Ethics approval and consent of participants
This study is approved by the Brandon University Ethical Approval Committee (approval # 23056) and the Brandon University Biosafety Committee (approval # 2020-BIO-02). Informed consent was obtained by the tumor banks before the collection of samples from participants.
Cell Culture
Cell lines MCF7, SKBR3 and Hs578T were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Stable miR-526b-, miR-655- and COX2-overexpressing cell lines (MCF7-miR526b, MCF7-miR655, MCF7-COX2, and SKBR3-miR526b) and empty vector controls (MCF7-Mock and SKBR3-Mock) were established using transfection of the overexpression and empty vector plasmids respectively (5, 6). These cells were cultured in RPMI media (Gibco, Mississauga, ON) supplemented with 10% fetal bovine serum (FBS) (Gibco, Mississauga, ON) and 1% penicillin-streptomycin (Gibco, Mississauga, ON). Additionally, transfected cells received Geneticin (Life Technologies ThermoFisher, Ottawa, ON, Canada) 200nM/ml. All cells were grown in an incubator at 37°C and 5% CO2.
RNA Extraction
Cells were grown until 90% confluent, and RNA was extracted from whole cells using miRNeasy Mini kit (Cat #217004, Qiagen, ON, Canada) following the manufacturer's protocol. For RNA extraction from cell secretions (conditioned media), cells were grown to 85% confluency and then grown for 24 hours in the basal media. After 24 hours, the conditioned media was collected and centrifuged to remove cell debris. The RNA was extracted from the clear supernatant using the miRNeasy Mini kit.
Biopsy tissues and plasma RNA were also extracted using miRNeasy Mini kit (Cat #217004, Qiagen, ON, Canada). We used 300–350 µL of plasma for miRNA and RNA extraction. For the biopsy tissues, 5mg of the flash-frozen sample was used to extract RNA and miRNA. This resulted in 500–600 ng of total RNA (23).
cDNA Synthesis
Around 1–2 µg of RNA and 200 ng of miRNA were reverse transcribed into complementary DNA (cDNA) using the SuperScript cDNA reverse transcription kit (ThermoFisher Scientific, ON, Canada).
Quantitative Real-Time PCR (qRT-PCR)
Quantitative Real-Time polymerase chain reaction (qRT-PCR) uses cDNA, RNAse-free water, TaqMan Universal PCR Master Mix (ThermoFisher Scientific, ON, Canada), and gene-specific probes. A ubiquitous control gene, RPL5 (Hs303044958), experimental YWHAB (Hs00793604) gene, pri-miR-526b (Hs03296227), and pri-has-miR-655 (Hs03304873) probes were used to conduct qRT-PCR.
Mass-Spec Data
Using previously generated liquid chromatography-mass spectrometry (LC-MS) data (9), secretory protein expression of all isoforms within the 14-3-3 family of proteins was isolated. Comparisons were made between low miRNA MCF7-Parental and miRNA overexpressed cell lines MCF7-miR526b and MCF7-miR655.
Western Immunoblotting
Total cellular protein was extracted by treating cells with 10X Cell Lysis Buffer (#9803, Cell Signaling Technology) and Protein Phosphatase Inhibitor (#58705, Cell Signaling Technology). Equal volumes of protein, ranging from 20ug-40ug among replicates, were electrophoresed on a 10% polyacrylamide gel. After separation, protein samples were transferred onto a Nitrocellulose membrane (Cytiva, Amersham, #10600006) and incubated in blocking solution (1% BSA in TBS), followed by overnight incubation in primary antibodies at 4℃ (1:300 anti-14-3-3-beta, Santa Cruz Biotechnology, Cat#sc-25276; 1:1000 anti-alpha-tubulin, Applied Biological Materials, Cat#Y054861). After 12–20 hours of incubation, membranes were washed and then probed with secondary antibodies (1:5000 AzureSpectra 700 anti-mouse, 1:5000 AzureSpectra 800 anti-rabbit) for 1 hour. Membranes were imaged on the Sapphire Biomolecular Imager (Azure Biosystems, Dublin, CA, USA).
In-Cell Western Immunoblotting
Approximately, 50,000 cells were seeded into a 96-well plate and grown to 80% confluency. Cells were fixed with 100% methanol for 15 minutes, then blocked with 1% BSA in PBS for 1 hour at room temperature. Following blocking, cells were incubated in primary antibodies (1:200 anti-14-3-3-beta, 1:1000 anti-TUBA1, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4℃, then incubated in secondary antibodies (1:5000 AzureSpectra 700 anti-mouse, 1:5000 AzureSpectra 800 anti-rabbit) for 1 hour at room temperature. The plate was imaged on the Biomolecular Imager and quantified with AzureSpotPro software.
