Apc Mutant Fare İntestinal Organoidlerinde Wnt Yolak Modülasyonunun Kök Hücreler Üzerine Etkilerinin Araştırılması

Abstract

One of the most important causes of colorectal cancer development is mutations of genes involved in the WNT signaling pathway. In the vast majority of cases, APC tumor suppressor gene mutations, which act as negative regulators of the WNT signaling pathway degradation complex, are encountered. In intestinal epithelial cells, which have a very rapid proliferation and regeneration process, the WNT signaling pathway shows high activity and takes an active role in the regulation of intestinal stem cells and tissue homeostasis. Most of the mutations of the APC gene occur at the AXIN binding site, which is another element of the degradation complex. Due to various mutations in the components of the destruction complex, it cannot fully function its task. When the β-catenin protein cannot be proteosomally degraded in the cytoplasmic region by the degradation complex, it translocates to the nucleus and thus increases the transcriptional activity of WNT target genes, which are involved in the regulation of the TCF/LEF transcription factor. In this thesis study, intestinal organoids produced from mutant ApcΔ750 mouse small intestine tissues specific to our laboratory were investigated at the RNA level of WNT-related genes Ctnnb1, Axin2 and Lgr5. As a result of the analysis, it was proved that the expression of these genes is high in ApcΔ750 intestinal organoids. As the primary aim of the thesis study, it has been shown that Ctnnb1, Axin2 and Lgr5 gene expressions are decreased at the RNA level when XAV939 and Niclosamide drugs, which are known to have anti-cancer effects, are applied to ApcΔ750 organoids. In addition, the level of Ctnnb1 protein in the crypt and villus domains was examined by applying the immunofluorescent staining technique. Thus, it was observed that XAV939 and niclosamide inhibited the Wnt signaling pathway in organoids that are precursors of CRC formation, and thus suppressed ICH activation. A secondary aim of the study is to show that the ApcΔ750 mouse model produced in our laboratory is also successful in in vitro studies.