Epigenetic Changes in The Genome of The Cellular Model of Colon Cancer Metastasis

Öz

In this study, identified variants of the colon cancer cell line were analyzed to reveal the cellular phenotypic pattern. The cell line at hand was analyzed phenotypically as epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT/MET), respectively. In the study, the tumor stroma structure, which was heavily exposed to IL6, was first induced by EMT. DLD1 colon cancer cell line was used in the study. After RNA and miRNA isolation, real-time PCR was applied to the cells and metastasis structure was examined. During the examination process, the induction process of the structures exposed to IL6 was observed. qPCR analysis of miRNA indicated in CHD1 cell types was performed and it was observed that SA and SPH cells showed significantly higher expression. In the study, fragments suitable for gene editing have been isolated from metastases with very low methylation in the cell structure. High expression was observed in mesenchymal-like cells associated with microRNAs (miR-17, miR-18a, miR-19). Demethylation of LncRNAs was observed in cells isolated as promoters in these metastatic processes. As a result, an increase in miRNA expression was observed in cells isolated from metastases due to the part of the DNA that did not undergo chemical change in the promoter region, and this result was found to be associated with the survival of colon cancer patients.

Anahtar Kelimeler

• miRNA
• epigenetic
• metastasis
• EMT

Destekleyen Kurum

Università degli Studi di Napoli Federico II

Etik Beyan

This study does not require ethics committee approval.

Teşekkür

I thank my supervisor Claudia Ciniglia for her contributions to the study.

Kolon Kanseri Metastazının Hücresel Modelinde Genomdaki Epigenetik Değişiklikler”

Öz

Öz Bu çalışmada, kolon kanseri hücre hattında tanımlanan varyantlar ayrıntılı biçimde incelenerek metastatik süreci yansıtan hücresel fenotipik örüntüler ortaya çıkarılmıştır. İncelenen hücre hattı, epitel-mezenkimal geçiş (EMT) ve mezenkimal-epitel geçiş (MET) süreçleri açısından kapsamlı bir fenotipik değerlendirmeye tabi tutulmuş; özellikle IL-6’ya yoğun şekilde maruz bırakılan tümör stromasında çalışmanın erken evrelerinde belirgin bir EMT indüksiyonu gözlenmiştir. Bu amaçla model olarak DLD1 kolon kanseri hücre hattı kullanılmıştır. RNA ve miRNA izolasyonlarının ardından hücrelere gerçek zamanlı PCR uygulanmış, böylece metastaza ilişkin moleküler ve yapısal değişiklikler ayrıntılı şekilde analiz edilmiştir. Değerlendirme sürecinde IL-6’ya maruz kalan hücresel yapılardaki indüksiyon dinamikleri takip edilmiş; CHD1 ile ilişkili hücre tiplerinde gerçekleştirilen qPCR analizleri, özellikle SA ve SPH hücre gruplarında çeşitli metastaz ilişkili miRNA’ların anlamlı düzeyde arttığını ortaya koymuştur. Ek olarak, metilasyon seviyesi oldukça düşük olan metastatik bölgelerden gen düzenlemesine uygun DNA fragmanları izole edilmiş ve mezenkimal fenotipe benzer hücrelerde miR-17, miR-18a ve miR-19 gibi mikroRNA’ların yüksek düzeyde eksprese edildiği belirlenmiştir. Metastatik süreçlerde promotör bölgesi olarak tanımlanan hücrelerde uzun kodlamayan RNA’ların (lncRNA) demetilasyona uğradığı saptanmıştır. Sonuç olarak, promotör bölgesinde kimyasal modifikasyona uğramayan DNA dizilerinin varlığına bağlı olarak metastazdan izole edilen hücrelerde artmış miRNA ekspresyonunun kolon kanseri hastalarının sağkalımı ile ilişkili olabileceği gösterilmiştir.

Anahtar Kelimeler

• miRNA
• epigenetik
• metastaz
• EMT

Destekleyen Kurum

Università degli Studi di Napoli Federico II

Etik Beyan

Bu çalışmada etik kurul onayına ihtiyaç yoktur.

Teşekkür

Çalışmaya katkılarından dolayı danışmanım Claudia Ciniglia'ya teşekkür ederim.

Kaynakça

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