2.1 Cell lines, cell culture and tissue specimens
Human renal cancer cell lines (786-O, CAKI-1 OS-RC-2, and CAKI-2) and human renal tubular epithelial cells (HK-2) were all purchased from the Cell Resources Center, Shanghai Academy of Life Sciences, Chinese Academy of Sciences. All cell lines were cultivated in the recommended medium supplemented with 10% heat-inactivated foetal bovine serum (Gibco, Beijing, China) and 1% streptomycin/penicillin (Keygen, Nanjing, China) at 37°C and 5% CO2. RCC tissue specimens were obtained from 40 patients who were treated by radical nephrectomy or preserved kidney surgery at the Department of Urology, First Affiliated Hospital of Harbin Medical University between 2010 and 2019. All diagnoses were confirmed by histopathological examination.
2.2 Antibodies and reagents
The following antibodies were used for Western blotting: SAP30 (#LS-C676447, Lsbio), MT1G (#323730, US Biological), P53 (#60283-2-Ig, Proteintech), Bax (#50599-2-Ig, Proteintech), Bcl2 (#12789-AP, Proteintech), CCNB1 (#28603-AP, Proteintech), CCND1 (#60186-I-Ig, Proteintech), and CCNL2(#LS-C749918, LSBio). And the anti-SAP30 antibody for IHC was purchased from Lsbio(#LS-C676447). The anti-SAP30 antibody for ChIP-PCR was purchased from Abcam (#ab231804). Sunitinib was purchased from MCE (#HY-10255A).
2.3 siRNA and plasmid transfection
The siRNAs for SAP30 and MT1G were purchased from GenePharma (Suzhou, China), and their sequences are shown in Supplementary Table S1. Transfection of the siRNA-SAP30 and siRNA-MT1G vectors was performed using jetPRIME (PolyPlus, Shanghai, China). The plasmids (pENTER-SAP30, pENTER-NC, pCMV3-SAP30 and pCMV3-NC) were purchased from Weizhen (Shandong, China) and SinoBiological (Beijing, China). Briefly, 1.2–1.3 x106 cells were loaded in a 6 cm culture dish to prepare for transfection and collect cells for subsequent experiments after 47–72 hours.
2.4 Quantitative real-time PCR (qRT‒PCR)
Total RNA from cells and tissues was extracted using TRIzol reagent (Ambion,USA). Reverse transcription kits (TianGen, Beijing, China) and PCR kits (SYBR Green) (TianGen, Beijing, China) were used to perform RT‒qPCR according to the manufacturer’s instructions. The sequences of primers were provided in the Supplementary Table S1. The results were analysed by the 2 -ΔΔCt method to quantify the fold change.
2.5 Western blotting analysis and immunohistochemical staining (IHC)
Proteins from nude mouse kidney cancer cells and tissue and tumor cell lines were extracted with RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Quantified proteins were separated by SDS‒PAGE and transferred to PVDF membrane. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with the corresponding antibodies. For the IHC, paraffin-embedded 4-µm-thick kidney cancer tissue sections were deparaffinized, subjected to antigen retrieval, blocked with 5% bovine serum albumin (BSA) for 2 hours at room temperature, and then incubated with antibodies against SAP30 (1:400) at 4°C. The next day peroxidase-conjugated polymer was added for 30 min. Finally, visualization was performed with DAB (Beyotime, Shanghai, China).
2.6 CCK-8 cell proliferation assay
We used the CCK-8 assay (MCE, China) to analyse cell proliferation. Transfected cells were grown in 96-well plates (3000 cells/well) for 12 hours. Then 10% CCK8 solution was added at different time periods (0/24/48/72 h) according to the CCK8 kit manufacturer's protocol. The OD absorbance values were measured at 450 nm using a 96-well plate reader.
2.7 Colony formation assay
Cell colony formation capacity was assessed by a plate colony formation assay. Transfected 786-O, CAKI-1 and OS-RC-2 cells (1 × 103 cells/well) were seeded in 6-well plates for 14 days to form colonies, which were subsequently fixed in paraformaldehyde and then stained with crystal violet.
2.8 Hoechst staining
A apoptosis of transfected cells was induced with H2O2. After fixation with paraformaldehyde, the cells were washed with PBS 3 times. Finally, the cells were stained with Hoechst dye (#C1017, Beyotime, China) for 5 min and photographed under a microscope.
