Molecular mechanisms involved in the regulation of certain miRNAs in breast cancer cells and cancer-associated fibroblasts

Abstract

MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate gene expression by inhibiting the translation of target mRNAs. It has been well demonstrated that miRNAs play a regulatory role in different pathophysiological conditions, including breast cancer. In this regard, a distinct miRNA may be up regulated in certain malignancies and downregulated in others, suggesting both an oncogenic and tumor suppressor role, respectively. It has also reported that in breast cancer estrogens may regulate miRNA expression through the involvement of the classic estrogen receptor (ER) alpha and the G protein coupled receptor, GPER. By using RT-PCR and western blotting assays, as well as by gene silencing approaches, in the present work we provide novel insights on the involvement of miRNAs in the breast tumor progression. In particular, we ascertained that miR-221 expression is up-regulated in breast cancer cells SkBr3 and MDA-MB 231 and in cancer-associated fibroblasts (CAFs). In addition, we found that the proliferative and migratory effects induced by miR-221 in these cell types are mediated by interfering the A20/c-Rel/CTGF pathway. These stimulatory effects are abolished silencing the expression of the NF-κB component, c-Rel and the Connective Tissue Growth Factor (CTGF), as well as using the specific Locked Nucleic Acid (LNA)-Inhibitor of miR-221 (LNA-i-miR-221). In a second work, we evaluated the expression of 754 miRNAs by TaqMan Array in ER-negative and GPER-positive SkBr3 breast cancer cells and in CAFs upon 17-β-estradiol (E2) treatment. Among the miRNAs down-regulated by 100nM E2 for 4 hours in these cell types, we identified miR-338-3p that is positively correlated with overall survival in breast cancer patients as ascertained by METABRIC database analysis. According to these data, a miR-338-3p mimic sequence decreased and a miR-338-3p inhibitor sequence rescued the proliferative effects and cell progression induced by 100nM E2 and G-1, selective ligand of GPER in SkBr3 breast cancer cells and CAFs. Overall, these results provide new insights on the miRNA action in breast cancer, suggesting their use as an innovative therapeutic approach in this malignancy.