Cell lines and tissue specimens
Human bladder cancer T24, EJ, BIU-87 and UMUC3 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). UMUC3 cells were maintained in DMEM (Gibco, Gaithersburg, MD, USA) and T24, EJ and BIU-87 cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS Gibco) and 1% penicillin G sodium/streptomycin sulfate. All of the cell lines were cultured at 37 °C and 5% CO2 in a humidified incubator. All cell lines were used at early passages.
The tissue samples used for this study were obtained from patients with a diagnosis of primary BCa in Nanfang Hospital, Southern Medical University. Freshly frozen samples from BCa patients were selected for real-time PCR. 56 formalin-fixed paraffin-embedded BCa tissues and 20 adjacent non-tumor tissues were used to investigate the expression of SLC25A21 protein using immunohistochemistry (IHC). Tumor grade and stage assessments were made according to the World Health Organization (WHO) 2004 standard and Union for International Control Cancer (UICC) TNM system.
RNA isolation and real- time PCR
Total RNA from tissues and cell lines were extracted using Trizol reagent (Takara, Dalian, China) according to the manufacturer’s protocol. The cDNA was synthesized with 500 ng total RNA by using PrimeScript RT Reagent Kit (Takara). Real-time PCR was carried out using SYBR® Rremix Ex TaqTM (Takara) as described previously[16]. GAPDH was used as an internal control. The assay was performed in triplicate. All primer sequences are listed in Supplement Information S1.
IHC staining and scoring
IHC was performed on 3-μm sections of paraffin-embedded tissue samples. The sections were deparaffinized with xylene and rehydrated with descending ethanol concentrations, and then were treated with Peroxidase Blocking Reagent (Dako, Glostrup, Denmark) for 5 min, followed by incubation with SLCA5A21 primary antibody (1:80, Affinity Biosciences, OH, USA, Cat# DF4172) at 4 °C overnight. After incubation with the secondary antibody at room temperature for 1 h. Immunodetection was performed with the diaminobenzidine (DAB) reagent (Dako) according the manufacturer's protocol, and the reaction time of each section was consistent, followed by counterstaining with hematoxylin.
The IHC-stained tissue sections were reviewed and scored separately in a double-blinded manner. Scores were determined based on both the intensity and proportion of SLC25A21-positive cells as described previously[17, 18]. The staining score ranged from 0 to 4, corresponding to the percentage of immunoreactive tumor cells (0%, 1-25%, 26-50%, 51-75%, or 76-100%). The staining intensity scores were as follows: negative (0), weak (score = 1), medium (score = 2) or strong (score = 3). A total score of 0 to 12 was calculated by multiplying the staining degree score by the intensity score. The final staining score of ≥ 3 was considered to indicate high level of SLC25A21.
Construction of cell lines with stably overexpressed SLC25A21
The full-length open reading frame of human SLC25A21 was amplified and cloned into the pcDNA3.1 vector. BCa cell lines EJ and T24 were transfected with pcDNA3.1-SLC25A21 using Lipofectamine 3000 reagent (Thermo Fisher Scientific, IL, USA). The empty pcDNA3.1 vector was used as the control. G418 was used to select stable SLC25A21-overexpressing cells. The expression levels of SLC25A21 were determined by real-time PCR and western blotting analysis.
Oligonucleotide transfection
The siRNAs targeting SLC25A21 and negative control siRNA (silencer negative control siRNA) were synthesized (GenePharma, Shanghai, China). Oligonucleotides transfection was performed with Lipofectamine 3000 following the manufacturer’s protocol. Target sequences for siRNAs were shown in Supplement Information S2.
Cell proliferation
Cell proliferation was assessed using the Cell Counting Kit-8 kit (Dojindo Laboratories, Kyushu Island, Japan) according to the manufacturer’s instructions. In brief, BCa cells were seeded in 96-well plates at 8 × 102 cells per well. The results are reflected in the form of the absorbance optical density at 450 nm using a microplate reader (BioTek, VT, USA). The experiments were conducted in triplicate.
Colony formation assay
Colony formation assays in vitro were performed according to standard protocols as previously described[19, 20]. In brief, BCa cells were seeded in 6-well plates at 2 × 102 cells per well and cultured in medium containing 10% FBS. After 2 weeks of culture, cell colonies were fixed with methanol for 30 min and stained with Giemsa for 15 min. The surviving colonies (≥50 cells per colony) were then counted under a microscope. The experiments were performed at least in triplicate.
Cell cycle analysis and apoptosis analysis
Apoptosis analysis was performed by flow cytometry by a BD FACSCanto II Flow Cytometer (BD Bioscience, San Jose, CA, USA). The cells were collected and washed twice with ice-cold PBS, resuspended in 200 μl binding buffer and stained with the Annexin V-FITC Detection Kit (Genechem, Shanghai, China) according to the manufacturer’s instructions. Early apoptosis was determined based on the percentage of cells with annexin V+/PI- staining, while late apoptosis was determine based on the percentage of cells with annexin V+/PI+ staining. The experiments were performed at least in triplicate.
For cell cycle, cells were harvested after 48 h transfection and fixed in ice-cold 70% ehanol at 4°C overnight. Cells were then resuspended in propidium iodide solution (Genechem) according to the manufacturer’s protocol and subjected to analysis using flow cytometry (BD).