Immunocytochemistry
Approximately, 200,000 cells were seeded onto sterile coverslips in a 6-well plate and grown to 80% confluency. Fixation was done using ice-cold 100% methanol for 10 minutes, and then the cells were blocked with 1% BSA in PBS for 1 hour at room temperature. The primary antibody (anti-14-3-3-beta, Santa Cruz Biotechnology) was incubated overnight at 4℃, followed by the secondary antibody (anti-m-IgGk BP-FITC, 1:1000, Cat# sc-516140, Santa Cruz Biotechnology) for 1 hour at room temperature followed by washing with PBS-T. Coverslips were mounted onto slides using Mounting Medium with DAPI (ab104139, Abcam) and imaged on the Eclipse Ti2 microscope (Nikon, Ontario, Canada).
Knockdown (KD) of YWHAB
PolyPlus INTERFERin siRNA/miRNA transfections kit (Ref# 409-01) was used following the manufacturer's protocol with a siRNA concentration of 1 nM. YWHAB siRNA and scrambled siRNA KD (Cat# AM16708, Ambion, TX, USA) were used to knock down following manufacturer protocol. We observed an 80% reduction in YWHAB gene expression compared to scramble KD after 24 hours of knockdown. All functional assays were conducted 24 hours post-knockdown.
Migration Assay
Cells were grown until 80% confluent, and using a 0.1-10uL pipette tip, one vertical and one horizontal line were scratched in the wells. Pictures of the scratch wounds were taken using an inverted phase contrast microscope at 0, 8, 16, and 24 hours and the width of the wound was analysed using ImageJ (version 1.53). Three lines were drawn across the wound, and the mean of three measures was considered for analysis.
Proliferation Assay
Cells were grown in 96-well plates. Gene KD was conducted in the 96-well plate. After 24 hours post KD, media with the siRNA cocktail was removed. Cell proliferation was measured using the CyQUANT NF Cell Proliferation Assay Kit (Ref#C35007, ThermoFisher Scientific, ON) following the manufacturer's protocol. Cell proliferation was measured using a multi-mode TECAN Microplate Reader, using a DNA binding dye with 485 nm for excitation and 535 nm for emission. The data was taken four times from 30 minutes once the dye was added up to an hour.
Biopsy Tissue Samples
Frozen human breast tissue samples were collected from the Ontario Institute for Cancer Research (OICR) Tumour Bank, tumour (n = 102) and adjacent non-cancerous tissues (n = 19) (Supplementary Table 1). Sample processing, RNA extractions, experiments, and data analysis were conducted as described above for plasma samples.
Blood Plasma Samples
Brandon University Ethical Committee (#21986) and the Biosafety Committee (2020-BIO-02) approved the project. Frozen human plasma samples were retrieved from the London Tumor Biobank (LTB), managed by the London Regional Cancer Program (LRCP). The LRCP collects matched human breast biopsy tissues and plasma samples from participants with written consent. From LTB, samples were transferred to Brandon University. The non-cancerous patients were aged 52 to 87, and breast cancer patients aged 36 to 91. Within breast cancer patients, there is only one stage 0 (n = 1), stage 1 (n = 30), stage 2 (n = 27), stage 3 (n = 5), and no stage IV sample. Patients were also broken down into hormone status; ER-positive (n-57), ER-negative (n = 7), PR positive (n = 54), PR negative (n = 10), HER2 positive (n = 14), HER2 negative (n = 46), HER2 equivocal (n = 4), and unknown (n = 1) (Supplementary Table 2).
Data Extracted from Databases: In silico analysis
The Enrichr online database was used to identify transcription factors resulting in a change in YWHAB expression (24). Predicted Targets of hsa-miR-526b and hsa-miR-655 were identified using TargetScanHuman V7.0 (25). YWHAB mRNA expression in breast cancer cell lines was obtained from the Gene Expression database of Normal and Tumor Tissues 2 (GENT2) database (26), and in patient cancer and healthy TCGA samples via cBioPortal (27) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) (28). YWHAB immunohistochemistry and survival data were accessed through the Human Protein Atlas (29). Liquid biopsy expression data for biomarker analysis was obtained from ExoRBase (30).
Receiver Operating Characteristic (ROC) Analysis
ROC analysis was used as the benchmark to evaluate the marker’s ability to classify two populations correctly; individuals with breast cancer and individuals in the control group - either benign samples in the case of blood plasma or non-adjacent disease-free tissue for tissue samples. For individual biomarker evaluation, RT-qPCR generated ΔCT values for each sample representing the relative expression of the chosen marker. The data was analyzed using GraphPad Prism 9.3.1.
When evaluating the combined effect of using two biomarkers, each sample was designated a predictive score through a binomial regression of the ΔCT values for each of the two markers. The binomial regression was done using the IBM SPSS V.28.0 statistics package. Then, GraphPad Prism was used to generate ROC statistics using the predictive scores of each sample as described above.
Statistical Analysis
All statistical data were collected using GraphPad Prism software used in t-tests to compare the mean of two groups and for Pearson correlation coefficient for correlation analysis. Differences were calculated with the relevance of p < 0.05 to determine statistical significance. Each data set was conducted with at least three biological replicates (N = 3) and three experimental replicates.