miR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice

miR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article miR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice Langsha Liu, Fanyan Luo This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3930815/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Genes & Nutrition → Version 1 posted 7 You are reading this latest preprint version Abstract Objective: Cardiac fibrosis is a significant contributor to atrial fibrillation (AF). Our aim is to identify biomarkers for AF using bioinformatic methods and explore the regulatory mechanism of miR-450a-2-3p in cardiac fibrosis in mice. Methods: Two datasets, GSE115574 and GSE79768, were obtained from the Gene Expression Omnibus (GEO) database and subsequently merged for further analysis. Differential gene expression analysis was performed to identify genes that were differentially expressed (DEGs) and genes that were differentially expressed in relation to miR-450a-2-3p (MRDEGs). To investigate the underlying mechanism of cardiac fibrosis, a mouse model was established by treating mice with isoproterenol (ISO) and miR-450a-2-3p agomir. Results: A total of 127 DEGs and 31 MRDEGs were identified and subjected to functional enrichment analysis using Gene Ontology (GO) and pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) to determine the functions and pathways involved in atrial fibrillation (AF). In animal experiments, histological staining using H&E and Masson's trichrome, as well as quantification of collagen volume fraction (CVF), were performed. The increased expression of α-smooth muscle actin (α-SMA), collagen type I (col1), collagen type III (col3), and extracellular signal-regulated kinase (ERK) 1/2 at both the mRNA and protein levels indicated the presence of significant myocardial fibrosis in mice induced with isoproterenol (ISO). However, after the overexpression of miR-450a-2-3p agomir through caudal vein injection, there was a notable improvement in the cardiac morphology of the treated group. The expression levels of α-SMA, col1, col3, and ERK1/2 also significantly decreased, as confirmed by Western blot and RT-qPCR analyses. Conclusion: Our study elucidates the mechanistic connection between ISO-induced myocardial fibrosis and the miR-450a-2-3p/ERK (1/2) signaling pathway, highlighting their role in the development of cardiac fibrosis. Modulating miR-450a-2-3p expression and inhibiting ERK (1/2) activation represent promising approaches for therapeutic intervention in atrial fibrillation. atrial fibrillation cardiac fibrosis microRNA mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Atrial fibrillation (AF) is one of the most prevalent sustained supraventricular arrhythmias and is associated with an increased risk of stroke[1]. The underlying mechanism is believed to involve electrical and structural remodeling of the atria, leading to the development of cardiac fibrosis[2]. Atrial fibrosis plays a significant role in the persistence of AF and is considered one of the key factors contributing to its resistance to rhythm control interventions[3]. The activation and proliferation of cardiac fibroblasts (CFs), which are responsible for excessive production of extracellular matrix (ECM) components such as collagen-1 (COL1) and collagen-3 (COL3), play a crucial role in fibrogenesis in patients with AF[4]. Additionally, CFs can differentiate into myofibroblasts, cells that exhibit a twofold higher capacity for collagen synthesis, including alpha smooth muscle actin (α-SMA)[5]. Similarly, in cardiac fibrosis, endothelial cells (ECs) can undergo endothelial-mesenchymal transition (EndMT), acquiring a mesenchymal phenotype and expressing characteristic markers of myofibroblast differentiation, including α-SMA, vimentin, and collagens[6]. The expression of α-SMA, a well-characterized cytoskeletal protein, serves as a hallmark of myofibroblast differentiation[7]. However, the precise molecular mechanisms underlying cardiac fibrosis and EndMT in AF remain largely unclear. In our previous study, we observed downregulation of miR-450a-2-3p in CFs and human umbilical vein endothelial cells (HUVECs) exposed to transforming growth factor (TGF)-β1, suggesting its potential as a microRNA (miRNA) that inhibits cardiac fibrosis[8]. In the present study, we employed established bioinformatic tools to investigate the underlying mechanism of miR-450a-2-3p in cardiac fibrosis. Two transcriptome datasets were selected from the Gene Expression Omnibus (GEO) database for analysis. Differential gene expression analysis was conducted to identify differentially expressed genes (DEGs), as well as miR-450a-2-3p-related DEGs (MRDEGs). Subsequently, functional enrichment analysis was performed, revealing the involvement of miR-450a-2-3p in multiple biological processes and signaling pathways related to cardiac fibrosis. Furthermore, we validated the anti-fibrotic effect of miR-450a-2-3p overexpression in a mouse model of isoproterenol (ISO)-induced myocardial fibrosis. Collectively, our findings unveil a previously unknown microRNA that holds potential as a target for future clinical treatment and diagnosis of AF patients. 