Circular RNAs (circRNAs) are stable non-coding RNAs characterized by a covalently closed loop structure lacking 5′- and 3′-ends. Due to this unique structure, they are resistant to exonuclease activity, which ensures their long-lasting stability in cells. In recent years, circRNAs have been shown to play important roles in regulating gene expression, controlling transcriptional and translational processes, and maintaining cellular homeostasis. Their functions are also associated with the development of diseases such as cancer and neurodegenerative disorders, making them increasingly recognized as potential biomarkers and therapeutic targets. The circadian rhythm is an internal biological mechanism that regulates numerous physiological processes in an approximately 24-hour cycle. It involves a complex network of genes and proteins forming transcriptional–translational feedback loops. While circadian genes are already well studied, the link between circRNAs and circadian rhythms remains relatively unexplored. Research in this area has recently gained momentum, as understanding the potential temporal regulation of circRNAs could contribute to discovering new mechanisms of cellular regulation. The aim of this thesis was to investigate whether selected circRNAs display circadian expression in the human osteosarcoma cell line U2OS, which is widely used as an in vitro model for circadian rhythm studies. In the experimental part, RNA was isolated at different time points from U2OS cells and the expression of selected circRNAs was determined using reverse transcription followed by quantitative polymerase chain reaction (qPCR). Selected circular RNAs were identified based on the performance of amplification products on an agarose gel. The circRNAs hsa_circ_0003638, hsa_circ_0032815, hsa_circ_0006041, and novel_circ_0012890 were successfully validated. ACTB was used as a reference gene for normalization, and the circadian gene DBP served as a positive control for rhythm validation. Data analysis was performed using the CosinorPy method, which provides a statistical evaluation of whether expression follows a 24-hour cycle. The results demonstrated that the selected circRNAs did not exhibit significant circadian expression under the given conditions, whereas DBP was confirmed as a positive circadian marker. These findings suggest that the selected circRNAs in the U2OS cell line are unlikely to play a direct role in the core circadian network. Nevertheless, this work contributes to a better understanding of circRNAs and opens possibilities for further research into their role in the temporal regulation of cellular processes.
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