Uspostava tumorske linije debelog crijeva s mutacijom u genu B7H3 i njena funkcionalna analiza

master's thesis

• Title (english): Generation of colorectal cancer cell line with mutation in B7H3 gene and its functional analysis
• Author: Lea Južnić
• Mentor: Sebastian Zeissig (mentor)
• Mentor: Maja Matulić (mentor)
• Committee member: Maja Matulić (predsjednik povjerenstva)
• Committee member: Martina Šeruga Musić (član povjerenstva)
• Committee member: Mirta Tkalec (član povjerenstva)
• Committee member: Petra Korać (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2017-12-12, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Tumori uspostavljaju različite mehanizme kako bi se zaštitili od protu-tumorskog odgovora imunološkog sustava. Ljudski imunološki sustav stoga sadrži niz različitih regulatornih molekula, koje mogu stimulirati ili inhibirati imunološke reakcije. Pri tom, receptori i ligandi iz CD28 i B7 obitelji imaju središnju ulogu u posredovanju interakcija između tumorskih i imunoloških stanica. B7 homolog 3 (B7-H3, Cd276) opisan je kao član obitelji B7 koji je aberantno eksprimiran u različitim tumorima, uključujući rak debelog crijeva (CRC). Prethodna istraživanja pokazala su kontradiktorne uloge B7-H3 unutar imunološkog sustava, upućujući na imunosupresivno i imunostimuliratorno djelovanje. Dodatno, nekoliko je studija pokazalo da je B7-H3 uključen u intrinzičnu regulaciju razvoja i progresije tumora, poput apoptoze, proliferacije, migracije, invazije i metastaza. Stoga, kako bi se razjasnila potencijalna uloga B7- H3 u CRC-u, ovaj je rad imao za cilj uspostaviti tumorsku liniju raka debelog crijeva bez ekspresije B7-H3 uz pomoć CRISPR/Cas9 sustava, te istražiti potencijalne autonomne uloge B7-H3 molekule neovisno o imunološkom sustavu. Analiza pomoću metoda qRT-PCR-a, Western blota i imunofluorescencije pokazala je nedostatak B7-H3 nakon što su mutacije uvedene. Nadalje, apoptoza i proliferacija nisu promijenjene u stanicama koje su deficijentne za B7-H3. Prema tome, rezultati ovog rada upućuju na to da B7-H3 ne regulira staničnu smrt i proliferaciju tumora na staničnoj razini, već potencijalno posredno djeluje putem kontrole aktivacije imunološkog odgovora.

Abstract (english)

Tumors establish a variety of different mechanisms to protect themselves from immunemediated recognition and rejection. The human immune system is orchestrated by a variety of different regulatory molecules, which can either stimulate or inhibit immune cell responses. Receptors and ligands of the CD28 and B7 family have a central role in regulating the interaction between tumor and immune cells. The B7 homolog 3 (B7-H3, Cd276) has been described as a member of the B7 family which is aberrantly expressed in various tumors, including colorectal cancer (CRC) where its expression has also been shown to correlate with poor prognosis. Previous studies showed contradictory roles of B7-H3 within the immune system, demonstrating both immunosuppressive and immunostimulatory actions. In addition, it was suggested that B7-H3 may regulate tumor apoptosis, proliferation, migration, invasion and metastasis in a cell-autonomous manner. To address the question of potential cellautonomous actions of B7-H3 in CRC, this work aimed at the generation of a CRC cell line with mutated B7-H3 using the CRISPR/Cas9 system and the investigation of potential cellintrinsic functions of B7-H3. Analysis via qRT-PCR, Western blot and immunofluorescence methods showed a lack of B7-H3 after mutations were introduced in the Cd276 gene coding for B7-H3. Furthermore, apoptosis and proliferation were unaltered in cells deficient for B7- H3. Therefore, the results of this work suggest that B7-H3 does not regulate CRC cell death and proliferation in a cell-intrinsic manner but rather acts indirectly through the control of immune cell activation.

• Keywords: rak debelog crijeva; imunološki sustav; B7 homolog 3; Cd276; B7-H3; CRISPR/Cas9
• Keywords (english): colorectal cancer; immune system; B7 homolog 3; Cd276; B7-H3; CRISPR/Cas9
• Language: croatian
• URN:NBN: urn:nbn:hr:217:170519
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2018-12-12
• Created on: 2018-01-31 10:18:51