2. Materials and methods 2.1 Data collection and download The AF gene expression datasets, GSE41177 and GSE79768, were obtained from the GEO database ( https://www.ncbi.nlm.nih.gov/geo/ ). GSE115574 (28 AF samples and 31 SR samples) and GSE79768 (14 AF samples and 12 SR samples) were generated using the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The R software packages "limma" and "SVA" were used to merge the GSE41177 and GSE79768 datasets, and batch effect was removed using the combat function. Data was adjusted using the FDR method. Differential expressed genes (DEGs) from the merged dataset were identified using the criteria |logFC| ≥ 0.5 and adj.P. Val. Filter < 0.05. The "heatmap" package and "ggplot2" package were utilized to generate heatmaps and volcano plots of the DEGs. Prediction of miRNA-to-mRNA interactions was performed using TargetScan ( https://www.targetscan.org/vert_72/ ). 2.2 Functional enrichment analysis To investigate the role of DEGs in AF patients, we utilized the Xiantao online tools ( https://www.xiantaozi.com/ ) to conduct GO and KEGG pathway analysis. Additionally, the Metascape database ( http://metascape.org ) facilitated a comprehensive understanding of the biological significance of MRDEGs through Gene Ontology enrichment analysis. 2.3 ISO‑induced atrial fibrosis model in mice and grouping Eight-week-old male C57BL/6 mice were utilized to establish an atrial fibrosis model through induction with ISO. A total of 24 mice were randomly divided into four groups: control group, ISO-induced model group, miR-450a-2-3p agomir group, and agomir-negative control (NC) group (n = 6 per group). In the model and control groups, ISO (0.1ml/10g) and normal saline, respectively, were injected into the abdominal cavity for a duration of 14 days. Throughout this period, the mice were weighed on days 1, 7, and 14. After the 14-day treatment, the hearts of the mice were harvested for hematoxylin-eosin (H&E) staining, Masson staining, and the detection of α-SMA, col1, col3, and ERK (1/2) expression levels. The miR-450a-2-3p agomir and agomir-NC groups received injections of miR-450a-2-3p agomir (5nmol/each) and agomir-NC (5nmol/each), respectively, for a total of four times over a period of 16 days (once every 5 days). The administration of miR-450a-2-3p agomir and agomir-NC (GenePharma, Shanghai, China) was performed via tail vein injection. 2.4 H&E staining and Masson staining The paraffin-embedded atrial tissue sections were dried in a 60°C incubator for more than 3 hours prior to staining. Standard protocols were followed for H&E staining and Masson staining. The slides were examined using an optical microscope and images were captured to evaluate histopathology and the extent of fibrosis. 2.5 Reverse transcription quantitative polymerase chain reaction (RT‑qPCR) Total RNA was extracted according to the manufacturer’s instructions. Briefly, take approximately 0.02g of tissue, add 1 mL of Trizol (Thermo Fisher Scientific, MA, USA) and mix well for 5 min. mRNAs were reverse transcribed using a reverse transcription kit (Cwbiotech, Beijing, China). RT-qPCR was performed using an UltraSYBR mixture (Cwbiotech, Beijing, China). The relative expression of genes was calculated using the 2 − ΔΔCT. The mmu-miR-450a-2-3p, col1 and col3 expression of samples was normalized with that of GAPDH, and the α-sma and ERK (1/2) expression of samples was normalized with that of U6. All primers used in this study were commercially obtained from Sangon (Shanghai, China) and are shown in Supplementary Table 1. 2.6 Western blot analysis 0.025 g of mouse heart tissues were lysed in 300 µL RIPA buffer (Abiowell, Changsha, China). After centrifugation at 12,000 × g for 15 min at 4°C, the supernatant was collected. The protein concentration was determined using the bicinchoninic acid assay with a commercial kit (Cwbiotech, Beijing, China). Next, 120 µL of protein was separated using a 5× loading buffer (30 µL) and then transferred to a nitrocellulose filter membrane. The membrane was incubated in 5% bovine serum albumin (BSA) at 4°C for 1.5 h and then incubated overnight with primary antibodies at 4°C. The primary antibodies used for Western blotting were as follows: β-actin (1:5000; Proteintech, IL, USA), α-SMA (1:3000; Proteintech, IL, USA), COL1 (1:2000; Proteintech, IL, USA), COL3 (1:500; Proteintech, IL, USA), and ERK (1:10000; Proteintech, IL, USA). After overnight incubation with the primary antibody, the membrane was incubated with the corresponding secondary antibodies (HRP goat anti-mouse IgG, 1:5000; HRP goat anti-rabbit IgG, 1:6000; Proteintech, IL, USA) at 25°C for 90 min. Detection was performed using an enhanced chemiluminescence (ECL) system (Abiowell, Changsha, China). Relative protein expression levels were analyzed using Quantity One v4.6.2 software. 2.7 Statistical analysis The two-tailed Student's t-test was employed to compare the two distinct conditions. In situations where the data did not exhibit a normal distribution, the Mann-Whitney test was utilized. For experimental comparisons involving three or more experimental groups, Tukey's multicomparison test or one-way ANOVA with Dunnett's posthoc test was conducted. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, SanDiego, CA, USA). A significance level of P < 0.05 was deemed statistically significant. 