Radiation-mediated effect on exosomal and non-exosomal-derived microRNA-21 (miR21) gene expression by Triple Negative Breast Cancer cell line MDA-MB-231

Background: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype which lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 receptor (HER2). TNBC is therefore not responsive to hormonal treatment and currently lacks targeted therapies. Overexpression of miR21 is routinely observed with TNBC and correlates with advanced tumor stage and lymph node metastasis. MDA-MB-231 is a metastatic human TNBC cell line with high recurrence rate via metastasis to secondary sites. Specialized extracellular vesicles called exosomes are involved in intercellular communication and have been postulated to have roles in tumor metastasis. Objective: In this study, we evaluate the effect of high-dose radiation on viability of MDA-MB-231 and identify changes in miR21 expression in cells and exosomes released in response to ionizing radiation. Methods: MDA-MB-231 cells (obtained from American Type Culture Collection) were cultured and irradiated with single dose exposure to 8.6 Gy (low dose) and 17.2 Gy (high dose). At 24h post-irradiation, cells were assessed for viability, proliferation, and wound healing. Exosomes were isolated from culture medium at 48h post-irradiation using differential ultracentrifugation method and evaluated for size and purity. Western blot confirmed isolation of exosomes by determining expression of established exosome membrane protein markers CD81 and TSG101. RT-qPCR evaluated expression of miR21 in cells and exosomes. Results: High-dose (17.2 Gy) radiation suppressed MDA-MB-231 proliferation based on MTT and wound healing assays. MDA-MB-231 cells exposed to 8.6 Gy showed marked upregulation of cellular miR21 and relative downregulation of exosomal miR21; exposure to 17.2 Gy resulted in downregulation of both cellular and exosomal miR21 relative to the control. Conclusion: This pilot study demonstrated that tumor cells may display compartmental differential expression of miRNA in response to radiation and suggests that miRNA expression in cells may not be predictive of exosomal cargo.