Estudo do efeito do silenciamento do gene ITGB3 em células tumorais de mama

Abstract

Breast cancer is the major disease that affects women, especially the triple negative phenotype, which has the worst prognosis and metastatic potential. Included, metastasis is still the main cause of death in cancer patients and its development involves several stages and characters that play important roles, such as the tumor microenvironment and the extracellular matrix (ECM). Integrins also contribute to metastasis, especially the αvβ3 integrin whose overexpression is associated with every step in tumor progression. Among the molecules that interact with integrins, there are disintegrins, which are small proteins derived from snake venom metalloproteases. DisBa-01 (Disintegrin of Bothrops alternatus-01) is a recombinant protein derived from the Bothrops alternatus venom (urutu-cruzeiro) snake, which has a high affinity for this integrin. Thus, this work aimed to study the effects of silencing the β3 subunit in tumor cells of the triple negative breast adenocarcinoma cell line (MDA-MB-231). Therefore, the methods adopted involved the cell culture of human kidney embryonic (293FT) and wild and modified triple negative breast adenocarcinoma (MDA-MB-231) cell lines; production of ITGB3 shRNA lentivirus as well as target cell transduction and the validation of silencing by RTqPCR and immunofluorescence. We also verified possible alterations in the expression of other integrin subunits; functional assays (inhibition of adhesion, migration by wound healing and transwell), and the identification of metalloproteases (MMP) (MMP-2 and MMP-9). We compared the cellular responses of the silenced lineage with the action of DisBa-01 (1000 nM). The silencing took to a 69% reduction in ITGB3 mRNA and a 49% reduction in FN rich microenvironment, with an increase in β1, α1 and α5 subunits; increased adherence to FN (58%) and COL I (83%) proteins; decreased transwell migration (69% inhibition) and MMP-9, responses different from those obtained with DisBa-01. The generation of this new cell line opens perspectives for its use as a tool in future studies of the basic biology of breast cancer.