Committee at Chongqing Medical University (project license number: 2017012) and animal care and use protocols adhered to national regulations for the administration of laboratory animals.
Data are expressed as the mean ± standard deviation (SD). Means were compared using Student’s t-test when comparing two groups or one-way analysis of variance (ANOVA) when comparing more than two groups. Correlations were assessed using the Spearman Rank Correlation test. Two-sided P values < 0.05 were considered statistically significant. Statistical analyses were conducted using GraphPad Prism 7.0 software (La Jolla, CA, USA).
Nucleotide sequence accession numbers
The RNAseq data generated in this study have been submitted to the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo) under the identifier GSE117822.