Breast cancer (BC) is a heterogeneous disease, and establishing biomarkers is essential to patient management. We previously described that extracellular vesicle–derived miRNAs (EV-miRNAs) miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p in serum discriminated BC from control samples, either alone or combined in a panel. Using these previously described markers, we intend to evaluate whether the same markers identified in EVs are also potential biomarkers in tissue and serum. Expression analysis using RT-qPCR was performed using serum of 67 breast cancer patients (BC-S), 19 serum controls (CT), 83 fresh tumor tissues (BC-T), and 29 adjacent nontumor tissue samples (NT). In addition, analysis from The Cancer Genome Atlas (TCGA) data (832 BC-T and 136 NT) was performed. In all comparisons, we found concordant high expression levels of miR-320a and miR-4433b-5p in BC-S compared to CT in both EVs and cell-free miRNAs (cf-miRNAs). Although miR-150-5p and miR-142-5p were not found to be differentially expressed in serum, panels including these miRNAs improved sensitivity and specificity, supporting our previous findings in EVs. Fresh tissue and data from the TCGA database had, in most comparisons, an opposite behavior when compared to serum and EVs: lower levels of all miRNAs in BC-T than those in NT samples. TCGA analyses revealed reduced expression levels of miR-150-5p and miR-320a-3p in BC-T than those in NT samples and the overexpression of miR-142-5p in BC-T, unlike our RT-qPCR results from tissue in the Brazilian cohort. The fresh tissue analysis showed that all miRNAs individually could discriminate between BC-T and NT in the Brazilian cohort, with high sensitivity and sensibility. Furthermore, combining panels showed higher AUC values and improved sensitivity and specificity. In addition, lower levels of miR-320a-3p in serum were associated with poor overall survival in BC Brazilian patients. In summary, we observed that miR-320a and miR-4433b-5p distinguished BC from controls with high specificity and sensibility, regardless of the sample source. In addition, lower levels of miR-150-5p and higher levels of miR-142-5p were statistically significant biomarkers in tissue, according to TCGA. When combined in panels, all combinations could distinguish BC patients from controls. These results highlight a potential application of these miRNAs as BC biomarkers.

Introduction: MicroRNAs (miRNAs) are regulators of gene expression in biological processes, mainly repressing translation or degrading messenger RNA (mRNA) from their target genes. Their deregulation is associated with a wide variety of diseases, including cancer. Breast cancer (BC) is the most common cancer among women worldwide. Understanding the mechanisms involved in this pathology is essential for the discovery of new diagnostic and prognostic markers. Objectives: Investigate the differential expression of selected miRNAs in luminal A (LA) and triple-negative breast cancer (TNBC). Methods: We evaluated the expression of miR-320a, miR-4433b-5p, miR-142-5p, and miR-150-5p in 31 BC samples (19 LA and 12 TNBC) and 29 adjacent non-tumor breast cancer (NT). The miRNAs were selected after in silico study. RT-qPCR was the method of choice, using RNU48 as endogenous control, and the BT-474 cell line was used as a calibrator between plates. The 2−ΔΔCt method was used to estimate miRNA expression level. Individual receiver operating characteristic (ROC) curves were calculated based on RQ values to investigate the diagnostic value of miRNAs. Results: miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p were downregulated in BC compared to NT samples. All studied miRNAs were able to discriminate between BC and NT samples with sensitivity and sensibility (AUC – Area under curve >0.7 and p <0.05). A panel including all miRNAs improved the AUC to identify TNBC patients compared to NT (AUC=0.9240, sensitivity 94.44%, specificity 100%). We found no difference comparing miRNAs between LA and TNBC BC subtypes. There was no association between expression levels and prognostic parameters (age, histological grade, size of the tumor, axillary lymph node status). Conclusions: Our data suggest that downregulation of miR-142-5p, miR-150-5p, miR-320a, and miR-4433b-5p can be involved in BC for not repressing the expression of target genes. Functional studies will contribute to elucidate their involvement in BC.