Ugotavljanje vpliva prekomernega izražanja izbranih krožnih molekul RNA na cirkadiani ritem celic linije U2OS in njihova karakterizacija

Trobec, Ana (author), Režen, Tadeja (mentor)

Krožne ribonukleinske kisline (circRNA) so evolucijsko ohranjene endogene kovalentno zaprte molekule s raznolikimi biološkimi funkcijami, ki so bile do danes opisane že pri različnih organizmih, tudi pri človeku. V nasprotju z evkariontskimi informacijskimi ribonukleinskimi kislinami (mRNA) so zelo stabilne in odporne proti eksonukleazam, kar jih postavlja v potencialno uporabno vlogo v diagnostične in terapevtske namene. Njihova aktivna vloga je bila pokazana že v številnih (pato)fizioloških procesih, kot so izločanje inzulina, imunski odziv, miokardni infarkt in razvoj raka, ne pa še pri uravnavanju cirkadianega ritma celic, zato smo želeli raziskati njihov potencialen vpliv na cirkadiani ritem. V ta namen smo v humani celični liniji U2OS s transfekcijo povišali izražanje izbranih circRNA in s pomočjo luciferaznega reporterja vstavljenega za promotor Bmal1 spremljali izražanje enega glavnih cirkadianih genov. Po statistični analizi smo izbranim circRNA z uporabo in silico orodij napovedali potencialne biološke funkcije in potencialne povezave s proteini cirkadianega ritma. Po pregledu napovedanih povezav v literaturi bi izpostavili hsa_circZFP91_003 v vlogi vezave mikro RNA, kar potencialno poviša izražanje dveh cirkadianih proteinov (PML, PIWIL2), potem hsa_circMAN1A2_009 in sicer v vlogi vezave mikro RNA, RNA vezavnega proteina in prevajanja v protein; ter hsa_circRBM23_004, ki kaže na vlogo v vezavi mikro RNA in prevajanja v dva proteina. Velja pa opozoriti, da so navedene ugotovitve le napovedi, ki zahtevajo eksperimentalno potrditev resničnih funkcionalnih povezav in vloge circRNA v uravnavanju cirkadianega ritma. Prve korake eksperimentalne potrditve smo v okviru magistrske naloge že začrtali sami.

• Language: Slovenian
• Keywords: krožne RNA, cirkadiani ritem, funkcije, mikro RNA, hsa_circMAN1A2_009, hsa_circZFP91_003, hsa_circRBM23_004, PIWIL2, PML, RNA vezavni proteini
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [A. Trobec]
• Year: 2023
• PID: 20.500.12556/RUL-149778
• UDC: 577.2(043.2)
• COBISS.SI-ID: 163969027
• Publication date in RUL: 10.09.2023

TROBEC, Ana, 2023, Ugotavljanje vpliva prekomernega izražanja izbranih krožnih molekul RNA na cirkadiani ritem celic linije U2OS in njihova karakterizacija [online]. Master’s thesis. A. Trobec. [Accessed 12 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=149778

Secondary language

• Language: English
• Title: Assessing the effect of overexpression of selected circular RNA molecules on the circadian rhythm of the U2OS cell line and their characterization

Abstract

Circular RNAs (circRNA) are evolutionarily conserved endogenous covalently closed molecules with diverse biological functions, described in various organisms, including humans. In contrast to eukaryotic messenger RNAs (mRNAs), they are highly stable and resistant to exonucleases, rendering them potentially valuable for diagnostic and therapeutic purposes. Their active role has been demonstrated in numerous (patho)physiological processes, such as insulin secretion, immune response, myocardial infarction, and cancer development. However, their involvement in circadian rhythm regulation remains underexplored. Therefore, we aimed to investigate their potential impact on the circadian rhythm by elevating the expression of selected circRNA in the human U2OS cell line through transfection and monitoring the expression of one circadian gene using a luciferase reporter inserted into Bmal1 promoter. Using in silico tools, we predicted potential biological functions and associations with circadian rhythm proteins for the selected circRNAs. Upon reviewing the predicted associations in the literature, we highlight hsa_circZFP91_003 for its potential role in microRNA binding, potentially enhancing the expression of two circadian proteins (PML, PIWIL2). Additionally, hsa_circMAN1A2_009 was implicated in microRNA and RBP binding, as well as translation into protein. Lastly, hsa_circRBM23_004 exhibited a potential role in mikroRNA binding and translation into two proteins. However, it is crucial to note that these findings are mere predictions, necessitating experimental validation to confirm real functional associations and roles of circRNA in circadian rhythm regulation. The first steps of experimental validation have already been outlined

• Keywords: circRNA, circadian rhythm, functions, micro RNA, hsa_circMAN1A2_009, hsa_circZFP91_003, hsa_circRBM23_004, PIWIL2, PML, RNA-binding protein