2.1 Patient samples
This study was approved by the Ethics Committee of Shanghai Ruijin Hospital, and all patients have provided written informed consent. ccRCC specimens (n = 280) histopathologically confirmed by three independent pathologists were made into a tissue microarray with matched normal sections. Six paired tumor and adjacent normal tissues were also obtained from ccRCC patients underwent radical nephrectomy in our institution. Clinical information was obtained from the medical records, including age, gender, tumor size, grade, stage. Tumor samples and adjacent normal tissues were placed into liquid nitrogen followed by storage at -80℃. In addition, computed tomography (CT) guided biopsies were collected from two ccRCC patients, and the patient derived organoids (PDO) were established and cultured.
2.2 Cell culture, materials and antibodies
HEK293T cells (ATCC CRL-11268) were cultured in DMEM (Gibco 12430-054) containing 10% FBS (Gibco 10099141), HeLa cells (ATCC CCL-2) and KMRC-20 cells (JCRB JCRB1071) were cultured in MEM (Gibco 11095-080) containing 1% NEAA (Gibco 11140-050) and 10% FBS, and Caki-1 cells (ATCC HTB-46) were cultured in McCoy 5’A (Gibco 11095-098) containing 10% FBS, RENCA cells (ATCC, CRL2947) were cultured in DMEM with 10%FCS (Gibco 10500-064). Cells were transfected with plasmids using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific Cat. No: 12566014). Wild type SIRT5, SIRT5Δ50 (lack the mitochondrion signal peptide, 1–36 AAs), wild type PDHA1, PDHA1 mutants were cloned into pcDNA3.1-Flag, pcDNA3.1-Myc and pcDNA3.1-HA vectors, respectively. The sequence of SIRT5 specific shRNA (5’-GCTGGAGGTTATTGGAGAA-3’, 5’-CAGCATCCCAGTTGAGAAA-3’) was used to knock down of SIRT5 and the non-silencing shRNA oligonucleotide was used as a negative control. Anti-pan-succinyllysine antibody and anti-succ-K351 of PDHA1 were homemade. The synthetic PDHA1 peptides containing succinylated K351 was used as antigens to immunize rabbits and the purified serum proteins were measured. Antibodies specific to pyruvate dehydrogenase E1-alpha subunit antibody (Abcam), SIRT5 (Sigma), β-actin (Genscript), Flag-Tag (Abmart), Myc-Tag (Abmart), HA-Tag (Cell Signaling Technology), Mitochondria (Abcam) were purchased. PDH activity was determined using PDH Enzyme Microplate Assay Kit (Abcam ab109902).
2.2 In Vitro Succinylation and Desuccinylation
To succinylate the lysines containing peptides (GL Biochem), 0.5 mM Succinyl-CoA (Sigma) and 50 ng/µl peptides were mixed in 30 mM HEPES (pH 7.4) in a 15 µl system and kept at 37 ℃ for 3 hours. The peptides were desalted using C18 ZipTip (Millipore) prior to MALDI-TOF MS analysis. The desuccinylation was carried out by incubating enzymes with SIRT5 (Sigma) at 37℃ for 3 hours in 30mM HEPES buffer (6 mM MgCl2, 1 mM DTT, 1 mM NAD+, pH 7.4).
2.3 Animal Studies
Four-week-old male nude BALB/c mice obtained from Shanghai Lingchang Biotechnology Co., Ltd. were used to establish orthotopic tumor model. Mice had ad libitum access to a standard diet and water. Cages were maintained in well‑ventilated racks in temperature and humidity controlled environment with a 12 h light/dark cycle. All animal experiments were approved by our institution Animal Research Ethics Committee. Luciferase-expressing Renca cells (1×105) stably transfected with shCtrl or shSIRT5 in 25 µL of 2:1 (v/v) PBS:Matrigel were injected into the sub-renal capsule of right kidney of BALB/c mice (5 mice/group). In vivo bioluminescence imaging (BLI) was performed to record the growth of tumor every 5 days and Living Image® software was used to quantify BLI signal. Mice were euthanized under anesthesia 28 days after implantation, tumor samples were fixed with 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4 µm paraffin sections for subsequent experiments.
An in vivo lung metastasis model was generated using male nude BALB/c mice aged 4 weeks by the tail vein injection of Luciferase stable-expressing Caki-1 cells. Lung metastatic progression was monitored using the IVIS-100 system (Caliper Life Sciences). In vivo BLI were performed every 7 days and Living Image® software was used to analyze the data. Mice were euthanized 28 days after implantation, and lung metastatic lesions were analyzed by BLI system.
2.4 Western blot analysis
Western blot analysis followed standard procedures. After cell harvest, protein concentration was determined using Quantity One software (Bio-Rad, Hercules, Calif., USA). Cell lysate were separated by 10% SDS-Page, transfer to PVDF membranes (Millipore, Bedford, MA, USA) and blocked with 5% w/v skim milk for 2 hours at room temperature. Anti-PDHA1, Succ-K351 PDHA1, SIRT5, GAPDH, Actin antibody were incubated overnight at 4℃. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibody (1:3000, Southern Biotech) for 2 hours at RT. Finally, they were visualized using an enhanced chemiluminescence system (ECL; Pierce Company Woburn, MA, USA). Western blot signals were obtained by detecting chemiluminescence on Typhoon fla9500 (GE Healthcare). ImageJ (NIH) was used to analyze the densities of the bands. Each blot shown in the figures is representative of at least three experiments.