3. Result 3.1 Screening of DEGs in AF The schematic flowchart of this study is presented in Fig. 1 . Differential expression analysis was performed between 42 atrial fibrillation (AF) samples and 43 sinus rhythm (SR) samples from GSE79768 and GSE115574. Box plots (Fig. 2 A-B) and PCA analysis (Fig. 2 C) effectively demonstrated the successful elimination of batch effects between GSE79768 and GSE115574. In the merged matrix, 126 differentially expressed genes (DEGs) were identified using the "limma" R package, including 50 upregulated genes and 76 downregulated genes. The heatmap and volcano plot of DEGs are depicted in Fig. 3 for the merged data cohort comparing AF and SR. 3.2 Functional enrichment analysis of DEGs and MRDEGs To explore the potential functions of the DEGs, a functional enrichment analysis of the DEGs was conducted based on GO and KEGG terms. The various GO functions and KEGG pathways are presented in Fig. 4 A, including muscle tissue development, gland morphogenesis, morphogenesis of an epithelial fold in the Biological Process (BP) category; collagen-containing extracellular matrix, sarcomere, myofibril in the Cellular Component (CC) category; extracellular matrix structural constituent, heparin binding, glycosaminoglycan binding in the Molecular Function (MF) category; and Aldosterone synthesis and secretion, and PI3K-Akt signaling pathway in the KEGG pathway. These results indicate a strong correlation between the selected DEGs and cardiac fibrosis, suggesting that these DEGs play a crucial role in the development of AF. Based on our previous study, overexpression of miR-450a-2-3p suppressed the expression of ERK(1/2) in cardiac fibroblasts (CFs), leading to the inhibition of α-SMA, COL1, and COL3 expression and the prevention of CF proliferation. In HUVECs, overexpression of miR-450a-2-3p upregulated the expression of VE-Cadherin (VE-Cad) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by inhibiting the expression of ERK(1/2), while the expression levels of vimentin, COL1, and COL3 decreased. Furthermore, we confirmed that ERK(1/2) is a direct target of miR-450a-2-3p using a Luciferase assay. Now, we aim to further explore the relationship between the target genes of miR-450a-2-3p and DEGs. The potential target genes of miR-450a-2-3p were predicted using TargetScan, a database for microRNA-target interactions, resulting in 4893 predicted target genes. Subsequently, the miR-450a-2-3p-related differentially expressed genes (MRDEGs) that overlapped with the miR-450a-2-3p-predicted genes and DEGs were identified. Finally, 31 meaningful MRDEGs were selected using two datasets, as indicated in the Venn plot (Fig. 4 B). Remarkably, approximately a quarter of the DEGs were predicted to be target genes of miR-450a-2-3p, highlighting its significant role in cardiac fibrosis. GO and pathway analyses were performed to determine the biological functions of the MRDEGs using the Metascape database. The MRDEGs were primarily associated with response to hormone, muscle tissue development, transmembrane receptor protein serine/threonine kinase signaling pathway, cell-cell adhesion, and other processes. Moreover, these MRDEGs were predominantly enriched in VEGFA VEGFR2 signaling and the Muscle contraction signaling pathway network map (Fig. 4 C-D). These findings suggest that the anti-fibrotic effect of miR-450a-2-3p is likely achieved through these mechanisms. 3.3 Increasing expression of miR-450a-2-3p and inhibiting ERK(1/2) reverse cardiac fibrosis in ISO induced mice After demonstrating that miR-450a-2-3p targets ERK(1/2) to mediate TGF-β-induced cardiac fibrosis in vitro, our objective was to assess whether miR-450a-2-3p could serve as a potential therapeutic target for reducing atrial fibrosis in vivo. The mice were randomly divided into four groups. The model group received intraperitoneal injections of isoproterenol (ISO), while the blank control group received an equal amount of normal saline. The experimental group and negative control group were injected with miR-450a-2-3p agomir and agomir NC via the tail vein, respectively. The heart weight to body weight ratio (HW/BW) was measured in each group, and the results showed no significant differences between the control group and the ISO-induced group. The miR-450a-2-3p agomir group and the ISO-induced group exhibited similar results (Fig. 5 A). HE staining results suggested that mice in the ISO group displayed cardiomyocyte hypertrophy, disordered arrangement, and a significant increase in myocardial interstitial cells compared to the control group. However, after injection of miR-450a-2-3p agomir, these pathological changes significantly improved (Fig. 5 B). Masson staining revealed collagen deposition and severe cardiac fibrosis in the ISO model group compared to the control group. However, mice injected with miR-450a-2-3p agomir showed a significant reduction in collagen deposition and collagen volume fraction (Fig. 5 B-C). These findings indicate the successful construction of a mouse myocardial fibrosis model at the tissue level, and miR-450a-2-3p can reverse the fibrosis process. Furthermore, we investigated the regulatory effect of miR-450a-2-3p on fibrosis markers in mice with myocardial fibrosis. We performed RT-qPCR assays to detect the expression levels of miR-450a-2-3p in myocardial tissue between the control group and the ISO-induced group. Compared to the blank control group, the expression of miR-450a-2-3p in myocardial tissue of mice induced by ISO modeling was significantly reduced. In contrast, the expression levels of miR-450a-2-3p increased significantly in myocardial tissue injected with agomir, while the expression levels decreased significantly in myocardial tissue transfected with agomir NC. These results indicate the successful establishment of the model for the injection of miR-450a-2-3p agonists (Fig. 5 D). At the mRNA level, overexpression of miR-450a-2-3p reduced ISO-induced upregulation of α-SMA, COL1, and COL3 (Fig. 5 E-G). Western blotting results also demonstrated that the expression of these collagen biomarkers at the protein level was significantly reduced after treatment with miR-450a-2-3p, consistent with the transcriptional level. However, although COL1 showed a downward