All tissue specimens were obtained from the Kidney Cancer Specimen Bank of the Department of Urology, Shanghai Changzheng Hospital, and the Urology Specimen Bank of Shanghai Changhai Hospital. The use of all tissue specimens involved in this study was approved by the ethics committee of Changzheng Hospital Affiliated with Naval Military Medical University and Changhai Hospital Affiliated with Naval Military Medical University. From December 2005 to January 2020, primary tumors were obtained after RCC surgery in Changzheng Hospital and the Department of Urology of Changhai Hospital Affiliated with Naval Military Medical University. After the specimens were obtained, they were immediately treated with liquid nitrogen and stored in a -80°C freezer or were fixed with formalin and embedded in paraffin blocks. The specimens were used for PCR, Western blot analysis, tissue chip preparation, and immunohistochemical analysis.
Female BALB/c nude mice (6-8 weeks old) were purchased from Shanghai Jihui Laboratory Animal Care Co., Ltd. (Shanghai, China) and maintained under specific pathogen-free (SPF) conditions in a controlled environment of 20-22°C and 50-70% humidity on a 12/12 h light/dark cycle. Food and water were provided ad libitum. Small animal euthanasia equipment was used for animal euthanasia. Mice were put into the euthanasia chamber filled with 99.9% CO2 gas for 10 min. All animal experiments were performed with approval from the Shanghai Medical Experimental Animal Care Committee.
Animal studies were approved by the Institutional Animal Care and Use Committee of the Second Military Medical University, Shanghai, China. A total of 5×106 cells of experimental group and control group were injected subcutaneously into the flanks of the mice. When the xenografts grew to 100 mm3, the mice were treated with sunitinib (40 mg/kg/day). Xenograft volumes were evaluated by caliper measurements of two perpendicular diameters and calculated individually with the following formula: Volume = a×b2/2 (where a represents the length and b represents the width). Xenograft samples were collected for histological evaluation (paraffin sections) or were snap frozen in liquid nitrogen.
Cell lines. All human RCC cell lines (786-O and ACHN) were purchased from the American Type Culture Collection (ATCC). 786-O cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS. ACHN cells were cultured in DMEM (Gibco) supplemented with 10% FBS. All cells were cultured at 37°C in 5% CO2.
To generate sunitinib-resistant cell lines, we used a low to high concentration gradient of sunitinib to stimulate 786-O and ACHN cells. When the 786-O and ACHN cells were adherent at a confluence of 50%, the culture medium was replaced with medium containing sunitinib at an initial concentration of 2 μM. After culture for 48 h, the medium was replaced with fresh drug-free culture medium, and cells were then passaged after the cell status was restored. After two passages in medium containing each concentration of sunitinib, the concentration was increased by 0.5 μM to the final concentration of 20 μM. The culture was maintained at this concentration. Resistant cells were cryopreserved in serum-free cryopreservation containing 5μM of sunitinib.
RT-PCR and qRT-PCR.
RNA was reverse transcribed using RT SuperMix for qPCR (+gDNA Wiper) (Takara, Japan). SYBR Green Master Mix (Takara, Japan) was used for qRT-PCR. The circRNA and mRNA levels were normalized to the levels of GAPDH. All primer sequences are listed in Supplementary Data 1.
The probes were designed and synthesized by Genepharma (Shanghai, China) and Cy3-labeled probes were specific to cSLC8A1. The probe sequences : 5’-Cy3-CAACCTAACAATTTCATCATTCTGG. 786-O and ACHN cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Subsequently, hybridization using cSLC8A1 probes was performed at 37°C in the dark overnight, and cells were rinsed in SSC buffer at 42°C. Then, cells were incubated with blocking buffer (PBST containing 5% bovine serum albumin) for 30 min at room temperature. FUS (ab124923, anti-rabbit, 1:200) was performed at 4°C in the dark overnight. Goat anti rabbit IgG FITC conjugated (MultiSciences Biotech) was used as the secondary antibody at 1:500. Subsequently, cells were incubated with DAPI (Vector Laboratories) for 30 min. Images were acquired using a confocal microscope.
RNase R treatment.
Two micrograms of total RNA was incubated for 30 min at 37°C in the absence or presence of 5 U/μg RNase R (Epicentre Technologies, Madison, WI, USA), and the resulting RNA was subsequently purified with an RNeasy MinElute Cleaning Kit (Qiagen) and analyzed by qRT-PCR.
Actinomycin D assays.
786-O, ACHN, Sun-7R and Sun-AR cells were seeded in six-well plates (105 cells per well). Twenty-four hours later, the cells were exposed to 2 μg/ml actinomycin D (Abcam) and collected at the indicated time points. RNA stability was analyzed using qRT-PCR, and the values measured in the treatment groups were normalized to the values measured in the mock treatment group (the 0 h group).
Vector construction and cell transduction.
Lentiviruses expressing human cSLC8A1 and FUS were constructed and produced by the Chinese Academy of Sciences (Shanghai). 786-O, ACHN, Sun-7R and Sun-AR cells were transduced following the manufacturer's instructions. After 72 h, puromycin was added to select the stably transduced cell lines.
Cell counting kit 8 (CCK-8) assay.
RCC cells were cultured in medium containing different concentrations of sunitinib (0 μM, 2 μM, 4 μM, 8 μM, 10 μM, 16 μM and 20 μM). Then, 100 μl of culture medium containing 10 μl of CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added to each well for another 2 h of incubation at 37°C. The absorbance was measured at 450 nm using a microplate reader (Varioskan Flash; Thermo Scientific, Waltham, MA, USA). Viability (%) was calculated based on the optical density (OD) values. All experiments were independently repeated in triplicate at separate times.
