2.1 Cell line authentication
The breast cancer cisplatin resistant cell line MCF-7/DDP was purchased from Chuanqiu biology in October, 2019, Shanghai, China. The cell line was tested and authenticated by STR and the last time the cell line was tested was in June, 2018. The cell was test by extracting DNA from MCF-7/DDP, then it was amplified by PowerplexTM16 ID System STR compound amplification kit. ABI 3130xl genetic analyzer was used to detect the STR locus and sex gene amelogenin, and the map and genotype were analyzed. The MCF-7 human breast cancer cell line was obtained from the library of affiliated hospital of Qingdao University in October, 2016. The cell line was tested and authenticated by STR and the last time the cell line was tested was in
August, 2020. Test method: took an appropriate amount of samples, extracted DNA with Microread Genomic DNA kit, amplified 20 STR sites and gender identification sites with MicroreaderTM21 ID Systemsystem, detected PCR products with ABI 3730xl genetic analyzer, analyzed the detected products with GeneMapperID-X software(Applied Biosystems), and compared them with ATCC and DSMZ databases.
2.2 Cell culture and chemical reagents
MCF-7 was cultured in DMEM (HyClone, USA) supplemented with 10% fetal bovine serum (ExCell Bio,China). The breast cancer cisplatin resistant cell line MCF-7/DDP was cultured in RPMI-1640 (HyClone, USA) supplemented with 10% fetal bovine serum (ExCell Bio,China) and 0.5uM DDP (Solarbio, China) at 37℃ in a tissue culture incubator with 5% CO2. Total RNA and protein were extracted from the cells in logarithmic phase. Real-time PCR and Western Blotting were used to detect mRNA and protein levels. Puromycin (Yeasen, China) was used at a concentration of 1 ug/ml. Protein synthesis inhibitor cycloheximide (CHX) was used at a concentration of 200 ug/ml. Proteosome inhibitor MG132 (Selleck, China) was used at a concentration of 10 µM.
2.3 Lentivirus transfection
Logarithmic growth cells with confluence of 80-90% were prepared single cell suspension with complete medium, and inoculate 2×105 cells per well into 6-well plate. RRS1-targeting sh-RRS1, RRS1-targeting OE-RRS1 and a non-targeting control were expressed via pSuper constitutive expression constructs (Genecard, China). The amount of lentivirus was accurately calculated according to the number of cells (The number of virus=the number of cells×MOI/the titer of virus, and the MOI of MCF-7 was 20). The solution was changed after transfection for 12h. Its efficiency was observed under fluorescence microscope after transfection for 48-72 h, and the total protein and mRNA were extracted for detection of RRS1.
2.4 Quantitative PCR
Total RNA was extracted from cells or tumors using a Trizol RNA isolation kit (Vazyme, China) and detected by RT-qPCR according to the manufacturer’s protocol (Vazyme, China). All samples were analyzed in triplicate. Primers for GAPDH were forward 5’-AGAAGGCTGGGGCTCATTTG-3’ and reverse 5’-AGGGGCCATCCACAGTCTTC-3’. The primers for RRS1 were forward 5’-CCCTACCGGACACCAGAGTAA-3’ and reverse 5’-CCGAAAAGGGGTTGAAACTTCC-3’. The primers for AEG-1 were forward 5’-CGAGAAGCCCAAACCAAATG-3’ and 5’-TGGTGGCTGCTTTGCTGTT-3’.
2.5 Sensitivity of Resistant Cell MCF-7/DDP to cisplatin
Cells were prepared into single cell suspension by trypsinization and seeded into 96-well plates at 3 × 103 cells per well. When cells grow to the logarithmic growth stage, DDP were added to final concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.5, 2.5, 3.5, 4.5, 6 µg/ml. Incubated under normal conditions for 24 h, then the viabilities were examined using CCK-8 (Solarbio, China) at 10 ul/well. The absorbance values were used to calculate the inhibition ratios and IC50 at 24 h.
