Optimizacija metode za određivanje transkripata RNA i miRNA u uzorcima egzosoma pacijenata sa kolorektalnim karcinomom

master's thesis

• Title (english): Optimization of method for determination of transcripts of RNA and miRNA in exosomes from colorectal cancer patients
• Author: Matej Barišić
• Mentor: Andrea Hulina Tomašković (mentor)
• Committee member: Andrea Hulina Tomašković (predsjednik povjerenstva)
• Committee member: Anita Somborac Bačura (član povjerenstva)
• Committee member: Kristina Radić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2022-09-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kolorektalni karcinom (CRC) multifaktorska je bolest složene etiologije i visoke prevalencije. U većini slučajeva nastaje iz na početku benignih adenoma koji postaju maligni nakon niza genskih mutacija u epitelnim stanicama kolona. Karakterizira ga spor razvoj i duga presimptomatska faza što predstavlja mogućnost za njegovo rano otkrivanje i liječenje. Tekuća biopsija neinvazivna je metoda za određivanje molekularnog profila tumora uključujući CRC, a egzosomalne RNA i miRNA obećavajući su biomarkeri za dijagnozu, prognozu i evaluaciju odgovora na terapiju u CRC-u. Cilj ovog rada bila je optimizacija metode za određivanje transkripata RNA i miRNA u egzosomima pacijenata sa CRC-om koji su u literaturi istaknuti kao potencijalni prognostički i terapijski biljezi ove bolesti. RNA je izolirana iz egzosoma iz plazme 18 bolesnika s potvrđenim CRC-om. Ekspresija C-MYC te referentnih gena B2M, PPIA i GAPDH, određena je pomoću qPCR tehnike i TaqMan eseja, a ekspresija miR-19a-3p, miR-92a-3p, miR-125-3p, miR-193a-3p, miR-210-3p te referentnih mir-103a-3p, mir 1228-3p i miR-520d-3p pomoću SYBR green tehnologije. Koncentracija RNA kretala se u rasponu od 2,44-51,20 ng/ μl sa medijanom od 11,48 ng/μl. Gen C-MYC kao i ispitivani referentni geni nisu dokazani niti u jednom uzorku što može značiti da su te mRNA prevelike za pakiranje u egzosome. S druge strane, od predloženih referentnih miRNA dokazana je ekspresija jedino miR-103a-3p zbog čega je kao standard s kojim se uspoređivala ekspresija ciljnih miRNA korištena sintetička UniSp6. U najvećem broju uzoraka (svih 18) dokazane su miR-19a-3p i miR-92a-3p dok je u najmanjem broju uzoraka (3) dokazana miR-125-3p. Iz dobivenih rezultata može se zaključiti da su uzorci unatoč niskim koncentracijama RNA dovoljno kvalitetni za određivanje većine miRNA od interesa u našem istraživanju ali ne i za mRNA.

Abstract (english)

Colorectal cancer (CRC) is multifactorial disease with complex etiology and high prevalence among population. In most cases it arises from initially benign adenomas that become malignant after series of gene mutations in colon epithelial cells. It is characterized by slow development and long presymptomatic phase which gives opportunities for early detection and treatment. Liquid biopsy is non-invasive method for determination of cancer molecular profile including CRC, while exosomal RNAs and miRNAs are promising biomarkers for diagnosis, prognosis, and evaluation of therapy response in CRC patients. The goal of this research was to optimize the method for assessment of RNA and miRNA isolated from exosomes of CRC patients found to be promising potential prognostic and therapeutic markers of this disease. RNA was isolated from exosomes from plasma of 18 patients diagnosed with CRC. Expression of C-MYC and reference B2M, PPIA and GAPDH genes was determined using qPCR and TaqMan assays, and expression of miR-19a-3p, miR-92a-3p, miR-125-3p, miR-193a-3p, miR-210-3p and three reference miRNAs: mir-103a-3p, mir 1228-3p and miR-520d-3p was determined using SYBR green technology. Concentration of RNA ranged from 2,44 ng/μl to 51,20 ng/μl with median of 11,48 ng/μl. C-MYC gene and three reference genes weren’t detected in any sample what can imply that these mRNAs are too big for packing in exosomes. On the other hand, only miR-103a-3p was detected of three potential reference genes, which is why UniSp6 was used as standard to normalise expression of target miRNAs. MiR-19a-3p and miR-92-a-3p were detected in most samples (18) while miR-125-3p was detected in lowest number of samples (3). We can conclude that despite low concentrations, our samples have sufficient quality for determination of the expression of most of the targeted miRNAs in this research, but the same cannot be said for mRNAs.

• Keywords: kolorektalni karcinom; tekuća biopsija; egzosomi; RNA; miRNA; PCR; qPCR
• Keywords (english): colorectal cancer; liquid biopsy; exosomes; RNA; miRNA; PCR; qPCR
• Language: croatian
• URN:NBN: urn:nbn:hr:163:036848
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-09-23 10:03:10