Plate colony formation assay.
RCC cells (500 cells) were seeded into 6-well plates in medium containing sunitinib (5 μM) and cultured in a 37°C incubator for 10 days until most single colonies contained more than 50 cells. The plates were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and stained with crystal violet. The number of colonies containing more than 50 cells was counted in each well.
RNA pulldown assay.
For the RNA pulldown assay, 1 × 107 cells were washed in ice-cold PBS, lysed in 500 μl of coimmunoprecipitation (co-IP) buffer (Thermo Scientific) supplemented with a cocktail of proteinase inhibitors, phosphatase inhibitors, and an RNase inhibitor (Invitrogen). Lysates were then incubated with 3 μg of biotinylated DNA oligo probes targeting the cSLC8A1 backsplice junction region (sense) or the corresponding complementary probes (antisense) for 2 h at room temperature. A total of 50 μl of washed streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated for another hour at room temperature. The beads were washed briefly with co-IP buffer five times. Finally, the retrieved proteins were used for mass spectrometry or western blot analysis. The probe sequences are listed in Supplementary Data 4.
Wound healing assay
RCC cells (5×105cells) were seeded into 6-well plates in medium containing sunitinib (5 μM). Then, a wound was made by using a 200 μl pipette tip on the cell monolayer and photographs were taken at the appropriate time to estimate the area occupied by migratory cells.
Transwell assay was used to evaluate the invasion and migration capacities of RCC cells in vitro. Cells at a concentration of 1×104 cells in 500 μl of serum-free medium with containing sunitinib (5 μM) were inoculated in the upper chamber, coated with (invasion assay )or without (migration assay) growth factor reduced Matrigel®, and medium containing 10% FBS was added into the lower chamber as a chemoattractant. After incubation for the appropriate time, cells on the upper surface of the membrane were removed by wiping with a Q-tip, and the invaded or migrated cells were fixed with formaldehyde and stained using 0.5% crystal violet. The numbers of invaded and migrated cells were counted in five randomly selected fields under a microscope.
RIP was performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Coprecipitated RNA was detected by qRT-PCR.
Western blot analysis. Cell lysates or retrieved proteins were analyzed by immunoblotting with primary antibodies and an IRDye 800 CW-conjugated secondary antibody (Rockland Immunochemicals, USA). The fluorescence intensity was determined with an Odyssey fluorescence scanner system (Li-Cor Biosciences, USA). GAPDH was used as the loading control. The other antibodies were as follows: anti-FUS (ab23439, anti-rabbit, 1:1000), anti-Ubiquitin (ab7780, rabbit, 1:1000), and anti-GAPDH (ab9485, rabbit, 1:2500).
Specimens were stained with an anti-FUS antibody (ab124923, 1:100). The sections were heated at 70°C for 1 h, dewaxed in xylene, and dehydrated through an alcohol gradient. After antigen retrieval, quenching of endogenous peroxidase activity with 3% H2O2 and blocking of nonspecific binding with normal bovine serum, the sections were incubated with the primary antibody overnight at 4°C. The slides were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 10 min at 37°C. Finally, staining was visualized with diaminobenzidine (DAB) solution and counterstaining with hematoxylin. Two pathologists blinded to the patient outcomes independently scored the staining intensities and percentages of positive tumor cells.
To detect protein–protein interactions, cells were lysed in 500 μl of co-IP buffer supplemented with a cocktail of proteinase inhibitors, phosphatase inhibitors, and an RNase inhibitor. The lysates were centrifuged at 12,000 × g for 30 min, and the supernatant was used for immunoprecipitation with beads, which were preincubated with the corresponding antibodies. After incubation at 4°C overnight, the beads were washed three times with co-IP buffer. SDS sample buffer was added to the beads, and the immunoprecipitates were used for western blot analysis.
All patients developed tumor metastasis or recurrence and received regular sunitinib treatment for more than 3 months. The starting point of the follow-up study was the time to receive regular sunitinib treatment, and endpoints included time to disease progression and death. The evaluation criteria for tumor response to drugs were based on Response Evaluation Criteria in Solid Tumors (RECIST). All patients developed tumor metastasis or recurrence and received regular sunitinib treatment for more than 3 months. We divided these patients into sunitinib sensitive group and resistant group according to RECIST1.1. Sensitive group included patients with complete response (CR) or partial response (PR) or patients with stable disease (SD) maintained for at least 6 months. Resistant group patients experienced progressive disease (PD).
All statistical analyses in this study were performed with SPSS 16.0 software (SPSS Inc., USA). Data are presented as the mean±s.d. values. The significance of differences between the mean values of two groups was analyzed by two-tailed Student’s t-test. Spearman correlation analysis was performed to evaluate correlations between two variables. The Pearson chi-square test was used to analyze the clinical variables. Kaplan–Meier survival analysis was performed, and the survival of ccRCC patients stratified by cSLC8A1 expression was compared by the log-rank test. Cox proportional hazards regression analysis was utilized to analyze the effects of clinical variables on patient survival. P values <0.05 were considered significant.
The gene expression profiles of the generated sunitinib-resistant RCC cell lines have been deposited in GEO with the accession code GSE173572. The mass spectrometry data that support the findings of this study are included in Supplementary Data 4. The authors declare that all other relevant data supporting the findings of this study are available upon request.