The microRNA-mediated gene regulatory network in the hippocampus and hypothalamus of the aging mouse

PLOS ONE RESEARCH ARTICLE The microRNA-mediated gene regulatory network in the hippocampus and hypothalamus of the aging mouse Choijamts Munkhzul ID1,2, Sun Shin Yi3, Junhyung Kim1,2, Seongsoo Lee4,5, Hyuntae Kim4, Jong-Seok Moon1,2*, Mihye Lee ID1,2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Soonchunhyang Institute of Medi-Bio Science, Soonchunhyang University, Cheonan, Korea, 2 Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan, Korea, 3 Department of Biomedical Laboratory Science, College of Medical Sciences, Soonchunhyang University, Asan, Korea, 4 Gwangju Center, Korea Basic Science Institute (KBSI), Gwangju, Korea, 5 Department of Systems Biotechnology, Chung-Ang University, Anseong, Korea * (ML); (JSM) Abstract OPEN ACCESS Citation: Munkhzul C, Yi SS, Kim J, Lee S, Kim H, Moon J-S, et al. (2023) The microRNA-mediated gene regulatory network in the hippocampus and hypothalamus of the aging mouse. PLoS ONE 18(11): e0291943. https://doi.org/10.1371/journal. pone.0291943 Editor: Tanja Grubić Kezele, University of Rijeka Faculty of Medicine: Sveuciliste u Rijeci Medicinski fakultet, CROATIA Received: May 15, 2023 Accepted: September 9, 2023 Published: November 9, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0291943 Copyright: © 2023 Munkhzul et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: RNA sequencing data generated in this study are available in the Gene Expression Omnibus (GEO) under GSE231582. Aging leads to time-dependent functional decline of all major organs. In particular, the aging brain is prone to cognitive decline and several neurodegenerative diseases. Various studies have attempted to understand the aging process and underlying molecular mechanisms by monitoring changes in gene expression in the aging mouse brain using high-throughput sequencing techniques. However, the effect of microRNA (miRNA) on the post-transcriptional regulation of gene expression has not yet been comprehensively investigated. In this study, we performed global analysis of mRNA and miRNA expression simultaneously in the hypothalamus and hippocampus of young and aged mice. We identified aging-dependent differentially expressed genes, most of which were specific either to the hypothalamus or hippocampus. However, genes related to immune response-related pathways were enriched in upregulated differentially expressed genes, whereas genes related to metabolism-related pathways were enriched in downregulated differentially expressed genes in both regions of the aging brain. Furthermore, we identified many differentially expressed miRNAs, including three that were upregulated and three that were downregulated in both the hypothalamus and hippocampus. The two downregulated miRNAs, miR-322-3p, miR542-3p, and the upregulated protein-encoding coding gene C4b form a regulatory network involved in complement and coagulation cascade pathways in the hypothalamus and hippocampus of the aging brain. These results advance our understanding of the miRNA-mediated gene regulatory network and its influence on signaling pathways in the hypothalamus and hippocampus of the aging mouse brain. Introduction Aging is an irreversible, time-dependent process marked by the decline of physiological functions responsible for survival and fertility [1]. This physiological decline leads to loss of organ PLOS ONE | https://doi.org/10.1371/journal.pone.0291943 November 9, 2023 1 / 24 PLOS ONE Additionally, the minimal data sets for Figs 4C, 4E, S4D, and S4E and minimal data set for Fig 4D are available as Supporting Information files. Funding: This work was supported by the National Research Foundation of Korea (Grant NRF2021R1A2C4002421 to M.L.; RS-2023-00219563 to M.L.; 2021R1C1C1007810 to J.M.) and Soonchunhyang University Research Fund (Soonchunhyang University Research Fund 2021 to J.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. miRNA-mediated gene regulatory network in the aging mouse brain and tissue function and an increased risk of cancer, diabetes, and cardiovascular disease [2]. Aging of the brain is often associated with a decline in motor, sensory, and cognitive functions, resulting in common neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease [3]. To understand the aging processes, the molecular mechanisms of aging, as well as morphological and physiological changes in the aging brain, have been extensively investigated [4]. In particular, analysis of gene expression changes associated with brain aging can characterize cellular events during aging and may allow prevention or treatment of age-related diseases [5]. Gene expression changes during aging have been reported at cellular, tissue and organism level using Drosophila [6, 7], Caenorhabditis elegans [8], and mouse models [9]. Several studies have identified differentially expressed genes (DEGs) by comparing young and aged mice, mostly focusing on specific brain regions [10]. Using RNA sequencing (RNA-seq), Shavlakadze et al. [11] identified 229 age-regulated genes with a significant proportion implicated in immune-response signaling pathways in the hippocampal region of rats. Li et al. [12] performed RNA-seq of the hippocampal regions of the mouse brain during aging and found DEGs associated with neuroinflammation. Ximerakis et al. [13] performed single-cell RNAsequencing (scRNA-seq) of whole brains of young and aged mice and found DEGs involved in protein synthesis, oxidative stress, inflammatory responses, and growth factor signaling. However, the underlying control mechanisms of genome-wide gene expression in aging are still not comprehensively understood. miRNAs are small non-coding RNAs composed of 19–24 nucleotides that regulate gene expression at the post-transcriptional level [14, 15]. miRNA-mediated regulation of gene expression is critical for most biological processes, including tissue development, cellular differentiation, cell proliferation, and cell death [16]. In humans, 60% of protein-coding genes are regulated by miRNAs [17]. Numerous studies have shown that miRNAs play an important role in regulating aging processes, such as cognitive decline, inflammation, and neurodegenerative diseases, in mouse brains [18–20]. Li et al. [21] analyzed miRNA expression datasets from human prefrontal cortex o (...truncated)