Konstrukcija vektora za ekspresiju proteina SHH u staničnoj liniji melanoma čovjeka CHL-1

master's thesis

• Title (english): Construction of vectors for SHH protein expression in CHL-1 human melanoma cell line
• Author: Josipa Čonkaš
• Mentor: Maja Sabol (mentor)
• Mentor: Sofia Ana Blažević (sumentor)
• Committee member: Sofia Ana Blažević (predsjednik povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Committee member: Tomislav Ivanković (član povjerenstva)
• Committee member: Maja Matulić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; Zagreb
• Defense date and country: 2021-01-25, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES

Abstract

U današnje vrijeme, melanom se smatra jednim od najsmrtonosnijih tipova tumora sa sve većom učestalosti u populaciji. Uslijed izuzetno složene patogeneze bolesti, koja je povezana sa promjenama u jednom ili više signalnih puteva, istraživanja se usmjeravaju prema razvoju kombiniranih inhibitora kao potencijalne terapije. U ovom istraživanju dizajniran je vektor za prolaznu i stabilnu ekspresiju proteina SHH u staničnoj liniji melanoma čovjeka CHL-1. Aktivni oblik proteina SHH djeluje kao ligand koji se veže za transmembranski receptor Patched i pokreće signalni put Hedgehog-Gli. Ključna uloga signalnog puta HH-GLI je regulacija embriogeneze, dok se poremećena funkcija istog povezuje s inicijacijom, progresijom, invazijom i preživljenjem tumora. Kloniranjem dizajniranog sintetskog genskog fragmenta gBlock SHH u ekspresijski vektor pcDNA3 uspješno je eksprimiran aktivni oblik proteina SHH-N. Westernskom metodom otiska dokazano je da prolazna transfekcija stanične linije melanoma CHL-1 vektorom pcDNA3.1 - SHH dovodi do povećanja ekspresije gena SHH i autokrine aktivacije signalnog puta HH-GLI ovisne o ligandu, što potvrđuje povećana proteinska ekspresija SHH-N, PTCH1, transkripcijskog faktora GLI1 i GLI3R te smanjenje razine ekspresije proteina GLI3FL, 48 sati nakon transfekcije. Isto tako, ekspresija proteina SHH stimulirala je aktivaciju signalnih puteva PI3K i MAPK, no nije pokazala učinak na regulaciju staničnog ciklusa. Važnost dokazane interakcije je izuzetno velika i predstavlja temelj razvoja modela za istraživanje terapija u liječenju melanoma.

Abstract (english)

Malignant melanoma is one of the most aggressive cancers nowadays with increasing incidence in the population. The focus of research today is on the development of combined inhibitors as potential therapy due to the extremely complex pathogenesis of the disease associated with mutations in one or a few signaling pathways. In this study, we designed vectors for transient and stable SHH protein expression in the CHL-1 human melanoma cell line. The active form of the SHH protein acts as a ligand that binds to the Patched transmembrane receptor and triggers the Hedgehog-Gli signaling pathway. The main role of the HH-GLI signaling pathway is the regulation of embryogenesis while its altered function is associated with initiation, progression, invasion and survival of tumor cells. The active form of the SHH-N protein was successfully expressed by cloning the recombinant gene fragment gBlock SHH into the pcDNA3 expression vector. Transient transfection of the CHL-1 melanoma cell line with the pcDNA3.1 - SHH vector increased the SHH gene expression and led to autocrine ligand-dependent activation of the HH-GLI signaling pathway. Increased protein expression of SHH-N, PTCH1, transcription factors GLI1 and GLI3R, as well as decreased expression of protein GLI3FL, was identified by Western blot analysis 48 hours after transfection. SHH protein expression also stimulated activation of the PI3K and MAPK signaling pathways, but it showed no effect on cell cycle regulation. There is a very high importance in this interaction and it represents the basis for the development of the research model in melanoma treatment.

• Keywords: signalni put HH-GLI; Sonic Hedgehog; kloniranje; melanom; transfekcija; PI3K/AKT; MAPK
• Keywords (english): HH-GLI signaling pathway; Sonic Hedgehog; cloning; melanoma; transfection; PI3K/AKT; MAPK
• Language: croatian
• URN:NBN: urn:nbn:hr:217:537217
• 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: 2023-01-25
• Created on: 2021-02-12 13:49:17