Patients and datasets
Microarray data of 200 breast cancer samples (GSE11121) and the survival information were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). RNA-seq data of 88 breast cancer samples without lymph node metastasis, including 44 cancer samples and 44 para-cancer samples, and their clinicopathological information were downloaded from the TCGA database (https://portal.gdc.cancer.gov/projects/TCGA-BRCA).
Identification of metastasis-related genes
The GSE11121 samples were processed using the R package WGCNA for removing outliers. After clustering, 199 breast cancer samples were included, GSM282518 was excluded. A univariable Cox proportional hazards regression model was used to select metastasis-related genes with |coefficient|> 0.50, P value < 0.05. Metastasis-related differentially expressed genes were analyzed by using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, and the GSEA with the clusterProfiler package of R. The KEGG pathways and GO terms regarding cellular component, molecular function, and biological process with P values and false discovery rates less than 0.05 were considered statistically significant.
Then, WGCNA was performed to identify RNF126-related signal pathways. The similarity matrix was transformed into an adjacency matrix with a network type of signed and a soft threshold of β = 5 and then transformed into a topological matrix with the topological overlap measure (TOM) describing the degree of association between genes. 1-TOM was used as the distance to cluster the genes, and then the dynamic pruning tree was built to identify the modules. We identified 5 modules by setting the merging threshold function at 0.20. The enrichment P values for the GSEA were based on 1000 permutations and adjusted by calculating the false discovery rates. The GSEA results were visualized using the R package enrichplot.
Cell culture and transfection
Human breast cancer cell lines (MCF7 and MDA-MB-231) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), and maintained at 37 °C in a humidified incubator with 5% CO2. All DNA-plasmid transfections were performed using Lipofectamine 2000 according to the manufacturer's recommendations (Invitrogen). The sequences of shRNA used were as follows: shRNF126#1 5′-TGCATGGTTTGTGGCGGAAGA-3′; shRNF126#2 5′-CAACGAGAACGCCACATGGTC-3′.
Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA was isolated with the TRIzol reagent (Invitrogen). Novogene (Beijing, China) completed the cDNA library construction. Experiments were carried out in triplicate for each data point. Reactions were performed using SYBR Green mix (Roche) and a MyiQ real-time PCR detection system (Bio-Rad). Relative mRNA levels were calculated using the comparative Ct method (ΔCt).
GAPDH forward primers: 5′-CTCTGCTCCTCCTGTTCGAC-3′; reverse primers: 5′-TTAAAAGCAGCCCTGGTGAC-3′.
RNF126 forward primers: 5′-TATCGAGGAGCTTCCGGAAGAGA-3′; reverse primers: 5′-AAAGCAAACTGTCCGTAGCCCT-3′.
CDK1 forward primers:5′-GGATGTGCTTATGCAGGATTCC-3′; reverse primers: 5′-CATGTACTGACCAGGAGGGATAG-3′.
CDK2 forward primers: 5′-TATTAACACAGAGGGGGCCA -3′; reverse primers: 5′-AAAGATCCGGAAGAGCTGGT-3′.
CDK5 forward primers:5′-GGAAGGCACCTACGGAACTG-3′; reverse primers: 5′-GGCACACCCTCATCATCGT-3′.
Scratch wound assay
The cells were inoculated in a 6-well plate, scraped through each hole with the tip of a sterile 10 μL pipette and washed with phosphate-buffered saline to remove any debris. After 24 h, the cells migrated to the empty space.
Cell invasion and migration assays
Approximately 2 × 104 cells in 300 μL DMEM medium without FBS were seeded into upper transwell chamber (8 μm pore size) to evaluate cell migration. The lower chamber was filled with 800 μL DMEM medium supplemented with 10% FBS. After 24 h, the cells attached to the lower surface of the membrane were fixed with 4% formaldehyde, stained with 0.5% crystal violet, and then counted under a microscope in five random fields. Invasion assays were performed under the same conditions as the migration assays, but in matrigel-coated transwell (Corning, NY, USA) inserts.