trend in the miR-450a-2-3p treatment group compared to the modeling group, the results were not statistically significant (Fig. 5 H-K). These findings suggest that miR-450a-2-3p inhibits ISO-induced collagen formation in myocardial tissue. Similarly, to confirm whether the in vivo improvement of miR-450a-2-3p on mouse myocardial fibrosis is achieved by inhibiting ERK(1/2), we observed changes in the nucleic acid and protein expression levels of ERK(1/2) under ISO and miR-450a-2-3p intervention. The results from both RT-qPCR and Western blotting assays demonstrated an increase in ERK(1/2) expression in the ISO-induced group, while miR-450a-2-3p inhibited ERK(1/2) expression, consistent with our previous cell experiments (Fig. 5 L-O). Discussion Atrial fibrillation (AF) is frequently associated with cardiac fibrosis[3], however, the specific molecular mechanisms underlying AF remain unclear. The primary treatment methods for AF patients include drugs for rate control, oral anticoagulants for stroke prevention, antiarrhythmic drugs, and catheter ablation for conversion[9]. Despite significant advancements in recent decades, the fundamental mechanisms of AF have yet to be fully understood, and effective therapies for combatting AF are still lacking[10]. In this study, we merged two gene expression datasets from the GEO database and conducted an integrated analysis. We detected 127 DEGs and found that muscle tissue development, gland morphogenesis, and morphogenesis of an epithelial fold were significantly correlated with cardiac fibrosis. EndMT is a transcriptional program that downregulates the expression of endothelial genes and upregulates the expression of mesenchymal genes. Endocardial endothelial cells can transdifferentiate into fibroblasts via EndMT and contribute to extracellular matrix deposition[11–14]. Pathway analysis showed that these 127 DEGs were mainly highly correlated with aldosterone synthesis and secretion and PI3K-Akt signaling. The renin-angiotensin-aldosterone system is known to play a crucial role in the structural and electrical remodeling of the heart[15]. Zhang et al. found that the principal signaling pathways of PI3K/AKT are mainly activated in various pathological states, such as fibrosis, apoptosis, and regeneration after myocardial infarction[16]. Upregulated PI3K and phosphorylation of AKT may be involved in the increased proliferation and migration of CFs[17]. In this study, we found that miR-450a-2-3p regulates nearly a quarter of differentially expressed genes (DEGs). Further functional and pathway analysis of miR-450a-2-3p was conducted using Metascape. Notably, the transmembrane receptor protein serine/threonine kinase signaling pathway, VEGFA VEGFR2 signaling, and Muscle contraction signaling pathway were identified as significantly enriched pathways. ERK1/2, a serine/threonine kinase involved in the MAP kinase signal transduction pathway, has been reported to play a critical role in various biological processes, including myocardial fibrosis[18–21]. Guo et al. demonstrated that H19 promoted cardiac fibroblast (CF) proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis[22]. Furthermore, studies have suggested that the relationship between tissue factor and VEGF and its receptor in atrial fibrillation may indicate a possible role for VEGF in the hypercoagulable state observed in AF[23]. It is evident that miR-450a-2-3p plays a crucial role in multiple functions and pathways related to cardiac fibrosis. Wu et al. reported reduced expression of miR-450a-2-3p in gastric cancer tissue, while the expression level of the oncogenic factor JHDM1D-AS1 was increased. Functionally, JHDM1D-AS1 depletion caused a significant reduction in cell proliferation and invasion both in vitro and in vivo, while the effects were nullified with the addition of miR-450a-2-3p inhibitor. Mechanistically, JHDM1D-AS1 promoted gastric cancer progression via the sponging of miR-450a-2-3p to increase PRAF2 expression[24]. Additionally, the expression of miR-450a-2-3p was also reduced in a study of tuberous sclerosis complex, indicating that miR-450a-2-3p may play a vital role in inhibiting cell proliferation[25]. However, the role of miR-450a-2-3p in myocardial fibrosis in patients with atrial fibrillation remains to be investigated. Our previous findings in cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs) demonstrated that miR-450a-2-3p can suppress cardiac fibrosis by downregulating ERK(1/2) signaling[8]. In CFs, overexpression of miR-450a-2-3p attenuated the upregulation of α-SMA, COL1, and COL3 induced by TGF-β1. The CCK8 assay revealed that TGF-β1 stimulation promoted CF proliferation, but this effect was reversed by treatment with the miR-450a-2-3p mimic. In HUVECs, TGF-β1 stimulation decreased the mRNA and protein expression levels of VE-CAD and PECAM-1/CD31, while increasing the expression levels of vimentin, COL1, and COL3. The miR-450a-2-3p mimic reversed these changes in gene expression levels. Scratch experiments demonstrated that TGF-β1 accelerated HUVEC migration, which was blocked by the miR-450a-2-3p mimic. Conversely, the miR-450a-2-3p inhibitor significantly facilitated HUVEC migration. Transfection with the miR-450a-2-3p mimic markedly reduced endogenous ERK(1/2) expression, while transfection with the miR-450a-2-3p inhibitor enhanced ERK(1/2) mRNA and protein expression levels in both CFs and HUVECs. Luciferase reporter gene assays confirmed that miR-450a-2-3p directly targeted ERK(1/2). Overexpression of ERK(1/2) abolished the inhibitory effect of miR-450a-2-3p on CFs. Furthermore, the scratch