Unveiling the Antitumor Potential of miR-224-3p and miR-224-5p in Triple-Negative Breast Cancer

Breast cancer is the most commonly diagnosed cancer type worldwide and the second cause of cancer-related death in women. Among its subtypes, triple-negative breast cancer (TNBC) represents the most aggressive form, accounting for about 15-20% of all cases. TNBC is characterized by the absence of estrogen (ER), progesterone (PR) and human epidermal growth factor receptor-2 (HER2), making it unresponsive to hormone or HER2-targeted therapies. As a result, TNBC is associated with poor prognosis and a high incidence of early metastasis. Several oncogenic drivers such as KRAS, FOXM1 and eEF2K and the immune checkpoint protein PD-L1 are frequently overexpressed in TNBC, contributing to tumor progression, therapy resistance, and immune evasion, and thus representing potential molecular targets. To date, radio- and chemotherapy in combination with surgery remain the mainstay of systemic treatment. However, the high recurrence rate (30-40%) within 3 years highlights the importance of developing innovative targeted therapies. MicroRNAs (miRNAs) are small natural non-coding RNA molecules, 20-25 nucleotides long, that regulate gene expression and key cellular processes. In the final stage of miRNA biogenesis, pre-miRNA matures into two strands: the 3-prime (3p) and the 5-prime (5p). In recent years, an increasing number of studies have highlighted the crucial role of dysregulated miRNAs in various cancer pathophysiological processes, such as uncontrolled cell proliferation, migration, invasion, drug resistance and tumor growth and progression. Due to their ability to degrade target messenger RNA (mRNA) or inhibit translation, strategies to restore tumor-suppressor miRNAs or inhibit oncogenic miRNAs represent significant potential for novel targeted therapies. Among the large number of existing miRNAs, expression profiling revealed that the miR-224-5p is significantly downregulated in TNBC tissues compared to healthy counterparts. Moreover, survival analysis correlated low levels of miR-224 with poor patient outcomes. These initial insights suggest that miR-224 may act as tumor suppressor and give hope as a promising therapeutic agent. This project aims to investigate and compare the roles of miR-224-3p and miR-224-5p in the context of TNBC. We will examine their impact on cell proliferation, viability, migration and invasion. Finally, we will evaluate their ability to suppress key oncogenic molecular targets. In vitro, both miR-224-3p and miR-224-5p inhibited proliferation, migration and invasion in various TNBC cell lines including MDA-MB-231, MDA-MB-436 and BT-549. Moreover, miR-224 treatments led to an increase in intracellular reactive oxygen species (ROS) levels, along with a higher number of dead cells undergoing both apoptosis and ferroptosis. Notably, protein expression analysis identified miR-224-3p and -5p as potential regulators of KRAS, FOXM1, eEF2K and PD-L1. These regulatory effects were confirmed by Western blot analysis, which showed significant downregulation of these proteins. In conclusion, the findings of this study highlight the dual therapeutic potential of miR-224-3p and miR-224-5p in TNBC and its clinical significance in patient survival and prognosis. Reintroduction of miR-224 to TNBC cells can suppress hallmark features of cancer such as uncontrolled proliferation, invasiveness and resistance to cell death, while also modulating immune escape mechanisms. These insights support further development of miRNA-based interventions as a promising strategy for treating TNBC.