Cell viability and calculation of half-maximal inhibitory concentration (IC50)
The cells were seeded in 96-well plates in 100 μL DMEM medium containing 10% FBS, at a density of 2 × 103 cells per well. The cells were exposed to various doses of inhibitors and assayed for viability at indicated times, using the MTT according to the manufacturer's instructions. In brief, MTT (20 mL of 5 mg/mL) was added to each well and cells were incubated for a further 3.5 h in an incubator. MTT solvent was added after removing the medium and the cells in plates were agitated on an orbital shaker for 15 min. The absorbance was read at 590 nm with a reference filter of 620 nm. The absorbance values were normalized with respect to those of untreated control cells. The IC50 was calculated using nonlinear regression analysis in GraphPad Prism 6.0.
In vivo studies
MCF7 and MDA-MB-231 cells (1 × 107) in 150 μL PBS were subcutaneously injected into the right flank of female nude mice. Tumor volume was measured by caliper and calculated as length × width2/2. When tumor volume grew up to 50–100 mm3, the mice were randomly divided into two groups (five mice per group), and then treated with PBS daily, AZD6738 (50 mg/kg, oral, daily). Tumor volume was measured every 3 days. In assays to measure formation of metastases, 107 breast cancer cells were injected into tail veins of mice. The number of metastases was assessed in 3 or 6 weeks, respectively. All the animal experiments were carried out with the approval of the guidelines of Guangxi Medical University Cancer Hospital.
Immunohistochemistry
The formalin-fixed mouse tumor, liver, or lung tissues were embedded with paraffin. The treated tissues were sectioned (3 μm) and stained with the hematoxylin and eosin (H&E). H&E-stained liver or lung sections were imaged using a microscope (Olympus). For immunostaining, slides were heated to 60 °C and then deparaffinized in xylene. The slides were rehydrated in descending alcohol concentrations. Antigen retrieval was performed by incubating slides in a retrieval solution of citrate buffer. Hydrogen peroxide was added to block endogenous peroxidase activity to decrease unwanted background staining. Primary antibody (RNF126, ab234812, Abcam) was added at 1:100 dilution. The substitution of primary antibody performed negative controls with phosphate-buffered saline (PBS). To guarantee consistent IHC evaluation, slides from a reference tumor previously determined as positive were included in each staining procedure. Evaluations of staining reactions were performed in accordance with the immunoreactive score (IRS) proposed by Remmele and Stegner: IRS = staining intensity (SI) X percentage of positive cells (PP). Staining intensity was marked as nongranulated (0); low grade (light yellow; 1); moderate (brownish yellow; 2); or strong (reddish brown; 3). The PP was scored as negative (< 5%; 0); weak (5–10%; 1); moderate (11–50%; 2); strong (51–80%; 3); or very strong (> 81%; 4). Specimens scoring beyond 3 were considered positive overexpression. Slides were studied with the microscopic (Olympus).
Immunoblotting
Cellular extracts were prepared by resuspending cells in radio immunoprecipitation assay (RIPA) buffer. After protein samples were separated by 5%, 12%, or 15% SDS-PAGE, they were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA). Around 5% BSA was used to incubate the PVDF membrane for 1 h at room temperature, and then at 4 °C overnight with antibodies specific to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AP0066, 1:10,000; Bioworld), RNF126 (C-1 SC-376005, 1:100; Santa Cruz Biotechnology); ATR (C-1 SC-515173, 1:100; Santa Cruz Biotechnology); p-ATR (S428 AB178407, 1:100; Abcam); γ-H2AX (Ser139 SC-517348, 1:100; Santa Cruz Biotechnology); p-RPA2 (S4/S8; rabbit polyclonal, BL647, 1: 1000; Bethyl Laboratories); CHEK1 (AB32531, 1:100; Abcam); CDK1 (AB265590, 1:500; Abcam); CDK2 (610,146, 1:200; BD Biosciences); CDK5 (AB40773, 1:500; Abcam); Cleaved PARP (AB4830, 1:200; Abcam). After washing in Tris-buffered saline with 0.1% Tween 20 (TBST) for three times (10 min each time), the PVDF membranes were incubated with secondary antibodies (Goat anti-mouse IgG-horseradish peroxidase (HRP)–conjugated (#7076S, 1:1,000; Cell Signaling Technology), goat anti-rabbit IgG-HRP–conjugated (#7074S, 1:1000; Cell Signaling Technology), and donkey anti-goat IgG-HRP–conjugated (A2216, 1:1,000; Santa Cruz Biotechnology)) for 1 h at room temperature. By using enhanced chemiluminescence blotting reagents, proteins were detected after three TBST washes (FUDE Biological, Hangzhou, China). Signal intensity was assessed by using a Tanon-5500 chemiluminescence detection system (Tanon Science & Technology Ltd, Shanghai, China).
