Patients and tissue samples
Tumor tissue specimens from 89 patients with prostate cancer who underwent surgery were acquired from the First Affiliated Hospital of Wenzhou Medical University between February 2011 and March 2016. The mean age of the patients was 70 years (range, 55–90 years). No patient underwent preoperative radiotherapy or chemotherapy before surgery. All patients were diagnosed with pathological type prostate adenocarcinoma. The patients’ Gleason scores were obtained from the American Joint Committee on Cancer (AJCC, 7th edition) data.
Tissue microarray (TMA) chips were obtained commercially (Superchip Inc., Shanghai, China). The TMA contained 89 prostate cancer patient specimens including tumor tissue samples and their corresponding adjacent non-neoplastic tissue specimens. A tissue cylinder (diameter, 2 mm) was stamped out from morphologically representative areas of each donor block and transferred to a recipient paraffin block. Tissue microarray blocks were 2.0 mm in diameter, and all points were covered with paraffin wax.
The immunohistochemical assay was performed on TMA chips. The chip was deparaffinized in xylene twice for 5 min at room temperature (RT) and then rehydrated in successively graded concentrations of ethanol at 100%, 95%, 85%, and 70%, respectively for 5 min. Antigen retrieval was performed by heating the sections at 100°C for 30 min in citrate buffer (0.05% Tween 20 and 10 mM citrate, pH 6.0) at 170 kPa at 120°C for 5 min. Subsequently, endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in Tris-HCl buffer for 15 min at RT. The chip was then washed 3 times and incubated with a polyclonal rabbit anti-SIRT4 antibody (HPA029692, 1:400, Sigma, USA) at 4°C in a refrigerator for more than 8 h. Subsequently, a secondary antibody was added to the chip using the GTVision Kit (Gene Tech Inc., Shanghai, China) and the chip was incubated, as per the manufacturer’s instructions. The microarray chip section was then consecutively stained with diaminobenzidine (DAB) and hematoxylin. Next, the chip was dehydrated and sealed with a coverslip. Tissues treated with only a diluent (without antibody) were used as negative controls.
Tissue wafer scanning and analysis method:
Tissue chip scanner model Pannoramic MIDI, manufacturer: 3D HISTECH (Budapest, Hungary): The tissue chip section gradually moves under the scanner lens. The moving edge is imaged, and the information on the tissue section is scanned and imaged to create a file. The file contains all the tissue information available on the tissue section. The file can be enlarged 1–400 times in the Pannoramic viewer software. Further, any part of the tissue section can be imaged. The Quant center is an analysis software program associated with the Pannoramic viewer. On scan completion, the TMA software of the Quant center analysis software can be run after setting the diameter of the wafer tissue and the number of rows and columns. The software then generates a number. The nuclei in the tissue sections that were strongly positive were colored dark brown, those moderately positive were brown yellow, those weakly positive were pale yellow, and those negative were blue using the densito quant software in Quant center. The area (unit: pixel), the percentage of positive staining, and final last histochemical score (H-SCORE) were analyzed for each tissue site. The formula for calculating the H-SCORE was as follows: H-SCORE = ∑(Pi × I) = (percentage of weakly positive cells × 1)(percentage of moderately positive cells× 2)(percentage of strongly positive cells × 3), with pi representing the percentage of the positive cells in the tissue section and i representing the intensity of the staining. The H-SCORE score ranged from 0 to 300, with a higher score indicating a stronger positive score [14, 15].
Cell lines and culture conditions
A human prostate cancer cell line, 22RV1, was procured from Shanghai Institute of Cell Biology, Chinese Academy of Sciences in June 2020. The cell bank use four pairs of primers DXS52, Apo-B, MD17S5 and D2S44 to monitor the variation of cell lines during passage. The cells were last tested in June 2020, and the cell experiment ends within half a year after the cell is purchased. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and penicillin/streptomycin (Gibco, USA), and incubated at 37°C with 5% CO2.
Vector and virus production
We design three siRNA sequences targeted to SIRT4, they were 5'-GCGTGTCTGAAACTGAATTCT', 5'-GCTCCTGATGGTGACGTCTTTCTCT-3' and 5'-GCGTTCAATGTGGAGGCCATCTGAA-3', respectively. The negative control sequence was 5′-TGTCACTCTCCGGAACGTT-3′. We purchased the lentivirus vector pHBLV-CMVIE-ZsGreen-T2A-Puro overexpressing SIRT4 and shSIRT4 from biotechnology company (Hanbio, Shanghai, China). The final titer of the lentivirus and negative control virus was 2 × 108 PFU/ml. Stable overexpression of SIRT4 or shSIRT4 was achieved by transfecting 22RV1 cells with lentivirus for 72 h; colonies were isolated using puromycin for two weeks. Lipofectamine2000 (Thermo Fisher Science) was used as the transfection reagent.
Reverse transcription (RT)-PCR
Total RNA from the tissues or cells was extracted using the TRIzol reagent (Invitrogen, USA). Further, 500 ng of cDNA was synthesized using a reverse transcription kit (PrimeScriptTM RT Master Mix, TaKaRa, Japan). Then, qRT-PCR was performed with thrice-diluted cDNA using the qRT-PCR Kit (SYBR® Premix Ex Taq™aqR KTaKaRa, Japan) on the DNA Engine Opticon 2 real-time detection system (BioRad). GAPDH was chosen as the internal reference gene. Primers for each gene were as follows: SIRT4 forward primer 5′- GCGAGAAACTTCGTAGGCTG − 3′, reverse primer 5′- TCAGGACTTGGAAACGCTCT − 3′; GAPDH forward primer 5′-TCAAGAAGGTGGTGAAGCAGG − 3′, reverse primer 5′- TCAAAGGTGGAGGAGTGGGT − 3′. The PCR reaction conditions were as follows: 2 min at 94°C; followed by 40 cycles of 30 s at 94°C, 30 s at 57°C, and 1 min at 72°C; and ending with 5 min at 72°C and cooling at 4°C. After the reaction was completed, the homogeneity of the PCR product was confirmed by analyzing the dissolution curve followed by analysis of relative gene expression by 2−∆∆CT.
