Patients and Cell lines
86 pairs of tumor and corresponding adjacent tissue specimens were obtained from RCC patients who underwent surgical therapy at China-Japan Union Hospital of Jilin University. Fresh tissue samples were rapidly stored in liquid nitrogen after surgical excision. Informed consent was obtained from all patients, and the study's design was reviewed and approved by the Ethics Committee of China-Japan Union Hospital of Jilin University.
The RCC cell lines used in the study were obtained from the Chinese Academy of Sciences. Specifically, 786-O cells were cultured in an RPMI-1640 medium, while ACHN, A498, and HK2 cells were cultured in a DMEM medium. The respective media were supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cells were cultured in a 37°C incubator with 5% CO2.
Western Blot
Total protein was extracted from tissues and cells using a potent cell lysate, and protein concentration was quantified using the BCA method. Equal amounts of protein were subjected to SDS-PAGE, followed by transfer to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% skim milk for one hour, PVDF membranes were incubated with primary and secondary antibodies. Protein expression was analyzed using electrochemiluminescence imaging (Bio-Rad, USA).
RT-qPCR assay
Total RNA was extracted from tissues and transfected cells using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription was performed on 1 µg of total RNA using a reverse transcription kit (Takara) to generate cDNA. SYBR Premix Ex Taq II (Takara) was used for qPCR analysis. Primer sequences are provided in supplementary table 2.
Cell Transfection
TRIM37 short hairpin RNA (shTRIM37), SMARCC2 small interfering RNA (siSMARCC2), pcDNA3.1 vectors expressing TRIM37 or SMARCC2, and their corresponding scramble controls were obtained from GeneChem (China). Cells were transfected with the respective siRNA or plasmids using Lipofectamine 3000 (Thermo Fisher Scientific) when cell density reached 70–80% in a six-well plate. Stable cell lines expressing TRIM37 and scramble control were generated by culturing in 0.5 µg/ml puromycin for ten days following infection. Sequences are provided in supplementary table 3.
Cell Counting Kit-8 (CCK8)
1×103 RCC cells that underwent transfection with shRNA or plasma were incubated in a 96-well plate and then cultured in a 37°C incubator. In brief, 10 µL of CCK-8 reaction reagent (Dojindo) was supplemented to each well and incubated for one hour at 37°C. Finally, the absorbance value (OD) was further analyzed at a wavelength of 450 nm.
Colony formation assay
RCC cells (1×103 cells/well) were seeded in a six-well plate and cultured at 37°C with 5% CO2 for 1–2 weeks. After fixing with 4% cell fixative, cells were stained with crystal violet (Solarbio) for one hour. Colonies with more than fifty cells were counted and analyzed.
Sphere formation assay
RCC cells were cultured at a density of 1000 cells/well in ultralow attachment six-well plates (Corning) for one week in DMEM/F12 medium (Invitrogen) supplemented with B27 (1:50, GIBCO), 20 ng/ml EGF (Sigma), and 20 ng/ml bFGF (Sigma). Primary spheres were collected, dissociated, and reseeded to produce secondary spheres for one more week. The number of spheres was counted under a microscope.
Co-immunoprecipitation (Co-IP) assay
Fresh cell protein was collected for immunoprecipitation. The target antibody was incubated with protein A/G magnetic beads (MCE, New Jersey) at 4°C overnight. The antibody-coated beads were then incubated with protein, followed by IP buffer washes. Eluted proteins were used for subsequent experiments.
Protein half-life assay
RCC cells were treated with cycloheximide (20 µM) to inhibit protein synthesis and collected at different time points for Western blot analysis.
The establishment of a subcutaneous xenograft model and patient-derived tumor xenograft model (PDX) in mice
Stably downregulated TRIM37 786-O and Caki-1 cells and their scramble controls were subcutaneously injected into nude mice. Tumor volume was monitored, and when tumors reached 200 mm3, mice were treated with sunitinib (40 mg/kg) twice a week. Tumors were collected after four weeks.
Fresh tumor fragments were subcutaneously transplanted into NOD scid gamma mice. After completing a sunitinib treatment cycle, tumor tissue was transplanted into new NOD mice, followed by sunitinib treatment. Third-generation xenografts were finally obtained.
Statistical Analysis
Data from three independent experiments were presented as mean ± standard deviation (SD). GraphPad Prism 9.5.1 was used for statistical analysis. Student's t-test and one-way analysis of variance were used for group comparisons. Pearson's correlation analysis was conducted to identify gene expression correlations. p-value < 0.05 was statistically significant.