Online data acquisition and analyses
RNA sequencing data of BC patients in the study were obtained from the TCGA database (https://cancergenome.nih.gov/) and analyses were performed using R software (v4.1.3). RAB10 RNA expression levels were analyzed using the TNMplot database (https://tnmplot.com/) for pan-cancer analysis and selected RNA-seq data from paired tumors and adjacent normal tissues of BC for differential expression analysis13. The UALCAN database (https://ualcan.path.uab.edu/) was used to access the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and the International Cancer Proteogenome Consortium (ICPC) dataset for pan-cancer analysis of RAB10 protein expression and differential expression analysis of BC and normal tissue14. Survival analysis using gene chip data was conducted in Kaplan–Meier Plotter (https://kmplot.com/) to plot the 5-year overall survival (OS) and recurrence-free survival (RFS) prognostic model of RAB10 with BC 15. Immunohistochemical images of RAB10 in normal breast tissue and BC tissue were obtained using The Human Protein Atlas data (https://www.proteinatlas.org/) as a control.
Correlation analysis of RAB10 expression and immune infiltration
We analyzed the correlation between RAB10 expression and immune cells in TCGA samples using the “CIBERSORT” algorithm available at https://cibersort.stanford.edu/. We also examined the differences in the level of immune cell infiltration between the high and low RAB10 expression groups. The analysis process utilized the R packages “ggplot” and “ggpubr.”
Enrichment analysis of RAB10 in BC
We divided the samples from TCGA into high and low expression groups based on the median RAB10 expression. Subsequently, we performed differential gene analysis on the two groups, using a threshold of |logFC| > 1 and FDR < 0.05. GSEA GO and GSEA KEGG16 enrichment analysis were conducted on the logFC values sorted by p value < 0.05 and false discovery rate (FDR) < 0.25 to identify significantly enriched functional or pathway terms. We employed the R package “clusterProfiler” for this analysis.
BC cell lines
The breast carcinoma cell lines MDA-MB-231, HCC1937 and SK-BR-3 were obtained from the American Type Culture Collection (ATCC). The MDA-MB-231 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 atmosphere. HCC1937 and SK-BR-3 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 0.1 mg/ml human recombinant insulin at 37 °C in a humidified 5% CO2 atmosphere.
Gene Chemistry (Shanghai, China) designed and synthesized a lentivirus containing short hairpin RNAs (shRNAs) targeting human RAB10. The RAB10-shRNA sequence used in this study was 5′-GCCTTCAATACTACCTTTATT-3′. MDA-MB-231, HCC1937 and SK-BR-3 cells were transduced with the lentivirus following the manufacturer’s instructions. The efficiency of RAB10 knockdown was determined using real-time quantitative fluorescence polymerase chain reaction (PCR), and stable transduced cells were expanded and harvested.
Quantitative real-time PCR
The RNA extraction from MDA-MB-231, HCC1937 and SK-BR-3 cells was performed using Trizol (Invitrogen), and the resulting RNA was reverse transcribed following the manufacturer’s instructions (Invitrogen). The detection of RAB10 was performed through quantitative real-time PCR, using the following primers: RAB10 forward: TTT CAC ACC ATC ACA ACC TCC, reverse: GGT ACA ACT CTT TTG TCG TCC ATA. GAPDH was used as a control, with the following primers: GAPDH forward: TGA CTT CAA CAG CGA CAC CCA, reverse: CAC CCT GTT GCT GTA GCC AAA.
BC cells were collected and lysed on ice for 30 min using a lysis solution containing 20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton x-100 and protease inhibitor, and the supernatant was collected by centrifugation at 12,000 r min−1 for 10 min at 4 °C with a radius of 10 cm. Total protein concentration was measured using the BCA Protein Assay Kit (Pierce). Nitrocellulose membranes were closed with 5% skim milk (diluted with 1 × PBS) for 2 h at room temperature. Rab10 antibody is then added at 4° overnight. Then 1 × PBS buffer was washed for 50 min, secondary antibody was added for 6 h at room temperature, and after 20 min of washing, enhanced chemiluminescence agent (Amersham) was added to observe the proteins in a dark room, and after 5 min at room temperature, exposure and development were performed.
