Validation of the effects of miR-145-5p application on the expression of SMAD-3 and smad-4 target genes in breast cancer

In this study, miR-145-5p tumor suppressor miRNA specific synthetic mimic, which was determined by Meta-analysis in breast cancer, was transfected to prevent or delay the metastasis mechanism in breast cancer. In-vitro transfected into the target cell by "Hi-Perfect Reagent Transfection Agent" (miRNA replecement therapy). The change in expression of SMAD-3 and SMAD-4 gene products (mRNA) in the TGF-β signaling pathway that control cell proliferation, differentiation and cell death of transfected miRNA was determined by real-time quantitative PCR (RT-qPCR) analysis. Western Blot analysis was then performed to see changes in the protein level of these target genes. In addition, migration test was performed to show the effect of mimic on cellular metastasis. After the mimic application, migration was stopped according to the control in the cells. Also in the RT-qPCR analysis, the expression of SMAD-3 and SMAD-4 gene products decreased compared to control in mimic transfected cells. Finally, in Western blot analysis, the SMAD-3 protein level was determined by the control band and the SMAD-4 protein was reduced by 86% and the SMAD-4 protein was reduced by 100%. This supports the results of the RT-qPCR analysis. Keywords: Breast cancer, miRNA Replacement Therapy, miRNA, miR-145-5p, TGF-β, SMAD-3, SMAD-4, RT-qPCR, Western Blot, mimic transfection, Migration test.