Silenciamento do gene itgb3 de células de tumor de mama murino triplo negativo (4T1BM2)

Abstract

Breast cancer is the most recurrent type of tumor in the world, surpassing lung cancer, according to data published in 2021 by the World Health Organization (WHO). When tumor cells spread or invade nearby or distant tissues, secondary tumors are formed and this process is called metastasis. αvβ3 integrin are heterodimeric receptors that connect cells to the extracellular matrix (ECM), they are crucial due to their role in signaling that contributes to cell motility, proliferation and cell behavior, being a therapeutic target of great relevance. The main objective of the present work was the production of a murine breast cancer cell line (4T1BM2) silenced for αvβ3 integrin and the analysis of the effect of this silencing. To obtain this silencing, lentiviral vectors produced from envelope and packaging plasmids were used together with the plasmid encoding the synthesis of interference RNA, which targets part of the β3 subunit gene, to be used in transfection. from the human kidney embryonic cell line (293FT). The viral particles were transduced into the tumor cell and the selection of silenced cells was performed using the antibiotic Puromycin. The verification of partial silencing of β3 integrin was obtained by western blotting and flow cytometry, certifying different levels of integrin in the parental and silenced cells. The evaluation of cellular behavior was carried out through functional migration assays in Boyden's chamber and wound healing. In addition, zymography assays were performed to compare the content of matrix metalloproteinases (MMPs) present in the parental cells and in the silenced cells. The results obtained in this work indicate the achievement of a silencing for the β3 subunit and the functional assays showed a lower migration rate of the silenced cells, as well as a decrease in MMP-9 and an increase in MMP-2 in these cells. These data contribute to improvements in knockdown procedures for this cell type and demonstrate the complex role of integrins in tumor progression.