Investigation of molecular changes occurring in adaptive immune system in breast tumors at miRNA level

Breast cancer, the most common type of cancer in women worldwide, is a complex and heterogeneous disease. The triple-negative breast cancer subtype accounts for approximately 15% of breast cancers and is still the most aggressive and poorly prognostic breast cancer subtype, despite the availability of systemic treatment options such as chemotherapy, PARP inhibitors, and immune checkpoint inhibitors. Recent studies on the relation between breast cancer and the immune system have indicated that breast cancer is not an immunologically silent cancer type and can be defined immunologically on the basis of subtypes. It is emphasized that the immune response against the tumor arises but is suppressed by various mechanisms in the triple negative breast cancer subtype. Non-coding small RNA molecules (microRNA or miRNA) play a regulatory role in various cellular pathways through the regulation of multiple target genes in the immune system cells. Although there are various studies on the relation between solid tumors, including breast cancer, and the immune system, miRNA profiling studies in breast cancer immunology in terms of immune cells are very limited. In this study, miRNA profiles of CD8+ T lymphocytes, which are the most effective immune system cell group in destroying tumor cells, were determined by microarray analysis to examine the changes in the immune system during the formation and development of triple negative breast cancer in a mouse model. The miRNA expression profiles were determined in four groups consisting of control, TNBC formation (primary tumor), regression and recurrence. It was found that mmu-miR-93-3p, mmu-miR-3470b, and mmu-miR-30b-3p were common significant differential expressed miRNAs. After the validation of these miRNAs by qPCR analysis, it was confirmed that their expressions increased especially in primary tumor and relapse groups while their expressions were low in the control and remission groups. miRNA target genes and enrichment analysis indicated that CD8+ T lymphocytes regulate the apoptosis, especially through targeting BCL-2 and BCL-XL anti-apoptotic genes. As a result of qPCR and Annexin-V experiments, the miRNA target genes BCL-2 and BCL-XL were decreased in CD8+ T cells in primary tumor group, and CD8+ T cells were removed by apoptosis in primary tumor group. In addition, it was shown that the expression of target genes increased, and the apoptosis process was suppressed in the remission group. In conclusion, mmu-miR-93 3p, mmu-miR-3470b and mmu-miR-30b-3p may play a role in the immune escape of tumor cells in the early and advanced stages of triple negative breast cancer through apoptosis and have the potential to be immunological biomarkers for CD8+ T cell response against tumor cells. Keywords: Triple negative breast cancer, Adaptive immune system, CD8+ T lymphocyte, miRNA, microarray