MDA-MB-231, HCC1937 and SK-BR-3 cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100 μl of cell culture medium (RPMI-1640 supplemented with 10% fetal calf serum). Each group was set up with three replicate wells and incubated in a 5% CO2 incubator at 37 °C for 2–5 days. After the incubation period, 10 µL of MTT solution (5 mg/mL) was added to each well, and the cells were further incubated for 4 h. The supernatant was then aspirated and discarded after horizontal centrifugation, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well. The plates were shaken at low speed for 10 min before measuring the optical density (OD) at 490 nm using an enzyme marker.
Wound healing assay
The experiments were conducted following the manufacturer’s guidelines. Briefly, cells were seeded in 24-well culture plates at a density of 2.0 × 105 cells per well and incubated at 37 °C in a 5% CO2 incubator until they formed a monolayer. Once the cells had formed a monolayer, a scratch was made using a 1000 μL pipette tip, and the cells were washed twice with PBS. The medium was then replaced with RPMI-1640 and the cells were further incubated at 37 °C in a 5% CO2 incubator.
The Transwell assay was conducted using a 24-well tissue culture plate and a 12-well cell culture plate that included an 8 μm pore size polycarbonate membrane. The cells were counted and adjusted to a concentration of 5 × 104 cells/mL in RPMI-1640 medium without 10% FBS. Next, 200 μL of the cell suspension was added to the upper chamber of the Transwell, while the lower chamber was filled with complete medium containing 10% FBS. The plates were then incubated in a 37 ℃, 5% CO2 incubator for 24 h. After incubation, the cells were fixed in a formaldehyde solution for 25 min, stained with 0.1% crystal violet for 20 min, and washed with PBS. Six random fields of view were selected and photographed under an inverted microscope.
A total of 164 tissue samples of invasive ductal carcinoma of the breast, along with 5 samples of paracancerous tissue, were included in the study. All samples were obtained from patients who had undergone surgical treatment for BC at the Affiliated Hospital of Hebei Engineering University between January 2014 and June 2016 and had been pathologically diagnosed with invasive ductal carcinoma of the breast. Neoadjuvant chemotherapy or any other systemic tumor treatment was not administered before surgery to any of the cases. All patients received systemic treatment after surgery and had follow-up information available for 5 years. This study is a retrospective study and will not have an impact on the treatment of patients. All methods were performed in accordance with the relevant guidelines and regulations. The study and experimental methods were approved by the Ethics Committee of the Affiliated Hospital of Hebei Engineering University and granted exemptions from patient informed consent.
Paraffin-embedded BC tissue and corresponding paracancerous tissue were sectioned and subjected to dewaxing in xylene, followed by gradient alcohol hydration. The tissue sections were then subjected to microwave heating in sodium citrate buffer (pH 6.0) to boiling for 10 min. Endogenous peroxidase was blocked using 3% hydrogen peroxide for 20 min, and 3% sheep serum was added and incubated at 37 °C for 20 min. Primary antibody was added and incubated overnight at 4 °C, followed by secondary antibody incubation at 37 °C for 30 min. Color development solution was used and hematoxylin re-staining was performed. The slices were dehydrated with gradient alcohol and sealed with neutral resin. Finally, two senior pathologists independently assessed the expression of RAB10 by reading the films under the microscope. The staining intensity was scored as: dark brown = 3, brown = 2, light yellow = 1, and colorless = 0. The percentage of positive cells to total cells was classified as: > 75% = 4, > 50% to 75% = 3, > 25% to 50% = 2, 5%-25% = 1, and < 5% = 0. The final two multiplied scores of 0–6 were considered negative and 6–12 were considered positive.
In this study, t tests were conducted to analyze the results of quantitative real-time PCR, MTT and Transwell assays. The correlation between RAB10 and clinicopathological characteristics of BC was evaluated using the one-way χ2 test. Kaplan–Meier curves and log-rank tests were used to analyze the correlation of RAB10 with OS and RFS of BC. Additionally, Cox regression univariate and multivariate analyses were performed to estimate the risk ratio (HR) and 95% confidence interval (CI), and the statistical significance was determined using the two-tailed test at P < 0.05. All clinical data were analyzed using SPSS 24.0 software (IBM, Chicago, USA).
Ethics approval and consent to participate
In this study, which involved all BC sample tissues, the Ethics Committee of the Affiliated Hospital of Hebei Engineering University approved the study number 2021[K]019.