Human HCC samples
This study was approved by the Institutional Review Board at Yonsei University Health System Severance Hospital (Seoul, South Korea), and the study was conducted using the current guidelines for ethical research (Yonsei IRB number: 4-2015-0904). The selection of patients was performed as described previously [29].
Chemicals
PD0332991 was purchased from TOCRIS Bioscience (Bristol, UK) and sorafenib was purchased from Santa Cruz (Dallas, TX, USA). PD0332991 and sorafenib were dissolved in DMSO (Sigma Aldrich, St. Louis, MO, USA) at a concentration of 10 mM. All reagents were stored at –80 °C.
Cell lines and cell culture
The human HCC cell lines HepG2, Hep3B, skHep1, and Huh7 were purchased from the Korean Cell Line Bank. HepG2 was cultured in RPMI, and Hep3B, skHep1, and Huh7 were cultured in Dulbecco’s modified Eagle’s medium (DMEM). All media were supplemented with 10% fetal bovine serum (FBS; Hyclone) and 1% penicillin streptomycin. Cells were maintained in a humidified incubator with 5% CO2 at 37 °C. Hep3B, skHep1, and Huh7 cell lines were plated and incubated for 24 h before transfection. Lipofectamine or RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) was used to perform siRNA transfection following the manufacturer’s instructions. The plasmids for hSIRT3 (sc-61555-SH) or scramble shRNA (sc-108060) were cotransfected into HepG2 cells using Lipofectamine 2000 (Invitrogen, 12566014). After 72 h of incubation, the cells were treated with puromycin (2 mg/mL) to generate stable cell line clones.
Cell proliferation assay and glucose measurement
WST-1 colorimetric assays (Roche, Mannheim, Germany) for cell viability were performed 48 h after treatment according to the manufacturer’s recommendations. Huh7 cells were placed in 96-well plates and being transfected with MOCK or pcDNA-SIRT3 plasmid. After 48 h of treatment, the glucose uptake was determined using Glucose Assay (Promega, Germany) according to the manufacturer’s recommendation. Absorbances at 440 nm and 640 nm were measured using a microplate reader (Molecular Devices, CA, USA).
RNA isolation and sequencing
Total RNA was isolated using TRIzol reagent (Invitrogen). RNA quality was assessed by Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and RNA quantification was performed using ND-2000 Spectrophotometer (Thermo Inc., DE, USA). For control and test RNA samples, library was constructed using QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer’s instructions. Briefly, 500 ng total RNA was prepared for each sample, an oligo-dT primer containing an Illumina-compatible sequence at its 5' end was hybridized to the RNA, and reverse transcription was performed. After degradation of the RNA template, second strand synthesis was initiated by a random primer containing an Illumina-compatible linker sequence at its 5' end. The double-stranded library was purified using magnetic beads to remove all reaction components. The library was amplified to add the complete adapter sequences required for cluster generation. The amplified library was purified, and high-throughput sequencing was performed as single-end 75 sequencing using NextSeq 500 (Illumina, Inc., USA).
Real-time PCR
Total RNA was extracted with TRIzol (Invitrogen) and cDNA was synthesized from 500 ng of total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Quantitative RT-PCR was conducted on C1000 a Thermal Cycler (Bio-Rad) using SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). Gene expression levels were normalized with beta-2 microglobulin (B2M) mRNA expression levels of corresponding cDNA samples. All PCR primers were purchased from Bioneer (Daejeon, Korea). The following primers were used: SIRT3 (Forward 5'-GAAACTACAAGCCCAACGTCA-3', Reverse 5'-AAGGTTCCATGAGCTTCAACC-3'), RB1 (Forward 5'-GAAGCAACCCTCCTAAACCAC-3', Reverse 5'-CTGCTTTTGCATTCGTGTTCG-3'), and B2M (Forward 5'-TTACTCACGTCATCCAGCAGA-3', Reverse 5'-AGAAAGACCAGTCCTTGCTGA-3').