assay showed that the inhibitory effect of the miR-450a-2-3p mimic on HUVEC migration was significantly blocked by ERK(1/2). The aim of this study was to investigate the function and mechanism of miR-450a-2-3p in the pathogenesis of atrial fibrillation (AF) using a mouse model. Our data demonstrate that overexpression of miR-450a-2-3p, a downregulated miRNA in AF, can alleviate heart fibrosis, which may be achieved through the inhibition of ERK1/2 signaling. The first major finding of this study was the observation of reduced expression of miR-450a-2-3p in mice treated with isoproterenol (ISO). Subsequent experiments revealed that overexpression of miR-450a-2-3p in vivo resulted in the suppression of heart fibrosis, as evidenced by a reduction in the levels of fibrotic markers such as α-SMA, COL1, and COL3. ERK(1/2), also known as MAPK1, belongs to the MAPK family and is involved in various cellular processes, including proliferation, differentiation, transcription modulation, and development. Numerous studies have demonstrated the significance of ERK(1/2) in fibrogenesis. For example, Thum et al. discovered that miR-21 modulates the ERK(1/2) signaling pathway, influencing cardiac fibroblast growth and the secretion of cytokines associated with interstitial fibrosis[20]. M. Harada et al. found that TRPC3 regulates myocardial fibrosis proliferation by influencing Ca2 + influx through the MAPK1/miRNA-26/NFAT pathway, thereby increasing TRPC3 expression in the myocardium[21]. Our study provides evidence for the in vivo regulation of miR-450a-2-3p on ERK(1/2). However, it is important to acknowledge the limitations of the present study. Firstly, we did not extensively utilize bioinformatics tools or include additional datasets to explore potential therapeutic targets. Secondly, our study focused solely on investigating the role of one specific miRNA (miR-450a-2-3p), and future studies should aim to elucidate the functions of other potentially important miRNAs based on microarray data. Thirdly, while our animal model successfully replicated cardiac fibrosis in mice, it would be beneficial for future studies to investigate the role of miRNAs or genes in other animal models as well. Lastly, despite including a total of 85 participants, the input data might still be insufficient to identify and validate key genes involved in atrial fibrillation development. It is worth noting that these participants came from various regions with different diets, levels of physical activity, genetic variations, and susceptibility to cardiovascular diseases, all of which may have influenced the occurrence of atrial fibrillation. Conclusion In summary, our study identified 127 differentially expressed genes (DEGs) and 31 miRNA-regulated DEGs (MRDEGs) as potential key biomarkers for atrial fibrillation (AF) using bioinformatics methods. Furthermore, our in vivo experiments demonstrated that administration of miR-450a-2-3p agonists effectively prevented AF development in mice with isoproterenol (ISO)-induced cardiac fibrosis. These findings provide insights into the molecular mechanisms underlying AF pathogenesis by elucidating the role of miR-450a-2-3p targeting the ERK(1/2) pathway. Abbreviations AF Atrial fibrillation α-SMA Alpha-smooth muscle actin CFs Cardiac fibroblasts CVF Collagen volume fraction ERK1/2 Extracellular regulated protein kinases1/2 HUVECs Human umbilical vein endothelial cells ISO Isoproterenol MAPK Mitogen-activated protein kinase MFB Myofibroblast RAAS Renin-Angiotensin-Aldosterone system TGF-β1 Transforming growth factor-β1 Declarations Acknowledgements Not applicable. Author contributions LLS performed the experiments, conducted data analysis, and drafted the manuscript. LFY contributed to the conception and design of the study and critically revised the manuscript. The initial draft of the manuscript was written by LLS and all authors provided feedback on earlier versions. All authors have read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (no. 82070352). Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. Ethical approval The study was approved by the Ethic Committee of Xiangya Hospital Central South University (201803209). The research was conducted according to the World Medical Association Declaration of Helsinki. Conflict of interest The data underlying this article will be shared on reasonable request to the corresponding authors. 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Beijing Da Xue Xue Bao Yi Xue Ban, 2017. 49 (4): p. 580-584. Additional Declarations No competing interests reported. Supplementary Files supplement.docx Cite Share Download PDF Status: Published Journal Publication published 19 Aug, 2024 Read the published version in Genes & Nutrition → Version 1 posted Editorial decision: Revision requested 10 Mar, 2024 Reviews received at journal 04 Mar, 2024 Reviewers agreed at journal 26 Feb, 2024 Reviewers invited by journal 25 Feb, 2024 Editor assigned by journal 25 Feb, 2024 Submission checks completed at journal 16 Feb, 2024 First submitted to journal 05 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3930815","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273148045,"identity":"df92fb01-fe7a-483b-8331-259cc1893cf9","order_by":0,"name":"Langsha Liu","email":"","orcid":"","institution":"The Third Xiangya Hospital of Central South