In vitro μελέτη επιγενετικών επιδράσεων σε μοντέλα καρκινικών κυτταρικών σειρών (Bachelor thesis)

Πανταζή, Χρυσούλα

The subject of the following dissertation is about the study of epigenetic actions of the agent demethylation 5-azacitidine on the HeLa cancerous cellular line, which de-rives from the human adenocarcinoma of the cervix, and evaluation of possible epige-netic alterations of the GATA3 gene on its genomic DNA (gDNA) and on the equiva-lent cell free DNA (ccfDNA). In that matter there is an effort to understand if the ep-igenetic phenomenon of methylation /demethylation which is present in the gDNA of the cells and is maintained in the corresponding ccfDNA after its release. Around the cancer of the cervix there a big scientific research interest, being the fourth most common type of cancer affecting women around the globe. The ccfDNA is a very promising and non-invasive biomarker. It is proven that the analysis of the ccfDNA of patients with cancer can attribute valuable information about its early diagnosis and the precise prognosis. Furthermore, the mechanism methylation of the DNA is one of the most common epigenetic phenomenon which take place on the mammal genome. It has been observed on cancerous cells, a deviant DNA methylation on the genes, which for that matter results in the loss of the protective role. The 5-azacitidine, which is a chemical equivalent of the nucleotide of the cytosine, found both in DNA and RNA, can be used in vitro for the removal of methylated groups from the DNA, thus weakening the impact of the gene silencing mechanism. On this particular study, at first we calculated in which grade of concentration the 5-azacitidine suspend for 50 % the survival of the HeLa (IC50 dosage) with the use of the MTT method. Afterwards we cultivated the HeLa cells for 24h, 48h and finally for 72h under the effect of the IC50 dosage of the 5-azacitidine, and at the end of every timeframe there was a col-lection of the supernatant and the cells. Continuing an extraction of the ccfDNA was conducted, from the supernatant and genomic DNA (gDNA) of the collected cells and was followed by elaboration of the DNA with the Bisulfite conversion. Lastly an analysis was conducted for the DNA methylation of the gene GATA3 with the use of PCR real time, specific for the methylation (Quantitative Methylation Specific PCR,-qMSP). According to the results of the MTT, regarding the survival of the HeLa after the effect of the 5-azacitidine, the IC50 was 3,52μΜ. The gene GATA3 was detect-ed, methylated on the genomic DNA of the cells and on the corresponding ccfDNA which was released, while after the effect of the 5-azacitidine no alteration of the methylation was observed on the genomic DNA, neither on the corresponding ccfDNA on all three incubation timeframes. Concluding, the DNA methylation is maintained on the ccfDNA after its release and imprints the profile of the genomic DNA of the cell. The ccfDNA could result in a valuable biomarker with clinical value on cancer, while the HeLa cells could be used as a reliable in vitro model for its study. Furthermore we did not observe the demethylation of the GATA3 after the effect with the 5-azacitidine, it is possible that higher concentration or bigger incubation timeframes could be proven necessary for the demethylation of the gene.
Institution and School/Department of submitter: Δημοκρίτειο Πανεπιστήμιο Θράκης. Σχολή Επιστημών Υγείας. Τμήμα Μοριακής Βιολογίας και Γενετικής
Subject classification: Epigenetics
Keywords: Epigenetics,Cancer,Cell culrure,Επιγενετική,Καρκίνος,κυτταροκαλλιέργεια
URI: https://repo.lib.duth.gr/jspui/handle/123456789/17197
http://dx.doi.org/10.26257/heal.duth.15931
Appears in Collections:ΤΜΗΜΑ ΜΟΡΙΑΚΗΣ ΒΙΟΛΟΓΙΑΣ & ΓΕΝΕΤΙΚΗΣ

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http://dx.doi.org/10.26257/heal.duth.15931
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