Western blotting
Western blotting was performed as described previously [29]. The primary antibodies in the present study were: SIRT3 (Cell Signaling Technology, Danvers MA, USA; clone C73E3; dilution 1:1000), CDK4 (DCS156, 1:1000), CDK6 (DCS83, 1:1000), Phospho-Rb (Ser807/811) (D20B12, 1:1000), Rb (4H1, 1:2000), PCNA (D3H8P, 1:2000), GLUT1 (1:2000) from Abcam (Cambridge, UK), and Ki67 (Santa Cruz, Dallas TX, USA; MIB-1, 1:500). Western blotting experiments from biological replicates showed similar expression data, attesting to the reproducibility of the results. For band quantification, images were analyzed using Image Lab software (Bio-Rad, Hercules, California, USA).
Flow cytometry analysis
For quantification of apoptosis, double staining was performed according to the manufacturer’s instructions using Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen™, NJ, USA) and propidium iodide (PI). After HepG2 and Huh7 cells were collected after incubation with indicated compound, cells were washed twice with ice-cold PBS and resuspended in 200 μL of binding buffer. Annexin V-FITC was added to the cells and incubated for 15 min in the dark at 25 °C. PI (10 mL) was added to the tube followed by 5 min of incubation at 4 °C in the dark. After incubation, the samples were analyzed by a flow cytometer using CELL Quest software (BD) and 1.0 × 105 events per sample were counted. The fraction of cell population in different quadrants was analyzed using quadrant statistics. Cells in the lower right quadrant (Annexin-V+/PI−) represented early apoptosis and those in the upper right quadrant (Annexin-V+/PI+) represented late apoptosis. For cell cycle analysis, after HepG2 and Huh7 cells were collected after incubation with indicated compound, the cells were incubated in 70% ethanol at 4 °C for 1 h. After washing with PBS, cells were incubated with PI at a concentration of 5 mg/mL and RNaseA at a concentration of 10 mg/mL for 30 min–4 h at 37 °C. The DNA contents of MG-63 were analyzed using FlowJo Software (Tree StarInc., Ashland, OR, USA).
Migration assay
Chemomigration assays were performed using 24-well plates with uncoated polycarbonate membrane inserts (BioCoat; BD Biosciences, Heidelberg, Germany). A total of 50,000 cells in medium containing 0.1% FBS and sorafenib, PD0332991, or combination of sorafenib and PD033291 were added onto the insert. The lower well was filled with a medium supplemented with 10% FBS. Twenty-four hours later, the cells that had migrated were fixed in 100% methanol and stained with 1.5% (w/v) toluidine blue in water. Images were recorded using an Olympus BX53 microscope with Olympus Cell Sens software (Carl Zeiss Microscopy, GmbH, Jena, Germany).
Immunostaining
Immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described previously [30]. After antigen retrieval, immunostaining was performed using various antibodies. The primary antibodies used were: SIRT3 (Cell Signaling Technology, Danvers MA, USA; clone C73E3; dilution 1:500); Ki67 (Dako, Glostrup, Denmark; MIB-1; 1:500); GLUT1 (1:500), and Ki67 (SP6, 1:500) from Abcam (Cambridge, UK). Images were recorded using an Olympus BX53 microscope with Olympus Cell Sens software (Carl Zeiss Microscopy, GmbH, Jena, Germany). The percentage of Ki67-positive cells and phosphorylated retinoblastoma protein (pRb) was calculated by counting the number of cells with DAPI-stained nuclei.
The Cancer Genome Atlas (TCGA) data analysis
mRNA levels of TCGA liver HCC data were obtained from the OncoLnc TCGA data portal (www.oncolnc.org). A set of 360 HCC samples with high and low gene expression groups (50-50 percentile) was used for correlation graphs of two different genes. GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used for mapping.
Statistical analysis
Statistical analyses were performed using GraphPad Prism Software (GraphPad Software, Inc., San Diego, CA). Results are expressed as mean ± SE (range). P values < 0.05 were considered statistically significant. Comparisons between groups were made using the Mann-Whitney test.