University","correspondingAuthor":false,"prefix":"","firstName":"Langsha","middleName":"","lastName":"Liu","suffix":""},{"id":273148046,"identity":"4eb50528-b63b-46ec-b3ff-e212ae624026","order_by":1,"name":"Fanyan Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYLCCBzYMcmzs7QdI0JKQxmDMx3MmgTQtifMkHAyIU21wI8dMIiHhcHqbBEMCw4+KbYS1SM6AaMltk248wNhz5jZhLfwSQC2JP4BaZA4kMDO2EaGFTQLqMDaJBAPitPBDtSQQr0Wy51mxRUJCumEbMJAPEuUXg+PJG298SLCWl29vP/jgRwURWhgEMkwkGBiawewDRKgHAv7jjz8wMNQRp3gUjIJRMApGJgAABJg8rbyLq1AAAAAASUVORK5CYII=","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Fanyan","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2024-02-05 12:00:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3930815/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3930815/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12263-024-00753-6","type":"published","date":"2024-08-19T15:57:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51335943,"identity":"9cf91871-1ee2-4f43-948a-4f1a4c8bd2f5","added_by":"auto","created_at":"2024-02-19 19:24:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111165,"visible":true,"origin":"","legend":"\u003cp\u003eThe overall protocol of this study.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/e30ab0ecbd0907b88571a6d8.png"},{"id":51335944,"identity":"3aae08d5-e6f0-4bfd-95ef-2f53a217b10e","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":499949,"visible":true,"origin":"","legend":"\u003cp\u003eThe normalized expression matrices \u003cstrong\u003e(A-B)\u003c/strong\u003e and PCA plots \u003cstrong\u003e(C)\u003c/strong\u003e of the GSE115574 and GSE79768 datasets are shown. PCA stands for principal component analysis.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/312842a13dbc62fb7d246166.png"},{"id":51335946,"identity":"642eb4b7-cfe3-45a9-b100-1e9eb0add8d1","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":269449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Heatmap visualization of differentially expressed genes (DEGs) between atrial tissue samples from patients with atrial fibrillation (AF) and sinus rhythm (SR). \u003cstrong\u003eB\u003c/strong\u003e Volcano plot visualization of DEGs between AF and SR atrial tissue samples, with red and blue dots representing genes with high and low differential expression, respectively. DEGs, diferentially expressed genes; AF, atrial fbrillation; SR, sinus rhythm\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/33d747d6fd55cb1d979d9926.png"},{"id":51335945,"identity":"09f08dbd-e30e-441f-9ee3-089ed391fe0d","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":339355,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis of DEGs and MRDEGs between AF and SR atrial tissue samples via GO and KEGG database. \u003cstrong\u003eA\u003c/strong\u003e Cord diagram shows the relationship between key DEGs and most enriched enriched GO terms. \u003cstrong\u003eB\u003c/strong\u003eVenn plot of the overlapping genes identifed by the DEGs algorithm and miR-450a-2-3p-predicted genes algorithm. \u003cstrong\u003eC\u003c/strong\u003e Gene ontology enrichment analysis and pathway analysis of MRDEGs. \u003cstrong\u003eD\u003c/strong\u003e Network relationship plots among all enriched terms. Colored by p-value, where terms containing more genes tend to have a more significant p-value. AF, atrial fbrillation; SR, sinus rhythm; DEGs, diferentially expressed genes; MRDEGs, miR-450a-2-3p-related differentially expressed genes\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/6eb5c2b464a366b1fa3829c8.png"},{"id":51335948,"identity":"ac7360ad-1a2f-4c9c-8415-929315b392f2","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1099428,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of miR-450a-2-3p attenuates the atrial fbrosis caused by ISO. \u003cstrong\u003eA\u003c/strong\u003e heart mass and body weight ratio (n=6) \u003cstrong\u003eB\u003c/strong\u003ePathology of the heart tissues in mice injected with miR-450-a-2-3p agomir and agomir NC was observed by H\u0026amp;E staining and Masson staining. \u003cstrong\u003eC \u003c/strong\u003eCollagen volume fraction (CVF). \u003cstrong\u003eD-G, L\u003c/strong\u003e Expression of miR-450a-2-3p, α-SMA, col1, col3 and ERK1 in the heart tissues of ISO-induced was determined using RT-qPCR. \u003cstrong\u003eH-K, M-O\u003c/strong\u003e Western blot analysis was conducted to examine the protein expression of key regulatory proteins of fibrosis (α-SMA, col1, col3 and ERK(1/2)) in ISO-induced mice injected with miR-450a-2-3p agomir and agomirNC.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/4041ab7ba507749c4b6e4f00.png"},{"id":51335947,"identity":"7b542aa5-74e8-4166-8800-81a29d23dfda","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":31078,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Materials and methods\u003cstrong\u003e \u003c/strong\u003esection.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/f25f8eb3f18420e3610e4822.jpeg"},{"id":63300624,"identity":"460f5356-9a65-4103-85ea-d0866906091c","added_by":"auto","created_at":"2024-08-26 16:15:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2840446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/91533310-3a74-4180-bf75-6dc8d7a770e9.pdf"},{"id":51335949,"identity":"2b0cc935-563e-46a0-9c26-5083cdd72cfa","added_by":"auto","created_at":"2024-02-19 19:24:04","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11780,"visible":true,"origin":"","legend":"","description":"","filename":"supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-3930815/v1/4c2cfe0962c0ee6d2c583272.