Silenciamento do gene HOXB7 em linhagem de câncer de mama utilizando nanopartículas híbridas contendo siRNA e co-tratamento com tamoxifeno

Abstract

Cancer is the second leading cause of death in the world and accounted for approximately 9.6 million deaths in 2018. Among the cases of cancer in women, breast cancer is the second most common type and the one that kills the most in Brazil and in the world. Among the conventional treatments, the most common are surgery, radiotherapy, chemotherapy and hormone therapy. However, these treatments promote several side effects, and therefore, targeted therapies are emerging. Among them, we can mention interfering RNA therapy (RNAi) consisting of gene silencing from messenger RNA degradation (mRNA) of the target gene by the specific pairing of small interfering RNA molecules (siRNA). Among the key genes of importance in cancer, the HOXB7 gene acts on several metabolic pathways of hormone receptor-positive breast cancer, such as cell survival and proliferation, and also acts on resistance to drugs used in hormone therapy such as tamoxifen. However, for the silencing of such a gene by the effect of RNAi, the use of nanocarriers is required to transpose the biological barriers. The objective of this study was to perform the silencing of the HOXB7 gene in the human mammary cancer MCF7 strain using hybrid nanoparticles (NPs) carrying siRNA and the efficacy of co-treatment with the drug tamoxifen in subtoxic concentrations. First, calcium phosphate nanoparticles stabilized by block copolymers containing polyethylene glycol (PEG) were formed by self-association from a stoichiometric mixture of the constituents. The NPs had mean hydrodynamic diameters of 87.9nm ± 0.9 and 104.9nm ± 0.4 and polydispersity index (PdI) of 0.100 ± 0.009 and 0.123 ± 0.008 for NP-siHOXB7 and NP-mock (without siRNA). respectively, with rounded and homogeneous morphology. The Zeta potential of both NPs was close to the neutral, indicating the presence of the PEG polymer on its surface. The siRNA encapsulation efficiency was approximately 65%. The stability test showed that the NPs were stable for 7 days when stored at 4 ° C. There was no reduction of the cell viability of breast cancer cells after treatment with the hybrid NPs carrying siRNA against the HOXB7 gene in 24h. However, there was a reduction in viability (p <0.05) of the same cells from 48h with treatment of NPs carrying final siRNA concentration of 150nM. The synergism of co-treatment of NPs-siHOXB7 and TAM was observed as early as 15h. Also, from these tests it was possible to verify that the nanostructured system used showed little or no cytotoxicity, since there was no reduction of viability in the treatment with the mock nanoparticle. The 65% (p <0.05) silencing was observed, as well as the internalization of the nanoparticles by confocal microscopy. Thus, it can be concluded that the nanoparticles used are efficient in the transfection of MCF7 cells and that the gene silencing of HOXB7 using as a delivery system the hybrid nanoparticles combined with tamoxifen is shown as a promising option for the treatment of positive breast cancer for estrogen receptor.