Uloga proteina ITIH5 u otpornosti na ciljanu terapiju vemurafenibom te proliferaciji i aktivnosti glavnih signalnih puteva staničnih linija melanoma čovjeka

master's thesis

• Title (english): The role of protein ITIH5 in resistance to vemurafenib targeted therapy, proliferation and activity of major signaling pathways in human melanoma cell lines
• Author: Doris Stepić
• Mentor: Ignacija Vlašić (mentor)
• Mentor: Nada Oršolić (komentor)
• Committee member: Nada Oršolić (predsjednik povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Committee member: Inga Urlić (član povjerenstva)
• Committee member: Duje Lisičić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-02-27, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Vemurafenib je selektivni inhibitor mutiranog proteina BRAF V600E koji se javlja u 60%
melanoma. Unatoč početnim obećavajućim rezultatima, ubrzo nakon liječenja
vemurafenibom dolazi do pojave stečene otpornosti stanica melanoma na ovaj lijek. Stečena
otpornost često je posljedica reaktivacije signalnog puta MAPK (engl. mitogen activated
protein kinase) ili aktivacije alternativnog signalnog puta PI3K/AKT (engl. phosphoinositide3-kinase/Akt). U mnogim je tumorima, uključujući melanom, uočena smanjena ekspresija
gena ITIH5. Unatoč tome, njegova uloga u stečenoj otpornosti stanica melanoma na
vemurafenib još nije istražena. Cilj ovog diplomskog rada bio je istražiti učinak utišavanja
ovoga gena na otpornost na vemurafenib, na signalne puteve MAPK i PI3K/AKT te na
proliferaciju staničnih linija melanoma čovjeka WM793B i A375M. Utišavanje gena ITIH5
provedeno je prolaznom transfekcijom stanica malim interferirajućim RNA koje ciljaju ITIH5
koristeći kalcijev fosfat. Provjera ekspresije i uspješnosti utišavanja provedena je
kvantitativnom polimeraznom lančanom reakcijom (qPCR). Nakon utišavanja ITIH5
provjerena je vijabilnost i proliferacija stanica metodom MTT te razina biljega aktivnosti
istraživanih signalnih puteva i proliferacije metodom Western blot. Navedene su analize
pokazale da tretman vemurafenibom znatno snižava ekspresiju gena ITIH5 te da snižena
ekspresija ITIH5 povećava proliferaciju u navedenim staničnim linijama melanoma čovjeka.

Abstract (english)

Vemurafenib is a selective inhibitor of the mutated protein BRAF V600E, which occurs in
60% of melanomas. Despite initial promising results, shortly after treatment with
vemurafenib, acquired resistance of melanoma cells develops. Acquired resistance is often
the result of the reactivation of the MAPK (mitogen activated protein kinase) signaling
pathway or the activation of the alternative PI3K/AKT (phosphoinositide-3-kinase/Akt)
signaling pathway. Decreased expression of the ITIH5 gene has been observed in many
tumors, including melanoma. However, its role in the acquired resistance of melanoma cells
to vemurafenib has not yet been investigated. The aim of this thesis was to investigate the
effect of ITIH5 silencing on the resistance to vemurafenib, on the activity of MAPK and
PI3K/AKT signaling pathways, and on the proliferation of human melanoma cell lines
WM793B and A375M. ITIH5 silencing was performed by transfecting cells with small
interfering RNA targeting ITIH5 using calcium phosphate. Verification of expression and
success of silencing was performed by quantitative polymerase chain reaction (qPCR). After
the silencing of ITIH5, cell viability and proliferation were checked using the MTT test,
while the levels of specific markers that represent the activity of signaling pathways or the
proliferation was checked using the Western blot method. The results showed that the
reduced expression of the ITIH5 increases the proliferation of the human melanoma cell lines
and is a consequence of vemurafenib treatment.

• Keywords: melanom; vemurafenib; otpornost; ITIH5; signalni putevi; proliferacija
• Keywords (english): melanoma; vemurafenib; resistance; ITIH5; signaling pathways; proliferation
• Language: croatian
• URN:NBN: urn:nbn:hr:217:672888
• 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: Embargoed access; Embargo expiration date: 2025-02-27
• Created on: 2023-03-28 11:04:09