Prispevek gastrina in genetske variabilnosti kandidatnih genov celičnega cikla k razvoju raka želodca

Pužar Dominkuš, Pia (author), Hudler, Petra (mentor)

V doktorskem delu smo preučevali molekularne in genetske dejavnike, ki prispevajo k nastanku in napredovanju raka želodca. Mednje uvrščamo hipergastrinemijo, ki dolgoročno spodbuja celično proliferacijo ter zavira apoptozo, zunajcelične vezikle, ki med celicami prenašajo onkogene, ter polimorfizme posameznega nukleotida (SNP-je) v genih celičnega cikla, ki prispevajo k nastanku kromosomske nestabilnosti. Na celični linij raka želodca smo preverili, kako gastrin vpliva na izražanje genov celičnega cikla in izločanje ter funkcijo zunajceličnih veziklov. Na kohortah pacientov z rakom želodca in zdravih posameznikov smo analizirali, kako so SNP-ji v genih celičnega cikla povezani s tveganjem za nastanek in klinično-histopatološkimi značilnostmi raka želodca ter ovrednotili njihove funkcionalne posledice. Celično linijo raka želodca smo izpostavili gastrinu. Izražanje kandidatnih genov celičnega cikla (AURKA, AURKC, ATM, CDC20, BUB1B, PLK1, PLK2, PLK3 in TTK ) smo analizirali z metodo kvantitativne verižne reakcije s polimerazo. Vpliv zunajceličnih veziklov, spodbujenih z gastrinom, na naivne celice raka želodca in normalne epitelijske celice smo analizirali s testom celične proliferacije. V študijo genotipizacije smo vključili 221 pacientov in 321 kontrol. Povezave SNP-jev v kandidatnih genih (ATM, CDC20, PLK2 in PLK3) s tveganjem za nastanek in klinično-histopatološkimi značilnostmi raka želodca smo analizirali z metodo splošnega linearnega modela. Vezavo kandidatne miRNA na polimorfni alel smo potrdili in vitro z luciferaznim testom. Spremenjeno izražanje kandidatne miRNA smo analizirali na naboru prostodostopnih podatkov iz podatkovnih zbirk TCGA in GEO. Gastrin je v primerjavi s kontrolo povišal izražanje gena AURKA za 1,121-krat (Padj = 0,002) in gena PLK1 za 1,065-krat (Padj = 0,019) ter povišal izločanje mikroveziklov za 1,802-krat (P = 0,047). Eksosomi, spodbujeni z gastrinom, so povečali proliferacijo naivnih celic raka želodca za 1,380-krat (Padj = 0,0001). Haplotip PLK2 Crs15009-Crs963615 je bil manj pogost v skupini z rakom želodca v primerjavi s kontrolno skupino (0,337 % proti 0,402 %; Pcorr = 0,050). Na polimorfni alel PLK2-rs15009 (C/G) sta se miR-23a-5p in miR-23b-5p vezali različno, odvisno od prisotnosti alela C oziroma alela G. Relativna luciferazna aktivnost v celicah, ki so izražale PLK2 3' UTR z varianto G, se je zmanjšala za 41 % ob ko-transfekciji z mimikom miR-23b-5p (P = 0,0097). Nizko izražanje miR-23b-5p pa je bilo povezano z daljšim 10-letnim preživetjem pacientov z rakom želodca (HR = 1,52; P = 0,007). Haplotip PLK2-Crs15009-Crs963615 bi lahko služil kot označevalec tveganja za razvoj bolezni, raven izražanja miR-23b-5p pa napovedni označevalec preživetja pacientov z rakom želodca.

• Language: Slovenian
• Keywords: rak želodca, gastrin, polimorfizem posameznega nukleotida (SNP), eksosomi, mikrovezikli, veliki onkosomi, zunajcelični vezikli, PLK2, miR-23b-5p, celični cikel
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2023
• PID: 20.500.12556/RUL-146120
• Publication date in RUL: 20.05.2023

Secondary language

• Language: English
• Title: Contribution of gastrin and genetic variability in candidate cell cycle genes to the development of gastric cancer

Abstract

In the doctoral thesis we studied molecular and genetic factors that contribute to the development and progression of gastric cancer. These include hypergastrinemia, which in the long-term promotes cell proliferation and inhibits apoptosis, extracellular vesicles that transport oncogenes between cells, and single nucleotide polymorphisms (SNPs) in cell cycle genes that contribute to chromosomal instability. We examined how gastrin affects the expression of cell cycle genes and the secretion and function of extracellular vesicles. We analysed the associations between single nucleotide polymorphisms in cell cycle genes with gastric cancer risk and clinical-histopathological features and evaluated their functional consequences. We exposed gastric cancer cell line to gastrin. We analysed candidate cell cycle gene expression (AURKA, AURKC, ATM, CDC20, BUB1B, PLK1, PLK2, PLK3 and TTK) with quantitative polymerase chain reaction. We evaluated the effect of gastrin-induced extracellular vesicles on the naïve gastric cancer cells and normal epithelial cells using cell proliferation assay. We included 221 patients and 321 controls in the genotyping study. Associations between SNPs in candidate genes (ATM, CDC20, PLK2 and PLK3) and the risk and clinical-histopathological features of gastric cancer were analysed with the generalized linar model. We confirmed the binding of the candidate miRNA to the polymorphic allele in vitro with the Luciferase reporter assay. We analysed the differential expression of candidate miRNA using the publicly available datasates within the TCGA and GEO databases. Gastrin increased the expression of AURKA gene 1.121-fold (Padj=0.002) and PLK1 gene 1.065-fold (Padj=0.019) as well as the secretion of microvesicles 1.802-fold (P = 0.047). Gastrin-induced exosomes increased the proliferation of naïve gastric cancer cells 1.380-fold (Padj=0.0001). The PLK2 Crs15009-Crs963615 haplotype was less frequent in the gastric cancer group compared to the control group (0.337% vs. 0.402%; Pcorr=0.050). The PLK2-rs15009 polymorphic allele (C/G) differentially bound miR-23a-5p and miR-23b-5p. Relative luciferase activity in cells expressing the PLK2 3' UTR with the G variant was decreased by 41% upon co-transfection with miR-23b-5p mimic (P=0.0097). Low miR-23b-5p expression was associated with longer 10-year survival in gastric cancer patients (HR=1.52; P=0.007). PLK2 haplotype Crs15009-Crs963615 could serve as a risk biomarker, whereas miR-23b-5p expression level could be used as prognostic survival biomarker in gastric cancer patients.

• Keywords: gastric cancer, gastrin, single nucleotide polymorphism (SNP), exosomes, microvesicles, large oncosomes, extracellular vesicles, PLK2, miR-23b-5p, cell cycle