Citation: Staszkiewicz, R.; Soba ´ nski, D.; Pulka, W.; Gładysz, D.; Gadzieli ´ nski, M.; Strojny, D.; Grabarek, B.O. Variances in the Expression Profile of Circadian Clock-Related Genes in Astrocytic Brain Tumors. Cancers 2024, 16, 2335. https://doi.org/10.3390/ cancers16132335 Academic Editor: Giulio Cabrini Received: 11 June 2024 Revised: 22 June 2024 Accepted: 25 June 2024 Published: 26 June 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Article Variances in the Expression Profile of Circadian Clock-Related Genes in Astrocytic Brain Tumors Rafał Staszkiewicz 1,2,3, * , Dawid Soba ´ nski 1,4 , Wojciech Pulka 5 , Dorian Gładysz 1,2,3 , Marcin Gadzieli ´ nski 1,2,3 , Damian Strojny 1,6,7 and Beniamin Oskar Grabarek 1 1 Collegium Medicum, WSB University, 41-300 Dabrowa Gornicza, Poland; drdsobanski@gmail.com (D.S.); gladyszdorian875@gmail.com (D.G.); gadzielinskimarcin@gmail.com (M.G.); strojny.ds@gmail.com (D.S.); bgrabarek7@gmail.com (B.O.G.) 2 Department of Neurosurgery, 5th Military Clinical Hospital with the SP ZOZ Polyclinic in Krakow, 30-901 Cracow, Poland 3 Department of Neurosurgery, Faculty of Medicine in Zabrze, Academy of Silesia, 40-555 Katowice, Poland 4 Department of Neurosurgery, Szpital sw. Rafala in Cracow, 30-693 Cracow, Poland 5 Department of Neurosurgery, Neurotraumatology and Spinal Surgery, Regional Hospital in Elblag, 82-300 Elblag, Poland; ns.wojciechpulka@gmail.com 6 Institute of Health Care, National Academy of Applied Sciences in Przemysl, 37-700 Przemysl, Poland 7 New Medical Techniques Specjalist Hospital of St. Family in Rudna Mała, 36-054 Rudna Mala, Poland * Correspondence: rafalstaszkiewicz830@gmail.com Simple Summary: This study investigates the role of circadian clock genes in the progression of astrocytic tumors, a common type of brain tumor. We aimed to understand how these genes, which control the body’s daily rhythms, behave differently in low-grade versus high-grade tumors. Our findings reveal that certain circadian clock genes are more active in advanced tumor stages, potentially driving tumor growth. Additionally, we discovered that changes in DNA methylation and microRNAs might regulate these genes. Understanding these molecular changes could help identify new biomarkers for tumor diagnosis and progression, offering new avenues for targeted treatments. This research provides valuable insights into the complex biology of brain tumors and highlights the importance of circadian genes in the development of cancer. Abstract: This study explores the role of circadian clock genes in the progression of astrocytic tumors, a prevalent type of brain tumor. The aim was to assess the expression patterns of these genes in relation to the tumor grade. Using microarray analysis, qRT-PCR, and methylation-specific PCR, we examined gene expression, DNA methylation patterns, and microRNA interactions in tumor samples from 60 patients. Our results indicate that the expression of key circadian clock genes, such as clock circadian regulator (CLOCK), protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1), protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2), protein kinase AMP-activated non-catalytic subunit beta 1 (PRKAB1), protein kinase AMP-activated non-catalytic subunit beta 2 (PRKAB2), period circadian regulator 1 (PER1), period circadian regulator 2 (PER2) and period circadian regulator 3 (PER3), varies significantly with the tumor grade. Notably, increased CLOCK gene expression and protein levels were observed in higher-grade tumors. DNA methylation analysis revealed that the promoter regions of PER1-3 genes were consistently methylated, suggesting a mechanism for their reduced expression. Our findings also underscore the complex regulatory mechanisms involving miRNAs, such as hsa-miR-106-5p, hsa-miR-20b-5p, and hsa-miR-30d-3p, which impact the expression of circadian clock-related genes. This underscores the importance of circadian clock genes in astrocytic tumor progression and highlights their potential as biomarkers and therapeutic targets. Further research is needed to validate these results and explore their clinical implications. Keywords: circadian clocks; brain neoplasms; astrocytic tumor; RNA; messenger; microRNAs; enzyme-linked immunosorbent assay; biomarkers Cancers 2024, 16, 2335. https://doi.org/10.3390/cancers16132335 https://www.mdpi.com/journal/cancers