Investigation of the relationship of wwox mediated miR-182 regulation with cisplatin resistance in breast cancer cells by CRISPR/CAS9 knockout method

The aim of the thesis project is to clarify whether cisplatin resistance developed in the TNBC cell line (MDA-MB-231) that does not express Wwox is due to miR-182 regulation. For this purpose, it was aimed to investigate the effect of cisplatin by knocking out miR-182, which is known to be an oncomiR, with the CRISPR method or knockdown with miRNA inhibitors, which we consider as an alternative method. For CRISPR knockout, which is not frequently used in miRNA knockout experiments but is frequently used in gene therapy studies today, guideRNAs specific to the miR-182 gene region were designed. No indel (insertion/deletion) was observed in the agarose gel, which would confirm knockout formation. In order to realize our aim, it was decided to knockdown using anti-miR (microRNA inhibitor), which we determined as an alternative method. Anti-miR182 inhibitor were transfected to MDA-MB-231 cells with lipofectamine RNAimax. It was shown that the miR-182 gene was knocked down by performing Real Time PCR with the cDNA synthesis. Apoptosis assay was performed by Annexin V. Analyzing the cells that were treated with anti-miR182 and cisplatin. Based on the data, 20.02% of the cells were in apoptosis and 20.10% were in late apoptosis. Analyzing the cells that were not treated with anti-miR182 and cisplatin. The data showed that 11.71% of cells were apoptosis and 18.37% of them are in late apoptosis. Then, cell cycle analysis studies were carried out by flow cytometry. Cells treated with miR-182 inhibitor and cisplatin had a 12.23% increase in G2 phase compared to MDA-MB-231 cells without cisplatin and anti-mir182 treatment. The results suggest that this link between cisplatin resistance and miR-182 in non-Wwox-expressing TNBC cells could be exploited in the development of a new gene therapy strategy for cisplatin resistance in breast cancer therapy.