Ação antiangiogênica da melatonina pela modulação do supressor tumoral miR-152-3p em linhagens de câncer de mama

Abstract

Breast cancer represents the second type of tumor that has the highest mortality rates, being the most common among women. The causes of these high mortality rates are related to high proliferation and metastasis, and for tumor progression, the growth of new blood vessels, angiogenesis is required. This event can be stimulated by several factors, such as insulin-like growth factor 1 receptor (IGF-1R), hypoxia-inducible factor 1-alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Several molecules are involved in the control of angiogenesis such as melatonin and microRNAs (miRNAs). The miRNAs can induce the gene silencing of genes related to angiogenesis by pairing with certain specific messenger RNA (mRNA), resulting in the degradation of this molecule. Melatonin (N-acetyl-5-methoxytryptamine), the main hormone produced and secreted by the pineal gland, has several physiological functions and a proven antiangiogenesis action. This hormone can regulate miRNAs and genes related to this process. Objective: To evaluate the ability of melatonin to modulate miR-152-3p and its targets in triple-negative breast cancer cells. Material and Method: After the determination of the melatonin concentration to be used, the differential expression of the miRNAs in the MDA-MB-468 strain after the melatonin treatment was evaluated using the plate RT² Profiler™ PCR Array Human Breast Cancer containing 84 miRNAs related to breast cancer. An in-silico analysis was performed to select a miRNA involved in angiogenesis and its potential target genes. Overexpression of miR-152-3p was performed on MDA-MB-468 and MDA-MB-231 cells by transient transfection and after relative quantification of their expression and their target genes IGF-1R, VEGF and HIF-1α was evaluated by real-time PCR. Quantification of the protein expression of the genes was verified by immunocytochemistry. Results: The cell viability assay in the MDA-MB-468 cell line demonstrated that cells treated with 1 mM melatonin had the lowest viability (p <0.05). Analysis of the miRNAs by PCR Array in the MDA-MB-468 cell line showed six positively regulated miRNAs and seven negatively regulated miRNAs after treatment with melatonin. Evaluation of gene expression demonstrated that miR-152-3p overexpression was influenced by melatonin, leading to increased expression of the genes IGF-1R, HIF-1α and VEGF in MDA-MB-468 cells. In the MDA-MB-231 cell line, melatonin did not influence the expression of miR-152-3p and decreased the expression of the target genes. Finally, immunocytochemistry revealed that melatonin and overexpression of miR-152-3p were able to decrease the protein expression of IGF-1R, HIF-1α and VEGF in the MDA-MB-468 and MDA-MB-231 cells. Conclusions: Melatonin was able to modulate the expression of miR-152-3p and its target genes involved in angiogenesis in triple-negative breast cancer. Therefore, this study confirms the action of melatonin on the important cellular event of angiogenesis, a determinant process for the progression of the disease and also indicates it as a potential therapeutic protocol for triple-negative breast cancer.