Ugotavljanje vpliva izbranih krožnih RNA na cirkadiani ritem celic

Slabe, Saša (author), Režen, Tadeja (mentor)

Cirkadiani ali dnevno-nočni ritmi uravnavajo različne biološke procese. Pri evkariontih temeljijo na transkripcijsko-translacijski negativni povratni zanki, sestavljeni iz genov centralne ure. Kratek razpolovni čas mRNA in proteinov vključenih v regulacijo skupaj z izmenjujočimi valovi aktivacije in represije generira cirkadiane oscilacije. Na ravni celice je cirkadiani ritem kompleksno uravnavan, tudi na nivoju RNA. Krožne RNA so skupina nekodirajočih kovalentno zaprtih RNA. Sprva je prevladovalo prepričanje, da je njihov nastanek posledica nepravilnega spajanja eksonov v procesu dozorevanja mRNA. Raziskave so pokazale, da imajo pomembno vlogo v celični biologiji, so stabilnejše, nekatere pa se pojavljajo celo pogosteje od linearnih RNA ekvivalentov. Krožne RNA so povezali že z več fiziološkimi in patofiziološkimi procesi, ne pa še z uravnavanjem cirkadianega ritma. Namen magistrskega dela je bil ovrednotiti potencialen vpliv izbranih krožnih RNA na cirkadiani ritem humane celične kulture. Preko pregleda podatkovnih zbirk smo izbrali 20 kandidatnih krožnih RNA, katerih zapise smo s pristopi molekulskega kloniranja vstavili v vektor pRR in jih čezmerno izrazili v celični liniji U2OS, ki ima dobro ohranjen cirkadiani ritem. V zadnji fazi smo z merjenjem luminiscence spremljali vpliv čezmernega izražanja krožnih RNA na cirkadiano oscilacijo in pridobljene podatke analizirali s prileganjem cosinor modela v programu CosinorPy. Iz začetnega izbora krožnih RNA smo uspešno pripravili konstrukte plazmida pRR z zapisi za pet različnih krožnih RNA, nadalje pa smo v celicah U2OS statistično potrdili izražanje štirih krožnih RNA, za katere smo ovrednotili vpliv na cirkadiano oscilacijo. Nepričakovano smo zaznali vpliv praznega pRR plazmida na cirkadiani ritem celic, katerega natančni vzrok bi bilo potrebno še podrobneje razjasniti. S prileganjem cosinor modela omejenemu naboru pridobljenih podatkov, smo kot najbolj obetavni krožni RNA, ki sta povzročili spremembe v amplitudi cirkadiane oscilacije celic U2OS, opredelili hsa_circ_0095570 iz zapisa za dolgo nekodirajočo RNA H19 in hsa_circ_0000119, ki izhaja iz starševskega gena MAN1A2. Magistrsko delo ponuja osnovo za nadaljnjo optimizacijo postopka transfekcije in metode vrednotenja cirkadiane oscilacije z meritvijo luminiscence, s čimer bi pridobili obsežnejši nabor podatkov, ki bi omogočal zanesljivejše statistično vrednotenje rezultatov. Ob potrditvi vpliva identificiranih najobetavnejših krožnih RNA hsa_circ_0095570 in hsa_circ_0000119 na cirkadiani ritem, bi sledila izvedba funkcijske analize, s katero bi podrobneje opredelili biokemijsko ozadje vpliva na celično cirkadiano oscilacijo.

• Language: Slovenian
• Keywords: krožne RNA, hsa_circ_0095570, hsa_circ_0000119, cirkadiani ritem, RNA vezavni proteini
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-144607
• COBISS.SI-ID: 144750339
• Publication date in RUL: 03.03.2023

Secondary language

• Language: English
• Title: Characterization of the effect of selected circular RNA on cell circadian rhythm

Abstract

Circadian rhythms regulate various biological processes. In eukaryotes, they rely on a transcriptional-translational negative feedback loop consisting of central clock genes. The short half-life of the mRNA and proteins involved in regulation and the alternating waves of activation and repression result in circadian oscillations. At the cellular level, circadian rhythms are regulated in complex ways, including at the level of RNA. Circular RNAs are a group of noncoding, covalently closed RNAs. Originally, the prevailing view was that their formation was the result of defective exon splicing during maturation of mRNA precursors. Modern research has shown that they play an important role in cell biology, are more stable, and in some cases are even more abundant than their linear RNA counterparts. The role of circular RNAs has been demonstrated in various physiological and pathophysiological processes, but not yet in circadian rhythm regulation. The aim of this master’s thesis was to investigate the possible influence of selected circular RNAs on the circadian rhythm of human cell cultures. Through a database search, 20 circular RNAs were selected, and their sequences were inserted into the pRR vector by molecular cloning and overexpressed in the U2OS cell line. In the final phase, we checked the effect of overexpression of circular RNAs on circadian oscillation by measuring luminescence and analyzed the results by fitting the Cosinor model to the obtained data in CosinorPy. Based on the initial selection of circular RNAs, we successfully made pRR plasmid constructs for five different circular RNAs and also statistically confirmed the overexpression of four circular RNAs in U2OS cells for which we performed a circadian experiment. Unexpectedly, we discovered an effect of the empty pRR plasmid on the circadian rhythm of the cells, the exact cause of which remains to be further elucidated. We identified hsa_circ_0095570, derived from the gene encoding the long non-coding RNA H19, and hsa_circ_0000119, derived from the MAN1A2 gene, as the two most promising circular RNAs that caused changes in the circadian oscillation of U2OS cells. The master's thesis provides the basis for further optimization of the transfection and the method for assessing circadian oscillation by measuring luminescence, which would provide us with a more comprehensive data set that would allow more reliable statistical analysis. Confirmation of the influence of the identified promising circular RNAs hsa_circ_0095570 and hsa_circ_0000119 on circadian rhythms would be followed by a functional analysis that could further define the biochemical background of their influence on cellular circadian oscillation.

• Keywords: circular RNAs, hsa_circ_0095570, hsa_circ_0000119, circadian rhythm, RNA binding protein