Exploring the Potential mRNA Targets of miR-2115-3p in Molecular Apocrine Breast Cancer

Abstract

Background: Breast cancer (BC) is the most prevalent cancer in women with around 2.3 million new cases reported on a yearly basis worldwide. Despite significant research efforts, BC incidence and mortality rates continue to rise. BC is characterized by substantial intra- and intertumoral heterogeneity, complicating diagnosis and treatment. One method of subtyping BC is based on the expression of three receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Triple-negative breast cancer (TNBC), an aggressive type of BC lacking expression of all three of these receptors, is itself a heterogeneous group overlapping with distinct subtypes such as molecular apocrine (MA) and luminal androgen receptor (LAR) BCs. These subtypes remain misdiagnosed and poorly treated. To better understand MABC, the discovery of underlying mechanisms that allow its development and tumorigenesis is required. Recent studies have highlighted the roles of microRNAs (miRNAs) which are small non-coding RNA molecules, in normal physiological processes and more importantly in the pathogenesis of various diseases, including BC. According to in-silico analyses and preliminary data from TNBC tumors, miR-2115-3p is a significantly upregulated miRNA in MABC. miR-2115-3p is a novel miRNA with limited reports linking it to diseases, making it a promising focus for study. The aim of this study was to identify and investigate potential mRNA targets of miR-2115-3p in MABC using in vitro BC cellular models.

Methods and results: Breast cancer cell lines representative of MABC (MDA-MB-453) were cultured under standard conditions and transfected with either miR-2115-3p mimics or inhibitors, along with their respective negative controls (NC) using Lipofectamine-based transfection methods. The transfection efficiency was assessed using the GUAVA flow cytometry and this showed significant average transfection efficiency with both inhibitors (93%) and mimics (91%), and their respective NC. After evaluating the transfection efficiency by flow cytometry, total RNA was extracted using TRIzol reagent, followed by complementary DNA synthesis performed using reverse transcription kits. Reverse-Transcriptase Quantitative PCR (RT-qPCR) was used to quantify the expression of miR-2115-3p inhibitors and mimics in modulated MDA-MB-453 cells, relative to their corresponding NC. RNU6B was used as the endogenous control. Moreover, data from our laboratory showed an increased expression of miR-2115-3p in MABC compared to TNBC; therefore, to select target genes, we used RNA-sequencing done on TNBC cells (MDA-MB-231) transfected with miR-2115-3p mimics. RT-qPCR was then conducted to evaluate the expression levels of the selected candidate target genes along with the endogenous housekeeping genes (TBP) used to normalize the data. Comparative analyses were performed between treated and control groups to identify differentially expressed genes potentially regulated by miR-2115-3p. As a result, BLMH, LRP12, and C8ORF33, were identified as potential target genes.

Conclusion: The findings of this project will help in identifying the functional role of miR-2115-3p in MABC. Identifying the mRNA targets of miR-2115-3p will enhance understanding of MABC tumorigenesis and may reveal novel biomarkers or therapeutic targets to improve clinical outcomes.