Abstract 2534: The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line
Authors: Moreland Nicole L. Retland , Aloufi Abrar , Alyahyawi Areej , Shakoori Afnan , Aubee Joseph , Thompson Karl , Abbas Muneer 
Published: September 2020

Abstract
In the United States, African American women are twice as likely to be diagnosed with triple-negative breast cancer (TNBC) than Caucasian women. African American women experience the highest death rates when diagnosed with breast cancer (Bca). TNBC is the most aggressive subtype of BCa and difficult to treat. Studies have demonstrated the expression of serotonin receptors (5-HTRs) on BCa cells and reported their role in tumor progression. The dysregulation of miRNAs has been associated with oncogenesis. miRNAs play a critical role in biological processes such as cell proliferation and death not only through targeting cell-cycle components but also by extensively regulating multiple signaling pathways. In the context of cancer, miRNAs have been observed to function as either oncogenes or tumor suppressors in cancer development. 5-Hydroxytryptamine receptor 2A (5-HTR2A), a G coupled-protein serotonin receptor, was implicated in cancer progression. In this study, we determined the miRNA expression profile related to 5-HTR2A biological activity in a TNBC cell line derived from an African American woman.
The MDA-MB-468 cell line was cultured and an MTS assay performed to identify the effective concentration of (±)-DOI hydrochloride (5-HTR2A agonist) for treatment. Following treatment with 5 µM DOI, total RNA was isolated using Qiagen's AllPrep DNA/RNA Mini Kit following modified protocol for including miRNA. Total RNA was subjected to the NanoString human v3 miRNA kit targeting 800 endogenous miRNAs using color-coded probe sets. nSolver was used for differential miRNA expression and statistical analysis. For differential miRNA analysis DOI treated cells were compared with untreated MDA-MB 468 cells. The miRNA counts for each triplicate set were averaged. nSolver Analysis Software created by NanoString was used to determine fold change and statistical significance. Quality-control measures and normalization of data were also performed using the nSolver analysis package.
After identifying significant (p&lt;0.05) miRNAs and selecting those with a fold change greater than three, fourteen miRNAs were differentially expressed between the DOI treated and untreated BCa cell line (MDA-MB 468). The greatest differential expression observed in has-mir-205-5p. This oncogenic miRNA has been associated with cellular differentiation, migration, and proliferation. When comparing these fourteen miRNAs to the normal breast cell line (MCF-10a), only one miRNA (hsa-miR-221-3p), was found to be significant (p&lt;0.05). In DOI treated TNBC (MDA-MB 468), hsa-miR-221-3p was downregulated by 3.44 and 1.81 compared to untreated TNBC (MDA-MB 468) and normal (MCF-10a), respectively. Reportedly, hsa-miR-221-3p (an oncogenic miRNA) is associated with TNBC and acts downstream of the oncogenic RAS-RAF-MAPK pathway.
This study elucidates miRNAs contributing to TNBC development and progression in correlation with 5-HTR2A activity in vitro. Specifically, two oncogenic miRNAs were identified, has-mir-205-5p and hsa-miR-221-3p. Although their involvement in breast cancer progression has been studied, has-mir-205-5p and hsa-miR-221-3p should be validated as a consequence of 5-HTR2A biological activity. These miRNAS may be used as prognostic biomarkers in breast cancer management.
Citation Format: Nicole L. Retland Moreland, Abrar Aloufi, Areej Alyahyawi, Afnan Shakoori, Joseph Aubee, Karl Thompson, Muneer Abbas. The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2534.