Wound healing assays
A wound healing assay was conducted to measure the migration of BCa cells. Cell migration was assessed by measuring the movement of cells into a scraped, acellular area by a 200 μL pipette tube in 6-well plates. The spread of wound closure was observed every 24 h. Migration was quantified by measuring the distances of cells that migrated toward the original wound field.
Transwell invasion assays
Cell invasion assays were performed in Ttranswell chambers (BD Biosciences, San Jose, CA, USA) containing 8-μm pores in 24-well plates. A total of 1× 105 cells were starved overnight and seeded into the upper chamber of the transwell, which had been pretreated with 60 μl Matrigel for 4 h, in 200 μl of serum-free medium. The lower chamber was supplemented with 500 μl of medium with 10% FBS. After incubation at 37 °C for 24 to 72 h, noninvading cells in the upper chamber were scraped off with a cotton swab, and invading cells stuck to the lower surface of the membrane were fixed in 100% methanol for 30 min and stained with Giemsa solution for 15 min. The cells were counted randomly for five fields of each membrane under a light microscope. The experiments were performed at least in triplicate.
In vivo functional assays in nude mouse models
Male, 4- to 6-week-old Balb/C-nu/nu nude mice were obtained from the Laboratory Animal Centre of Southern Medical University. Stably overexpressing SLC25A21 EJ cells and control cells were used to produce subcutaneous xenograft models. The nude mice were dIvided randomly into two groups (5 mice per group). A total of 2 × 106 SLC25A21-overexpressing EJ cells or control cells were subcutaneously injected into the right flank of mice, respectively. Tumor growth was measured every 3 days with a digital caliper. Tumor volume was calculated according to the formula: V (volume) = (D × d2)/2 (D indicates the longest diameter and d indicates the shortest diameter). After 30 days of monitoring, the mice were sacrificed by cervical dislocation, and the xenografts were removed and subjected to histological examination. This investigation was carried out in strict accordance with ethical standards in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Southern Medical University.
Mitochondria isolation and determination of α-KG
Mitochondria extraction was conducted according to the instructions of the Mitochondria Isolation Kit (Beyotime Biotechnology, Jiangsu, China). Briefly, at least 2 × 107 cells were harvested and then centrifuged at 600 × g at 4 °C for 5 min. Mitochondrial isolation buffer was used to resuspend the pellet. The mixture was placed on ice for 15 min and then ground and centrifuged at 1000 × g at 4 °C for 10 min. The supernatant was collected and centrifuged at 3500 × g at 4 °C for 10 min. The pellet contained the mitochondria. The supernatant was collected and centrifuged at 12,000 × g at 4 °C for 10 min. The supernatant contained the cytosolic fraction. Mitochondria were resuspended in mitochondria isolation buffer and centrifuged again at 3500 × g at 4 °C for 10 min to obtain purified mitochondria. The levels of α-KG in the mitochondria and cytosolic fraction were determined using α-ketoglutarate (α-KG) enzyme-linked immunosorbent assay (ELISA) kits according to the documents of the manufacturer (CUSABIO Technology, Wuhan, China).
Mitochondrial membrane potential (Δψm)
Changes in the Δψm during the early stages of apoptosis were assayed using the Mitochondrial Membrane Potential Assay kit with JC-1 (Beyotime, Shanghai, China) as described previously [21]. Briefly, 5 × 104 cells were harvested and incubated with JC-1 at 37 °C for 20 min in the dark. The stained cells were washed with ice-cold working solution twice and then analyzed by flow cytometry (BD). JC-1 aggregates in the polarized mitochondrial matrix and forms J-aggregates, which emit red fluorescence at 595 nm when excited at 525 nm. However, JC-1 cannot aggregate in the depolarized mitochondrial matrix and exists as JC-1 monomers, which emit green fluorescence at 525 nm when excited at 485 nm. Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
ROS production
The ROS levels were detected with the Human ROS ELISA Kit and analyzed with a microplate reader according to the documents of the manufacturer (JianglaiLab, Shanghai, China).
Western blotting
Cells were lysed in prechilled RIPA buffer containing phosphatase inhibitors, protease inhibitors and PMSF. The protein extracts were loaded on each line of a 10% SDS-PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The membranes were blocked in 5% skimmed milk in 1 × PBS-T (0.5% Tween-20) and incubated overnight at 4 °C with the following primary antibodies: anti-SLC25A21 (1:500; Affinity Biosciences), anti-cytochrome C (1:500; Affinity Biosciences), anti-caspase-9 (1:800; Affinity Biosciences), anti-cleaved caspase-9 (1:800; Affinity Biosciences), anti-caspase-3 (1:800; Affinity Biosciences), and anti-cleaved caspase-3 (1:800; Affinity Biosciences). Anti-tubulin (1:1000; Proteintech Group, Wuhan, China) was used as protein-loading control. Blots were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and visualized with ECL Western Blotting Substrate (ThermoFisher Scientific).
Statistical analysis
All statistical analyses were performed using SPSS version 16.0 software (SPSS, Chicago, Illinois, USA). Differences between groups were identified using a two-tailed Student’s t-test. Associations between SLC25A21 expression and clinicopathological characteristics were determined by the χ2 test. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The significance of various variables for survival was analyzed by the Cox proportional hazards model for multivariate analyses. A probability value of 0.05 or less was considered to be significant.