Posttranskripcijsko utišavanje stresnih proteina pomoću siRNA nanoterapije

doctoral thesis

• Title (english): Posttranscriptional silencing of heat shock proteins using siRNA nanotherapy
• Author: Mirela Matokanović
• Mentor: Karmela Barišić (mentor)
• Committee member: Jelena Filipović-Grčić (predsjednik povjerenstva)
• Committee member: Dušica Maysinger (član povjerenstva); MBZ: XXXXXXX
• Committee member: Sonja Levanat (član povjerenstva); MBZ: 113154
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2016-06-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Stresni proteini (HSP) pripadaju skupini unutarstaničnih proteina koji su eksprimirani konstitutivno i
kao odgovor na fizikalni odnosno biološki stres. U staničnom odgovoru na stres HSP imaju ulogu stabiliziranja
proteina i peptida čime promoviraju preživljenje stanice. Hsp27 i Hsp70 inhibitori su različitih čimbenika
apoptoze, dok je glavna uloga Hsp90 osiguravanje aktivnost čimbenika uključenih u proliferaciju stanica.
Sposobnost stresnih proteina da zaustave proces programirane stanične smrti i potaknu proliferaciiju predstavlja
bitan aspekt njihove povezanosti s malignom proliferacijom.
Glavni cilj ovog istraživanja bio je procijeniti ulogu Hsp70 u zaštiti od programirane stanične smrti u
tumorskim stanicama. U tom smislu ispitana je uloga inducibilnog oblika Hsp70 u fiziološkim i stresnim
uvjetima. Hsp70, induciran stresom, pokazao je značajnu ulogu u zaštiti tumorskih stanica od stanične smrti
izazvane staurosporinom. U svrhu utišavanja ekspresije Hsp70 pri fiziološkim uvjetima korištena je tehnologija
koja počiva na RNA interferenciji, specifičnoj razgradnji ciljne mRNA pomoću kratke siRNA uklopljene u
nanočestice. Dizajnirane su kitozanske nanočestice za dostavu Hsp70 sljedno-specifične siRNA. U uvjetima in
vitro kitozanske nanočestice pokazale su nisku toksičnost, učinkovito uklapanje Hsp70 siRNA te učinkovito
utišavanje Hsp70. U staničnim linijama Jurkat i U251N utišavanje Hsp70 omogućeno pomoću Hsp70 siRNA
uklopljene u kitozanske nanočestice, prouzročilo je smanjenje vijabilnosti ovih tumorskih staničnih linija.
U radu je ispitana i antitumorska učinkovitost istovremene inhibicije Hsp90, pomoću celastrola, i
Hsp70 pomoću kitozanskih nanočestica s uklopljenom Hsp70 siRNA. Učinkovitost ovog pristup bila je značajna
u dvodimenzionalnim modelima tumorskih staničnih linija, dok su u trodimenzionalnim modelima bile potrebne
povećane koncentracije oba spoja/sustava kako bi se postiglo značajno smanjenje vijabilnosti tumorskih stanica.
Rad predstavlja temelje za daljnja istraživanja optimalnih nanosustava za dostavu sljedno-specifičnih
siRNA za HSP te moguća ispitivanja istovremene inhibicije Hsp70 i Hsp90 u tumorskim modelima in vivo.

Abstract (english)

Stress proteins (HSP) are intracellular proteins expressed constitutively but can also be induced by
various types of stress including environmental changes and non-stress conditions such as cell cycle, growth
factors, development and differentiation. Under physiological conditions these proteins function as molecular
chaperones that enable the function of different proteins. Hsp27 and Hsp70 appear to function at key regulatory
control points in apoptotic process, whereas the major role of Hsp90 is protection from degradation for the major
factors responsible for cell proliferation. The promotion of tumor cell survival is closely related to the ability of
Hsp to inhibit programmed cell death during malignant proliferation.
The purpose and the main goal of the proposed study was to explore Hsp70's role in protection against
programmed cell death in tumor cells. The role of stress induced Hsp70 and the role of Hsp70 within
physiological levels was explored. Stress induced Hsp70 was found to efficiently protect tumor cells against cell
death promoted with staurosporin. Hsp70 silencing was accomplished using the technology based on RNA
interference, specific degradation of mRNA after binding with siRNA delivered into the cells with nanoparticles.
Chitosan based nanoparticles for Hsp70 siRNA delivery were prepared. These showed low toxicity, efficient
siRNA entrapment and efficient Hsp70 silencing. In Jurkat and U251N cell lines Hsp70 silencing with Hsp70
siRNA delivered in chitosan nanoparticles significantly decreased cell viability.
The antitumor efficiency of simultaneous inhibition of Hsp90 with celastrol and Hsp70 with chitosan
nanoparticles entrapping Hsp70 siRNA was also explored. Significant reduction in cell viability was observed in
two-dimensional cell cultures, while for the induction of comparable effect in three-dimensional cell cultures,
U251N spheroids, higher concentration of both celastrol and Hsp70 siRNA were necessary.
The results of this work present important findings that could lead into the development of optimal
delivery systems for siRNA and possible in vivo research of Hsp70/Hsp90 inhibition approach.

• Keywords: tumor; stresni proteini; Hsp70; RNA interferencija; kitozanske nanočestice
• Keywords (english): tumor; stress proteins; Hsp70; RNA interference; chitosan nanoparticles
• Language: croatian
• URN:NBN: urn:nbn:hr:163:304528
• Study programme: Title: Pharmacy and biochemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor znanosti (doktor znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-11-18 10:10:36