Povezanost metilacije promotora s ekspresijom gena BRCA1 i BRCA2 u seroznom raku jajnika visokog stupnja malignosti

master's thesis

• Title (english): Association between promoter methylation and BRCA1 and BRCA2 gene expression in high-grade serous ovarian cancer
• Author: Tina Miloš
• Mentor: Petar Ozretić (mentor)
• Mentor: Nada Oršolić (sumentor)
• Committee member: Nada Oršolić (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Romana Gračan (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-07-07, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Serozni rak jajnika visokog stupnja malignosti (HGSOC) karakterizira visoka stopa smrtnosti budući da se, zbog izostanka jasnih simptoma i nedostatka testova za ranu detekciju, često otkriva u uznapredovanoj fazi bolesti. Cilj ovog rada je istražiti povezanost metilacije promotora gena BRCA1 i BRCA2 s njihovom ekspresijom u tkivima HGSOC kako bi se omogućilo stvaranje novih dijagnostičkih i terapijskih pristupa. Uzorci HGSOC i zdravih jajovoda su analizirana metodom kvantitativne lančane reakcije polimerazom u stvarnom vremenu (qRT-PCR) kako bi se odredila ekspresija gena BRCA1 i BRCA2, dok je stupanj metilacije njihovih promotora određen metodom analize krivulje mekšanja visoke rezolucije osjetljive na metilaciju (MS-HRM). Naposljetku, stanična linija OVCAR-3 je tretirana demetilirajućim spojem, 5-aza-2′-deoksicitidinom, kako bi se istražio utjecaj globalne demetilacije DNA na razinu ekspresije gena BRCA1 i BRCA2. Rezultati su pokazali da je ekspresija gena, osobito BRCA2, značajno pojačana u raku jajnika te da su ekspresije gena BRCA1 i BRCA2 međusobno u korelaciji što ukazuje da je regulacija njihove ekspresije međusobno povezana. Metodom MS-HRM utvrđeno je da je u određenom broju uzoraka prisutna hipermetilacija gena BRCA1, dok je BRCA2 pokazao potpunu hipometilaciju. Tretman stanica 5-aza-2′-deoksicitidinom utjecao je na pojačanu ekspresiju gena BRCA1 i BRCA2, a stupanj metilacije promotora gena BRCA1 pokazao se nižim u odnosu na netretirane stanice.

Abstract (english)

High-grade serous ovarian cancer (HGSOC) is often diagnosed in its late stage due to the absence of symptoms and a general lack of early detection tests, resulting in a high mortality rate. The goal of this work was to study the relationship between BRCA1 and BRCA2 gene expression and their promoters’ methylation in HGSOC to understand the molecular mechanisms underlying its occurrence that would aid in the development of new diagnostic and therapeutic approaches. Healthy and cancerous tissue samples were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) to determine the BRCA1 and BRCA2 gene expression. Methylation-sensitive high-resolution melting (MS-HRM) was employed to determine the level of BRCA1 and BRCA2 promoter methylation. Finally, OVCAR-3 cells were treated with demethylating agent, 5-aza-2′-deoxycytidine, to study the effect of DNA demethylation on BRCA1 and BRCA2 gene expression. The results presented in this work demonstrate that the expression of both genes, especially of BRCA2, is significantly increased and corelated in cancerous tissues indicating their common expression regulation. MS-HRM analysis showed that BRCA1 promotor is hypermethylated in cancerous tissues while BRCA2 promotor exhibited complete hypomethylation. The 5-aza-2′-deoxycytidine treatment of OVCAR-3 cells induced reduced methylation of BRCA1 promoter and increased expression of both BRCA1 and BRCA2 genes.

• Keywords: HGSOC; BRCA1; BRCA2; metilacija; MS-HRM; homologna rekombinacija
• Keywords (english): HGSOC; BRCA1; BRCA2; methylation; MS-HRM; homologous recombination
• Language: croatian
• URN:NBN: urn:nbn:hr:217:472113
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-07-07
• Created on: 2021-07-22 12:50:26