Immunofluorescence analysis
Cells growing on slides were fixed directly in 3–4% paraformaldehyde. Cells were extracted for 5 min on ice with 0.5% Triton X-100 in cytoskeletal (CSK) buffer (10 mmol/L PIPES, 300 mmol/L sucrose, 100 mmol/L NaCl, 3 mmol/L MgCl2; pH = 6.8) supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 mmol/L sodium vanadate, and proteasome inhibitor for 10 min at 4 °C. Then, extracted cells were fixed with 3–4% paraformaldehyde. The cells were permeabilized for 10 min with PBS containing 0.5% Triton X-100 for 15 min at room temperature, followed by blocking with 1% BSA, and then incubated with primary antibodies specific to CDC45 (H-300 clone, SC20685, 1:50; Santa Cruz Biotechnology). The bound antibodies were revealed with chicken anti-rabbit IgG Alexa Fluor 488. Slides were viewed at 1,000 magnifications with a NIKON 90i fluorescence microscope (photometric cooled mono CCD camera, NIKON, Tokyo, Japan).
DNA fiber assays
DNA fiber assays were performed as published with some modifications [23]. Cells were pulse-labeled with 50 mmol/L IdU (Sigma-Aldrich #I7125) for 40 min and then pulse-labeled with 200 mmol/L CldU (Sigma–Aldrich #C6891) for 40 min in the presence or absence of ATR inhibitor. At the end of the CldU pulse, cell suspensions (2.5 mL) were mixed with 7.5 mL of lysis buffer (0.5% SDS, 200 mmol/L Tris–HCl (pH 7.4), 50 mmol/L EDTA). Each mixture was dropped on the top of an uncoated regular glass slide. Slides were inclined at 25° to spread the suspension on the glass. Once dried, DNA spreads were fixed by incubation for 10 min in a 3:1 solution of methanol-acetic acid. The slides were dried and placed in precooled 70% ethanol at 4 °C for at least 1 h. DNA was denatured with 2.5 mol/L HCl for 30 min at 37 °C. The slides were blocked in 1% BSA in PBS for 30 min at room temperature and then incubated with mouse anti-BrdU antibody (BD Biosciences #347,580) at a 1:200 dilution and rat anti-CldU antibody (Abcam #ab6326) at a 1:400 dilution. The slides were incubated with secondary fluorescent antibodies [goat anti-mouse IgG (H + L) Alexa Fluor 594 secondary antibody (A-11032, 1:400; Thermo Fisher Scientific); or chicken anti-rabbit IgG (H + L) Alexa Fluor 488 secondary antibody (A-21441, 1:400); Thermo Fisher Scientific]. Replication fibers were viewed at 1000 magnifications on a NIKON 90i fluorescence microscope (photometric cooled mono CCD camera, NIKON, Tokyo, Japan). Signals were measured using ImageJ software (NCI/NIH), with some modifications made specifically to measure DNA fibers.
Statistical analysis
Statistical analyses were undertaken using the statistical software package, R version 4.0.4. Univariate Cox regression analysis was performed to select genes with P < 0.05 for metastasis. Univariate survival analysis was performed by Kaplan–Meier survival analysis with the log-rank test. Paired t-test, one-way or two-way ANOVA analysis of variance were used to determine the significance of differences between groups. Continuous variables were expressed as mean ± SD. In all the statistical analyses, P < 0.05 was considered as statistically significant.