Ação antimetastática da melatonina pela modulação de microRNAs candidatos em linhagens de câncer de mama

Abstract

Breast cancer presents high incidence and death rates worldwide. Metastasis is one of the main causes of high mortality rates by this neoplasm. MicroRNAs (miRNAs) are small non-coding RNA molecules with nearly 18-22 nucleotides, involved in gene expression regulation. Studies demonstrate the role this molecules in tumor progression, including breast cancer. Melatonin, a hormone secreted mainly in pineal gland, has been presenting several effects in cancer regulation, acting on the modulation of proteins and miRNAs. Aim: The aim of the present study was to investigate the action of melatonin on miRNA-10a-5p modulation in triple-negative breast cancer cells (TNBC), and associating with tumor progression. Materials and Methods: The MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide) assay was performed to verify cell viability of MDA-MB-468 cells at different concentrations of melatonin for 24 hours. The differential expression of 84 miRNAs associated with breast cancer was verified by PCR-Array. The miR-10a was selected for its involvement with tumor process and its gene expression and its target HOXD10 were verified by real-time PCR in breast cancer cell lines. The following groups were considered: cells treated or not with melatonin; transfected with inhibition of miR-10a or negative control of reaction. The invasion and migration assay using matrigel inserts was performed and protein expression of epithelial-mesenchymal transition (EMT), Ecadherin, claudin 7 and vimentin markers were investigated, as PIK3CA proliferation marker, by Western blotting. Results: Our results showed that 1 mM melatonin significantly decreased cell viability. The results of PCR Array showed thirteen miRNAs modulated by melatonin (six overexpressed and seven inhibited). The gene expression showed a decrease in relative amount of miR-10a and its target HOXD10 after melatonin treatmet. The matrigel assays showed that melatonin and inhibition of miR-10a decreased the cell invasion and migration. According to the protein analysis, melatonin was able to reduce the expression of vimentin and claudin 7 proteins and increase E-cadherin expression. On the other hand, inhibition of miR-10a reduced the vimentin protein and did not modulate claudin 7 and E-cadherin. Conclusion: In this study was verified the ability of melatonin modulating miRNAs expression by decreasing miR-10a, affecting tumor invasion and migration, and proteins involved with EMT. In this way, we support the idea of the potential role of melatonin in the regulation of metastasis.