2.5 Immunofluorescence analysis
Standard procedures were followed for immunofluorescence analysis. Cells were seeded in 24-well plates, fixed with 4% paraformaldehyde, and then permeabilized with 1% Triton. Cells were incubated overnight at 4 ℃ with anti-PDHA1 and SIRT5 antibodies and detected the next day with Alexa-Fluor555 goat anti-mouse IgG antibody. The nuclei were stained with DAPI (Sigma). Immunofluorescence images were observed on a fluorescence microscope (Leica, DMI4000B).
2.6 Measurement of the activity of PDHA1 complex
PDHA1 activity was measured in a reaction buffer containing 50 mM KH2PO4 (pH 7.0), 1 mM MgCl2, 2 mM sodium pyruvate, 0.2 mM thiamine diphosphate and 0.1 mM 2,6- dichlorophenolindophenol (2,6-DCPIP). Purified PDHA1/PDHB complex was added to start the reaction. The reaction was maintained at 30 ℃. The course of the reaction was monitored by measuring the reduction of 2,6-DCPIP at 600 nm on a Roch spectrophotometer.
2.7 Oxygen Consumption Rates (OCR)
KMRC-20 cells or Caki-1 cells were seeded on XFe24 cell culture microplates (Seahorse Biosciences) at rates of 40000 or 15000 cells/well, respectively. The analysis was performed using the XF cell Mito stress test kit (Seahorse Bioscience) according to the manufacturer's protocol. Culture medium were replaced with the assay medium (XF Base Medium containing 5.5mM glucose, 2mM glutamine, 1% FBS, 1nM insulin, 100nM dexamethasone, pH7.4) (Seahorse Bioscience) 1 hour before analysis. Oligomycin, FCCP, Rotenone and Antimycin A used in assays were at final concentrations of 2 µM, 1 µM, 1 µM and 1 µM, respectively. The results were normalized by cell numbers.
2.8 Measurement of the growth curves
PDHA1 knockout cell were transfected with SIRT5, PDHA1, PDHA1 + SIRT5, PDHA1K351Q or PDHA1K351Q + SIRT5, respectively, and seed in 96-well plates. Cell morphology was observed under an inverted microscope, the plate and its contents were allowed to equilibrate at room temperature for about 30 minutes, a transparent bottom was affixed with a white back cover, and the luminescence was recorded with EnSpire.
2.9 Soft agar colony formation assay
For soft agar colony formation assays, a base of 2 ml of medium containing 10% FBS with 0.7% agar was used. Cells were seeded in 2 ml of medium containing 10% FBS with 0.35% agar at the density of 1×105 cells per well and incubated for 21 days at 37 ℃. Then, the number of colonies developed in soft agar was counted using Image J, and images were taken under an Olympus IX5 microscope.
2.10 Wound-healing assay
Cell migration ability was assessed by a wound-healing assay. Briefly, Caki-1 cells were transfected with PDHA1, PDHA1 + SIRT5, PDHA1K351Q or PDHA1K351Q + SIRT5, respectively. Approximately 5×106 cells were seeded into 24-well plates and cultured for 24 hours. Then a yellow plastic pipette tip was used to create a wound by scraping the cells. Cell migration was monitored under Nicon Eclipse microscope and photographed at 100×.
2.11 Transwell invasion assay
Cell invasion experiments were carried out using 24-well Transwell plates with 8 µm pore polycarbonate Matrigel-coated membrane inserts according to the manufacturer’s instruction. After 20 h of incubation, non-invading cells in the upper insert were removed and cells that had invaded into the lower matrigel surface were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, and 10 random fields of view were quantified.
Formalin-fixed and paraffin-embedded specimens were prepared for histological section. Antigen recovery was performed on renal carcinoma specimens incubated with Tris-EDTA buffer (ph8.4) at 99 ℃ for 60 minutes. Endogenous peroxidase was inactivated in methanol and 3% H2O2 solution. Slides were incubated with primary antibody for 60 minutes, secondary antibody for 8 minutes, and DAB developer for 8 minutes. All procedures were performed using the Ventana BenchMark XT automated stainer and the slides were scanned by Ventana iScanCoreo scanner. Quantification of IHC results was performed by experienced pathologists. The intensity was calculated according to the positive area and the positive degree. The sections were stained with SIRT5 (1:100), PDHA1 (1: 100), Succ-K351-PDHA1 (1: 100) and Ki67 (1: 100) antibodies using ultraView Detection Kit.
2.13 Statistical analysis
Data were expressed as the mean ± standard deviation (SD), and bars in the graph represent standard deviation. Statistical analyses were conducted using SPSS 22.0 software (IBM, Corp., Armonk, NY, USA). Significant differences between groups were determined using Student’s T test. The significance level for statistical testing was set at P < 0.05.