2.9 Flow cytometry
First, the transfected cells were trypsinized with EDTA-free trypsin and washed with cold PBS according to the manufacturer's instructions of the ANNEXIN V-FITC/PI Apoptosis Kit (Solarbio, Beijing, China). Second, apoptosis was examined in the dark with Annexin V-FITC/propidium iodide (PI) staining. Finally, cell cycle profiles were analysed by PI staining and flow cytometry according to the manufacturer's instructions of the Cell Cycle Detection Kit (Beyotime, Shanghai, China).
2.10 Chromatin immunoprecipitation assay (ChIP)
The chromatin immunoprecipitation assay with 786-O and CAKI-1 cells was performed according to the protocol for the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads). First, 1x107 cells were fixed with 1% formaldehyde and 1.25 M glycine was added to terminate the reaction. The cells were washed with cold PBS containing protease inhibitor and collected into a centrifuge for the next step. The last collected cells were sonicated in IP mixed medium and centrifuged at 14, 000 × g for 30 min at 4°C. Protein A/G agarose beads were mixed with the above supernatant and centrifuged, and the chromatin in the supernatant was immunoprecipitated overnight with antibodies against SAP30 or IgG. The protein A/G agarose beads were then washed with mixed wash buffer, LiCl/detergent and TE buffer. The beads were heated overnight at 65°C in a buffer mixture containing 1% SDS, 0.1 M NaHCO3 to crosslink the beads in reverse. The beads were then treated with 1 ml of RNaseA for 15 min at 37°C and digested with 2 ml of proteinase K solution for 1 hr at 37°C. Finally, DNA was purified by extraction with LiCl, phenol/chloroform and ethanol, and the specific MT1G promoter region was amplified with DNA template. Supplementary Table S1 lists the primer sequences designed to detect specific promoters.
2.11 Luciferase reporter assay
The SAP30 overexpressing plasmid pcDNA3.3-SAP30 and the plasmid pGL4.18WT + binding site (BS1, BS2 and BS3) containing the promoter region of MT1G were constructed. Binding site sequences for promoter reporter plasmid construction are listed in Supplementary Table S1. 293T Cells cultured in 6-well plates were transfected with overexpression plasmids or empty vectors. Luciferase activity was measured using a dual luciferase reporter system according to the manufacturer's instructions (E1910, Promega, USA).
2.12 Detection of EdU incorporation
Cells (3 x 105 cells/well) were seeded on coverslips in 6-well plates for 24 hours. Cells were then cultured in culture medium containing EdU for 2 h after 48 h of transfection. The cultures were subsequently stained with Azide 488 and Hoechst 33342. EdU-positive cells were observed under a fluorescence microscope and photographed. The EdU binding rate was expressed as the ratio of EdU-positive cells (green cells) to total Hoechst-labelled cells (blue cells).
2.13 Immunofluorescence (IF)
Cells (3 × 105 cells/well) were inoculated in 6-well plates with coverslips for 24 hours. First, after washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.05% Triton for 30 min, blocked with 5% BSA for 2 h, and incubated with antibodies against SAP30 overnight at 4°C. The next day, the type A fluorescent secondary antibody was incubated for 1 hour at room temperature. Finally, nuclear staining was performed with DAPI and subsequent imaging was performed with fluorescence microscopy.
2.14 Xenograft tumor growth
786-O cells (3 × 107 cells/ml) were injected into the posterior ventral sides of 6-week-old male nude mouse, purchased from Charles River Laboratories (Beijing, China). After 6 days, visible tumors were visible, their volume was measured, and transfection was performed according to the instructions of the EntransterTM-In Vivo kit (Engreen, Beijing, China) and repeated every 3 days. The transfection was repeated every 3 days. Forty-eight days later, the nude mice were sacrificed, the tumors were removed, and their weights and volumes were measured. The protocol complied with the regulations of the Animal Ethics Committee of Harbin Medical University.
2.15 Statistical analysis
Statistical analysis was carried out by GraphPad software (version: 7.0). Data are expressed as the mean ± standard deviation (SD). A t test was used to assess statistical significance, and a p value < 0.05 was considered significant.