Cell lines and cell culture
The ACHN and Caki-1 cell lines were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Caki-2 cell lines were provided by Guangzhou Cell cook Biotech Co. Ltd. ACHN, Caki-1 and Caki-2 cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS). All the cells were cultured in a humidified incubator containing 5% CO2 at 37°C and were used in further experiments.
Transfections
The plasmids were transfected into cells with Lipofectamine 2000 according to the manufacturer’s instructions. The following day, the cells were cultured with media containing neomycin and selected for two weeks to obtain stably transfected cells. The SPOP plasmid or shRNA plasmid was packaged and transfected into retroviral packaging cells. Retroviral supernatants were added to the cells, spun for 45 min at 1800 rpm and incubated for 4 h at 37 ℃. The cell medium was switched to medium supplemented with puromycin for one week to select stable cell lines. The overexpression and knockdown efficiency of SPOP in the cells was tested by western blot and qPCR analyses.
Western blot analysis
As previously described[19], cell protein samples were harvested using RIPA buffer, and 20 μg of the protein sample was separated on 12% SDS-polyacrylamide gels followed by wet transfer at room temperature. The blots were then blocked with non-fat milk, followed by incubation with diluted primary antibody overnight at 4°C. The membranes were washed three times with TBST for 5 min and subsequently incubated with the appropriate secondary antibody conjugated to IRDye800 at room temperature for 2 h. Protein bands were visualized with the ECL detection system and analysed using the ImageJ software.
Quantitative real-time PCR analysis
Total RNA from cells was extracted and reverse transcribed using a cDNA synthesis kit (Invitrogen). Real-time PCR analysis was performed using a LightCycler 96 (Roche). The peak of the melting curve was defined as the criterion for amplification specificity. The relative expression levels of mRNAs were determined by normalization to the expression levels of the internal control gene GAPDH, and the data were analysed by the ΔΔCt method.
Cell invasion assay
The capacity of cell invasion was evaluated by the Transwell assay. A total of 2×105 cells in serum-free medium were plated on top of the Transwell chamber, which was coated with Matrigel matrix (Corning 354230). Medium supplemented with 10% FBS as the chemoattractant was added to the bottom of the chamber. The cells were then incubated in Transwell plates at 37°C in 5% CO2 for 48 h. The non-invading cells at the top of the chamber were carefully removed with a cotton swab. The cells on the lower surface of the Transwell chamber were stained with crystal violet for 30 min after fixation with paraformaldehyde. The inserts were washed three times with PBS, and the number of invading cells was counted under a microscope.
Cell migration assay
Cell migration was determined by the wound-healing assay. A total of 1.2×105 cells were plated in a 12-well plate at 37°C and 5% CO2 overnight. A horizontal scratch was then made in the plate using a sterile pipette tip, followed by washing with PBS three times to remove the floating cells. Finally, the cells were incubated in serum-containing medium at 37°C in 5% CO2 for 24 h. The scratch migration area was calculated using the ImageJ software after 0 h and 24 h.
Cell proliferation and apoptosis assays
For the proliferation assay, 1.5×105 cells were cultured in 96-well plates with regular medium at 37°C and 5% CO2 for 24 h. The next day, the culture medium was replaced with medium supplemented with IFN-α2b (20, 80, 4000 and 5000 IU/ml) or sunitinib (2.50, 5.01, 7.00 and 10.05 µmol/L) and incubated for 48 h. During culture, 10 ml CCK-8 chromogenic agent and 100 ml DMEM without FBS were added to the wells and incubated for 1 h. The absorbance (A) at 450 nm was analysed using a microplate reader. The inhibitory rate of cell growth (%) was quantified as follows: (1-(Atreated)/(Acontrol)) × 100%. For the apoptosis assay, 1x105 cells were seeded into 6-well plates, allowed to attach overnight and then treated with 10% FBS (control), IFN-α2b (20, 40 and 80 IU/ml), or sunitinib (5.0, 5.5 and 6.0 µmol/L) for 48 h. The cells were collected by centrifugation at 1200 rpm for 5 min at 37°C. The collected cells were washed with PBS and 50 µl 1x binding buffer. Then, the cells were stained with Annexin V-APC and 7-AAD and incubated at room temperature in the dark for 15 min. Subsequently, 50 µl 1x binding buffer was added, and the samples were tested using an AccuriTMC6 PLUS flow cytometer (BD Biosciences). The sum of early apoptosis and late apoptosis was defined as total apoptosis.
Tissue microarrays (TMA) and normal kidney tissues
TMAs of formalin-fixed paraffin-embedded human renal tumour and adjacent normal tissues from the Shanghai Outdo Biotech Company were evaluated. The TMA comprised 180 tissue samples (90 tumour tissue samples and 90 adjacent normal tissue samples) collected from patients who underwent nephrectomy between 2006 and 2008. The 90 RCC specimens included clear cell renal cells (n=88) and papillary cells (n=2). In addition, normal kidney tissues (n=10) obtained from The People’s Hospital of Guangxi Zhuang Autonomous Region were used. Related clinical data, including follow-up time, sex, and tumour stage, were recorded in detail for all patients who signed the informed consent form.
Immunohistochemical staining
Immunohistochemical staining of the SPOP protein in the cytoplasm of TMA and kidney tissues was performed with appropriate antibodies according to the methods of a previous study[20]. Briefly, paraffin-embedded sections were subjected to deparaffinization, rehydration, and heat-induced antigen retrieval. The sections were incubated with primary SPOP antibody overnight at 4°C after blocking endogenous peroxidase activity with 3% hydrogen peroxide. Rabbit IgG antibody was used for the isotype control. 3,3'-Diaminobenzidine (DAB) was added as a chromogen followed by counterstaining with haematoxylin. The staining intensity and positive staining rate were assessed by two independent pathologists according to the histologic scoring system (H-score). SPOP expression was scored comprehensively based on the positive staining rate and staining intensity. The positive staining rate was scored as follows: 0 (negative), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%), and 4+ (76-100%). The intensity of cytoplasmic staining was classified as follows: 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong). The above two scores were multiplied to obtain the final score. A total score of SPOP immunohistochemical staining ≥ 6 was defined as high expression; otherwise, it was considered low expression.
Statistical analysis
Statistical analyses and figure preparation were performed using the SPSS 24.0 (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 7.0 (San Diego, California, USA) software. Values of the in vitro cell experiments are presented as the mean ± standard deviation based on results obtained from at least three independent experiments. Comparisons were made between homogeneous experimental groups using the t-test or ANOVA, as appropriate. The Mann-Whitney U test was used to analyse the differences in SPOP expression between tumour tissues and adjacent normal tissues. Survival analysis was determined by the Kaplan-Meier method and compared by the log rank test. A p-value less than 0.05 was considered to be statistically significant.