Silenciamento de metaloproteinases de matriz 9 e 13 por RNA de interferência em modelo de câncer de mama murino

Abstract

Cancer encompasses a set of diseases characterized by abnormal cell growth and invasion in organs and tissues that can spread to other parts of the body. It is currently considered a worldwide public health problem with high mortality rates. Breast cancer is the second most prevalent type of cancer in women with high mortality rates due to metastases. Proteins involved in the process of tumor progression have been targeted by therapeutic approaches. In this context, matrix metalloproteinases (MMPs) are genes that are overexpressed in several types of cancers and act on tumor progression through the proteolysis of components of the basement membrane and extracellular matrix, acting on the invasion and metastasis of tumor cells. Current therapies such as hormonal, chemotherapy, radiotherapy and immunotherapy have not been fully effective, making it necessary to seek new therapeutic approaches. In this context, the silencing of superexpressed genes in tumors is an alternative. The use of interfering RNAs (siRNA) has been described as a potential method for silencing, but the free siRNA molecules offer some limitations, such as the difficulty to enter passively into the cells and their molecular weight, being necessary the use of nanocarreadores that this molecule to the target cell. In view of this, the main objective was to use hybrid calcium phosphate nanoparticles for delivery of siRNA in vitro to 4T1 metastatic breast cancer cells to silence the MMPs genes 9 and 13. The results obtained showed that the siRNA sequences constructed at functional criteria were adequate for the proposed use. In addition, the hybrid nanoparticles used are suitable size for delivery of siRNA to target tumor cells and efficiency of incorporation of siRNA into nanoparticles of approximately 80%. The cytotoxic capacity (MTT and Tripan Blue assays) of the nanoparticles and siRNAs in 4T1 cells was evaluated and there was no significant difference in cell viability. Next, we evaluated whether the nanostructured complex was effective by reducing the expression of the target genes. We obtained a satisfactory silencing that allowed us to analyze whether silencing MMP9 and MMP13 would interfere with fundamental stages of tumor progression, such as cell proliferation (clonogenic assay) and cell migration (wound healing and transwell assay). The results obtained corroborated the literature data showing MMP9 and MMP13 involved in the migration process of tumor cells in breast cancer. Therefore, we did not obtain differences in terms of cell proliferation with the silencing of MMPs 9 and 13 but there was a significant reduction in the migration processes of tumor cells with silencing. In conclusion, MMPs 9 and 13 are involved in the migration of breast cancer cells and the silencing by RNAi has shown promise for new therapeutic approaches and for the better understanding on the metastases in breast cancer.