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Hepatocelularni karcinom (HCC) je najpogostejši tip primarnega raka jeter in je drugi najpogostejši vzrok smrti, povezane z rakavim obolenjem na svetovni ravni. Glavni razlog za nizek odstotek preživetja je pozno odkritje bolezni, pri čemer igra glavno vlogo pomanjkljiva diagnostika predvsem v zgodnjih stadijih bolezni. Krožne RNA (circRNA) so medtem že pokazale pomembno vpletenost v patologijo HCC. Osrednji namen doktorskega dela je bila opredelitev diagnostičnega potenciala ter vloge izbranih krožnih RNA v hepatocelularnem karcinomu. Z analizo objavljenih transkriptomskih podatkov bolnikov s HCC smo identificirali obogatene prepoznavne motive za RNA vezavne proteine (RBP) v okolici spojitvenih mest spremenjeno izraženih circRNA. Identificirani RBP bi lahko predstavljali potencialne regulatorje izražanja circRNA v HCC. Poleg tega smo z analizo objavljenih transkriptomskih podatkov identificirali povišano in znižano izražene circRNA v HCC, med drugim tudi povišano izraženo hsa_circ_0062682. S prehodno in stabilno spremembo izražanja hsa_circ_0062682 v modelnih celičnih linijah HCC smo pokazali vpletenost le-te v celične procese, kot so proliferacija, migracija, tvorba kolonij in invazija. Na modelnih celičnih linijah s spremenjenim izražanjem hsa_circ_0062682 smo izvedli transkriptomske analize ter identificirali spremenjeno izražene gene. Analizi obogatitve poti in genskih skupin sta identificirali obogatene poti (celični cikel, izrezovanje in spajanja RNA, metabolizem steroidov,…), signalne poti (TGF-beta, MAPK, PI3K-Akt, FoxO, TNF signalna pot) in transkripcijske faktorje (E2F1, Sp1, HIF-1?, NF?B1), ki bi lahko bili vpleteni v s hsa_circ_0062682-posredovano onkogenezo HCC. S testom »pull down« z biotiniliranimi oligonukleotidi, sklopljenim z masno spektrometrijo, smo identificirali nove potencialne proteinske vezavne partnerje. V nadaljnjem delu smo se osredotočili predvsem na interakcijo z YBX1, z znano onkogeno vlogo v HCC, in potrdili njuno interakcijo z RNA imunoprecipitacijo. Pri testih celične migracije, rezistence na multikinazni inhibitor sorafenib in lokalizacije proteina hnRNP K smo odkrili, da je vloga hsa_circ_0062682 odvisna od celičnega konteksta. Dodatno smo izmerili statistično značilno znižano izražanje hsa_circ_0062682 v tumorjih bolnikov s HCC, zdravljenih v slovenskih ustanovah, kar je v nasprotju s povišanim izražanjem hsa_circ_0062682 v bolnikih iz objavljenih transkriptomskih študij. Obe objavljeni študiji zajemata drugo populacijo bolnikov in najverjetneje druge etiološke dejavnike bolezni. Pridobljeni rezultati so v skladu s kompleksno heterogenostjo patologije HCC, kjer smo predpostavili, da sta izražanje in vloga hsa_circ_0062682 odvisni od podtipa tumorjev HCC. Diagnostičnega potenciala hsa_circ_0062682 v plazmi bolnikov s HCC v naši raziskavi nismo potrdili. V naši raziskavi smo tako ovrgli hipotezo, da izbrana circRNA izkazuje diagnostični potencial in omogoča razlikovanje med osebami s hepatocelularnim karcinomom ter kontrolnimi osebami v preučevani populaciji. Potrdili smo hipotezo, da izbrana circRNA deluje kot onkogen v modelnih celičnih linijah in vpliva na celično proliferacijo, invazijo in migracijo. Prav tako smo potrdili hipotezo, da izbrana circRNA vpliva na molekularne mehanizme, povezane z onkogenezo v hepatocelularnem karcinomu. Raziskava je tako identificirala nove potencialne regulatorje izražanja circRNA v HCC, ovrednotila vpliv hsa_circ_0062682 v modelnih celičnih linijah HCC, identificirala sistemske spremembe v transkriptomu in nove potencialne proteinske vezavne partnerje. Dodatno je izpostavila pomembnost vključevanja različnih modelnih sistemov HCC in kohort bolnikov z različnim podtipom tumorjev HCC. Hepatocellular carcinoma (HCC) represents the most common type of primary liver cancer and the second leading cause of cancer-related deaths worldwide. The main reason for the low survival rate of HCC is the late discovery of the disease since there is a lack of efficient diagnosis, especially in the early stages of the disease. Circular RNAs (circRNAs) have already been shown to be implicated in the pathology of HCC. The aim of the doctoral thesis was the characterization of a diagnostic potential and role of selected circRNAs in HCC. By performing transcriptomic analyses of datasets from HCC patients we identified enriched motifs of RNA binding proteins (RBPs) in the sequences around the splice sites of differentially expressed circRNAs. Identified RBPs could represent potential regulators of circRNA expression in HCC. Alongside the performed transcriptomic analyses we identified upregulated and downregulated circRNAs in HCC, also an upregulated hsa_circ_0062682. By performing transient and stable perturbations of hsa_circ_0062682 expression in HCC model cell lines, we showed its implication in cell processes such as cell proliferation, migration, colony formation and invasion. We further conducted transcriptomic analyses on model cell lines with perturbed hsa_circ_0062682 expression and identified differentially expressed genes. Pathway enrichment analysis identified enriched pathways (cell cycle, RNA splicing, steroid metabolism,…), signalling pathways (TGF-beta, MAPK, PI3K-Akt, FoxO, TNF signalling pathway) and transcription factors (E2F1, Sp1, HIF-1α, NFκB1) which could be implicated in hsa_circ_0062682-mediated HCC oncogenesis. We identified new potenital protein binding partners by performig pull down test with biotinylated oligonucleotides followed by mass spectrometry. In our further work, we focused on the interaction with YBX1, which has a known role in the oncogenesis of HCC, and confirmed the identified interaction with RNA immunoprecipitation. in the tests of cell migration, resistance to mutikinase inhibitor sorafenib and hnRNP K localization, we identified that the role of hsa_circ_0062682 is dependent on the cell type specific context. Additionally, we detected statistically significant downregulated expression of hsa_circ_0062682 in tumour tissues of HCC patients treated in Slovenian medical institutions. This is in discordance with identified upregulated expression of hsa_circ_0062682 in tumour tissues of HCC patients from the published transcriptomic analyses. Patients from both published studies represent different populations and most likely also have different aetiological causes of the disease. Obtained results are in concordance with the complex heterogeneity of HCC pathology and therefore we proposed that the expression and role of hsa_circ_0062682 are dependent on the HCC tumour subtype. In our study, we did not confirm the diagnostic potential of hsa_circ_0062682 in plasma samples of HCC patients. We rejected the hypothesis that the selected circRNA exhibits diagnostic potential and enables differentiation between HCC patients and the control group in the studied population. We confirmed the hypothesis that the selected circRNA can act as an oncogene in model cell lines and affects cell proliferation, invasion and migration. Additionally, we confirmed the hypothesis that the selected circRNA can affect molecular mechanisms linked to oncogenesis in HCC. Our study, therefore, identified new potential regulators of circRNA expression in HCC, characterized the role of hsa_circ_0062682 in HCC model cell lines, identified systemic changes in the transcriptome of HCC model cell lines and identified new potential protein binding partners. Additionally, the study shed a light on importance of including different HCC models and patient cohorts with different subtypes of HCC tumours. |