Cells were lysed with Ripa lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail (Beyotime, China). Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The primary antibodies used were rabbit anti-human SIRT4 polyclonal antibody (clone HPA029691, Sigma, USA), rabbit anti-human caspase3 (35/18 KDa) polyclonal antibody (9662, CST, USA), rabbit anti-human caspase9 (46 KDa) polyclonal antibody (10380-1-AP, Proteintech, China), rabbit anti-human p65 (65 KDa) monoclonal antibody (8242, Cell signaling, USA), rabbit anti-human p-p65 (65 KDa) monoclonal antibody (3033, Cell signaling, USA),rabbit anti-human matrix metalloproteinase 9 (MMP9) (78 KDa) polyclonal antibody (10375-2-AP, Proteintech, China), rabbit anti-human n-cadherin (99 KDa) monoclonal antibody (14472, CST, USA), rabbit anti-human e-cadherin (135 KDa) monoclonal antibody (Ab124397, Abcam, England), goat anti-rabbit detection antibody (ab97200, Abcam, England), rabbit anti-human Lamin B1 (67 KDa) polyclonal antibody (BA1228, Boster Biological Technology, China), and rabbit anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (AB-P-R 001, Goodhere, China).
For the analysis of GDH activity, the biochemical activity of these cell lines was analyzed using the Human Glutamate dehydrogenase (GLDH) test kit (A123, Jiancheng Bioengineering Institute, Nanjing, China).
Cell proliferation activity
Cells were inoculated in 96-well plates with a density of 1000 cells/well. Subsequently, 10 µL of cell counting kit-8 (CCK-8) reagent (Dojindo, Japan) was added in each well followed by culturing in a CO2 incubator for 2 h and absorbance determination. The final concentration of the bis-2-(5-phenylacetamide-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES; SML0601, Sigma, USA) was 10 µmol/L. The final concentration of dimethyl a-ketoglutarate (DM-αKG; 349631, Sigma, USA) was 8 mmol/L.
Wound healing assay
Wound healing assay was carried out to determine cell migration. Briefly, the SIRT4 overexpression, shSIRT4, and the corresponding negative virus group cells were seeded in a 6-well plate with a density of 5 × 105 per well and cultured for 10 h. Following this, a 200-microlite tip perpendicular to the bottom of the plate was used to make a scratch line. Subsequently, the detached cells were washed with PBS and cultured in a serum-free medium. DM-αKG at a concentration of 4 mM or 8 mM was added after scratching in the DM-αKG group. In the experimental group, cell images in the scratched area after 0 and 24 h of culture, obtained using an optical microscope, were used to calculate the distance migrated by the cells.
Cell Migration and invasion test
In the migration test, cells were suspended in a serum-free medium with a density of 3 × 105 ml, and a 0.2-ml suspension was inoculated into the upper chamber of the Transwell plate (Corning Inc., Conning, New York, USA). The cells were filled with 0.6 ml culture medium containing 10% FBS. The cells were then incubated at 37°C for 18 h. The cells in the upper compartment were then wiped with a sterile cotton swab and the membrane was soaked in 4% paraformaldehyde to immobilize the cells in the lower compartment, followed by staining with 0.1% crystal violet. The cells were counted under an optical microscope (Olympus). In the invasion test, the superior chamber was precoated with BD Matrigel™ Basement Membrane Matrigel (BD biosciences) before cell inoculation. The rest of the procedure was the same as that followed for the migration test. For DM-ɑKG group, the culture medium with a 4 mM or 8 mM DM-ɑKG was adjusted after seeding.
Flow cytometric analysis for apoptosis rate and cell cycle
Cells were harvested by trypsinization, pelleted by centrifugation, and resuspended in PBS containing 3% fetal bovine serum. The cell apoptosis was performed with flow cytometry (C6, BD, USA) using annexin V-allophycocyanin (annexin V-APC) and 7-aminoactinomycin D (7-AAD) (BD, USA) staining. Apoptosis rate was only calculated at early apoptosis. The survival rate was calculated by cells not stained with annexin V-APC or 7-AAD. The apoptotic cells were analyzed by flow cytometry analysis using the Accuri C6 software program (BD, USA). Cell cycle was measured with Propidium Iodide (PI)/RNase kit (BD, USA). The results were examined with the ModFit analysis software program (Verity Software House, Topsham, ME, USA).
Immunofluorescence analysis were performed as described previously , using rabbit anti-human NF-κB p65 polyclonal antibody (10745-1-AP, 1:50 dilution, Proteintech Group, China), rabbit anti-human Phospho-N F-κB p65 polyclonal antibody (3033S, 1:50 dilution, CST, USA), and an Cy3-conjugated goat anti-rabbit IgG secondary antibody (BA1032, 1:100 dilution, Boster Biological Technology, China). DAPI was used for staining of nuclei and to assess gross cell morphology.
The SPSS software package version 22.0 (SPSS, Inc., IBM, USA) was used for statistical analysis. All in vitro experiments were performed in triplicates. Data from three or more independent experiments are presented as the mean ± standard deviation. The final scores of tumor tissue and non-tumor tissue were analyzed using a paired student’s t tests. Experiments unless specified were analyzed using the non-paired t-test. P < 0.05 (two-tailed) was considered statistically significant.