2.6 Cell proliferation
The cells, treated according to the experimental group requirements, were inoculated into 96-well plates at 3 × 103 cells per well, added CCK-8 (10ul/well) (Solarbio, China) in different time periods, the cells were maintained at 37℃and 5% CO2 for 2 h, and then absorbance (optic density, OD) at 450 nm was measured.
2.7 Cell cycle and apoptosis
Cell cycle analysis was conducted with FlowJo software following staining with propidium iodide 25 ug (Solarbio, China) and flow cytometric( Cytoflex S, Beckman). Apoptosis was also evaluated by flow cytometric analyses of Annexin V-APC binding (Elabscience, China).
2.8 Co-Immunoprecipitation
Co-IP experiment was performed using a protein A/G kit (Epizyme, China). Beads were activated though incubating overnight with 25 ul lysis buffer. MCF-7/DDP cells were lysed in cell lysis buffer at 4℃ for 30 minutes. Protein concentration was measured with BCA assay (Solarbio, China). The equal resulting lysate, about 500 ul with antibody against AEG-1 (1 ug, Abcam, ab227981) or IgG (1 ug, Bioss, bs-0297R), was incubated overnight with activated beads for immunoprecipitants, immunoprecipitants were separated by SDS-PAGE after washing with lysis buffer and were analysed by immunoblotting with the indicated antibodies.
2.9 Protein stability assay
OE-CON and OE-RRS1 cells of 80% confluence were treated with 200 ug/ml cycloheximide (CHX), and protein was collected at the indicated time and analyzed with Western Blot.
2.10 Nude mice xenograft study
Four-week-old male nude (BALB/c) mice (vitalriver, China) were used in the experiments. MCF-7 cells (5×106 cells/0.2 ml liquid/mouse, medium/matrix=1) were injected into the mice subcutaneously to generate the mouse models. Xenografts were allowed to grow to approximately 100 mm3 about 1 week and randomly divided into two groups (n=5 in each group) as follows: control group (100 ul, saline solution) and DDP group (15 mg/kg). Cisplatin and saline were administered by intraperitoneal injection every one week for four weeks. The longest diameter (a) and the shortest diameter (b) of the tumor were measured using digital calipers, and the tumor volume (V=a×b×b/2) was calculated. At the end of the experiments, tumor tissues were harvested from these mice for Q-PCR or Western Blot analysis.
h2>2.11 Western Blot
Cells were lysed in RIPA buffer (Sparkjade, China) with protease inhibitor cocktail (Servicebio, China) and phosphatase inhibitor A and B on ice for 30 minutes and then centrifuged for 15 min at 12,000 rpm. After being resolved on 10% SDS-PAGE gels, proteins were transferred to a PVDF membrane and blocked with 5% BSA in TBST for 2 h at room temperature. The membranes were subsequently incubated overnight at 4 ℃ with the corresponding antibody GAPDH (Abclonal, AC002), RRS1 (Abcam, AB188161), AEG-1 (Abcam, ab227981), ABCG2 (ZenBio, R26465), MDR1 (Proteintech, 22336-1-AP), ERK (ZenBio, 340373), p-ERK (ZenBio, 340767), BAX (CST, #89477), Bcl-2 (CST, #3498), BAD (Abcam, ab32445), p-BAD (Abcam, ab129192), Ubiquitin (Proteintech, 10201-2-AP). After being washed with TBST three times, the blots were incubated with 1:1000 dilution of the HRP-conjugated secondary antibody (Bioss, bs-0295G-HRP), and washed again three times with TBST. The transferred proteins were visualized by ECL (MDBio,China).
2.12 Statistical analysis
The data was analysed by statistical software SPSS13. All data was expressed as mean ± standard deviation (SD), and student’s t test was used to compare the difference between two groups. P < 0.05 was considered as statistical significant.