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"miR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAtrial fibrillation (AF) is one of the most prevalent sustained supraventricular arrhythmias and is associated with an increased risk of stroke[1]. The underlying mechanism is believed to involve electrical and structural remodeling of the atria, leading to the development of cardiac fibrosis[2]. Atrial fibrosis plays a significant role in the persistence of AF and is considered one of the key factors contributing to its resistance to rhythm control interventions[3]. The activation and proliferation of cardiac fibroblasts (CFs), which are responsible for excessive production of extracellular matrix (ECM) components such as collagen-1 (COL1) and collagen-3 (COL3), play a crucial role in fibrogenesis in patients with AF[4]. Additionally, CFs can differentiate into myofibroblasts, cells that exhibit a twofold higher capacity for collagen synthesis, including alpha smooth muscle actin (α-SMA)[5]. Similarly, in cardiac fibrosis, endothelial cells (ECs) can undergo endothelial-mesenchymal transition (EndMT), acquiring a mesenchymal phenotype and expressing characteristic markers of myofibroblast differentiation, including α-SMA, vimentin, and collagens[6]. The expression of α-SMA, a well-characterized cytoskeletal protein, serves as a hallmark of myofibroblast differentiation[7]. However, the precise molecular mechanisms underlying cardiac fibrosis and EndMT in AF remain largely unclear.\u003c/p\u003e \u003cp\u003eIn our previous study, we observed downregulation of miR-450a-2-3p in CFs and human umbilical vein endothelial cells (HUVECs) exposed to transforming growth factor (TGF)-β1, suggesting its potential as a microRNA (miRNA) that inhibits cardiac fibrosis[8]. In the present study, we employed established bioinformatic tools to investigate the underlying mechanism of miR-450a-2-3p in cardiac fibrosis. Two transcriptome datasets were selected from the Gene Expression Omnibus (GEO) database for analysis. Differential gene expression analysis was conducted to identify differentially expressed genes (DEGs), as well as miR-450a-2-3p-related DEGs (MRDEGs). Subsequently, functional enrichment analysis was performed, revealing the involvement of miR-450a-2-3p in multiple biological processes and signaling pathways related to cardiac fibrosis. Furthermore, we validated the anti-fibrotic effect of miR-450a-2-3p overexpression in a mouse model of isoproterenol (ISO)-induced myocardial fibrosis. Collectively, our findings unveil a previously unknown microRNA that holds potential as a target for future clinical treatment and diagnosis of AF patients.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Data collection and download\u003c/h2\u003e \u003cp\u003eThe AF gene expression datasets, GSE41177 and GSE79768, were obtained from the GEO database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). GSE115574 (28 AF samples and 31 SR samples) and GSE79768 (14 AF samples and 12 SR samples) were generated using the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array platform. The R software packages \"limma\" and \"SVA\" were used to merge the GSE41177 and GSE79768 datasets, and batch effect was removed using the combat function. Data was adjusted using the FDR method. Differential expressed genes (DEGs) from the merged dataset were identified using the criteria |logFC| \u0026ge; 0.5 and adj.P. Val. Filter\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The \"heatmap\" package and \"ggplot2\" package were utilized to generate heatmaps and volcano plots of the DEGs. Prediction of miRNA-to-mRNA interactions was performed using TargetScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.targetscan.org/vert_72/\u003c/span\u003e\u003cspan address=\"https://www.targetscan.org/vert_72/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Functional enrichment analysis\u003c/h2\u003e \u003cp\u003eTo investigate the role of DEGs in AF patients, we utilized the Xiantao online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.xiantaozi.com/\u003c/span\u003e\u003cspan address=\"https://www.xiantaozi.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to conduct GO and KEGG pathway analysis. Additionally, the Metascape database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://metascape.org\u003c/span\u003e\u003cspan address=\"http://metascape.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) facilitated a comprehensive understanding of the biological significance of MRDEGs through Gene Ontology enrichment analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 ISO‑induced atrial fibrosis model in mice and grouping\u003c/h2\u003e \u003cp\u003eEight-week-old male C57BL/6 mice were utilized to establish an atrial fibrosis model through induction with ISO. A total of 24 mice were randomly divided into four groups: control group, ISO-induced model group, miR-450a-2-3p agomir group, and agomir-negative control (NC) group (n\u0026thinsp;=\u0026thinsp;6 per group). In the model and control groups, ISO (0.1ml/10g) and normal saline, respectively, were injected into the abdominal cavity for a duration of 14 days. Throughout this period, the mice were weighed on days 1, 7, and 14. After the 14-day treatment, the hearts of the mice were harvested for hematoxylin-eosin (H\u0026amp;E) staining, Masson staining, and the detection of α-SMA, col1, col3, and ERK (1/2) expression levels. The miR-450a-2-3p agomir and agomir-NC groups received injections of miR-450a-2-3p agomir (5nmol/each) and agomir-NC (5nmol/each), respectively, for a total of four times over a period of 16 days (once every 5 days). The administration of miR-450a-2-3p agomir and agomir-NC (GenePharma, Shanghai, China) was performed via tail vein injection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 H\u0026amp;E staining and Masson staining\u003c/h2\u003e \u003cp\u003eThe paraffin-embedded atrial tissue sections were dried in a 60\u0026deg;C incubator for more than 3 hours prior to staining. Standard protocols were followed for H\u0026amp;E staining and Masson staining. The slides were examined using an optical microscope and images were captured to evaluate histopathology and the extent of fibrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Reverse transcription quantitative polymerase chain reaction (RT‑qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted according to the manufacturer\u0026rsquo;s instructions. Briefly, take approximately 0.02g of tissue, add 1 mL of Trizol (Thermo Fisher Scientific, MA, USA) and mix well for 5 min. mRNAs were reverse transcribed using a reverse transcription kit (Cwbiotech, Beijing, China). RT-qPCR was performed using an UltraSYBR mixture (Cwbiotech, Beijing, China). The relative expression of genes was calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT. The mmu-miR-450a-2-3p, col1 and col3 expression of samples was normalized with that of GAPDH, and the α-sma and ERK (1/2) expression of samples was normalized with that of U6. All primers used in this study were commercially obtained from Sangon (Shanghai, China) and are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e \u003cp\u003e0.025 g of mouse heart tissues were lysed in 300 \u0026micro;L RIPA buffer (Abiowell, Changsha, China). After centrifugation at 12,000 \u0026times; g for 15 min at 4\u0026deg;C, the supernatant was collected. The protein concentration was determined using the bicinchoninic acid assay with a commercial kit (Cwbiotech, Beijing, China). Next, 120 \u0026micro;L of protein was separated using a 5\u0026times; loading buffer (30 \u0026micro;L) and then transferred to a nitrocellulose filter membrane. The membrane was incubated in 5% bovine serum albumin (BSA) at 4\u0026deg;C for 1.5 h and then incubated overnight with primary antibodies at 4\u0026deg;C. The primary antibodies used for Western blotting were as follows: β-actin (1:5000; Proteintech, IL, USA), α-SMA (1:3000; Proteintech, IL, USA), COL1 (1:2000; Proteintech, IL, USA), COL3 (1:500; Proteintech, IL, USA), and ERK (1:10000; Proteintech, IL, USA). After overnight incubation with the primary antibody, the membrane was incubated with the corresponding secondary antibodies (HRP goat anti-mouse IgG, 1:5000; HRP goat anti-rabbit IgG, 1:6000; Proteintech, IL, USA) at 25\u0026deg;C for 90 min. Detection was performed using an enhanced chemiluminescence (ECL) system (Abiowell, Changsha, China). Relative protein expression levels were analyzed using Quantity One v4.6.2 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe two-tailed Student's t-test was employed to compare the two distinct conditions. In situations where the data did not exhibit a normal distribution, the Mann-Whitney test was utilized. For experimental comparisons involving three or more experimental groups, Tukey's multicomparison test or one-way ANOVA with Dunnett's posthoc test was conducted. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, SanDiego, CA, USA). A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Screening of DEGs in AF\u003c/h2\u003e \u003cp\u003eThe schematic flowchart of this study is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Differential expression analysis was performed between 42 atrial fibrillation (AF) samples and 43 sinus rhythm (SR) samples from GSE79768 and GSE115574. Box plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B) and PCA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) effectively demonstrated the successful elimination of batch effects between GSE79768 and GSE115574. In the merged matrix, 126 differentially expressed genes (DEGs) were identified using the \"limma\" R package, including 50 upregulated genes and 76 downregulated genes. The heatmap and volcano plot of DEGs are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e for the merged data cohort comparing AF and SR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Functional enrichment analysis of DEGs and MRDEGs\u003c/h2\u003e \u003cp\u003eTo explore the potential functions of the DEGs, a functional enrichment analysis of the DEGs was conducted based on GO and KEGG terms. The various GO functions and KEGG pathways are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, including muscle tissue development, gland morphogenesis, morphogenesis of an epithelial fold in the Biological Process (BP) category; collagen-containing extracellular matrix, sarcomere, myofibril in the Cellular Component (CC) category; extracellular matrix structural constituent, heparin binding, glycosaminoglycan binding in the Molecular Function (MF) category; and Aldosterone synthesis and secretion, and PI3K-Akt signaling pathway in the KEGG pathway. These results indicate a strong correlation between the selected DEGs and cardiac fibrosis, suggesting that these DEGs play a crucial role in the development of AF.\u003c/p\u003e \u003cp\u003eBased on our previous study, overexpression of miR-450a-2-3p suppressed the expression of ERK(1/2) in cardiac fibroblasts (CFs), leading to the inhibition of α-SMA, COL1, and COL3 expression and the prevention of CF proliferation. In HUVECs, overexpression of miR-450a-2-3p upregulated the expression of VE-Cadherin (VE-Cad) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by inhibiting the expression of ERK(1/2), while the expression levels of vimentin, COL1, and COL3 decreased. Furthermore, we confirmed that ERK(1/2) is a direct target of miR-450a-2-3p using a Luciferase assay.\u003c/p\u003e \u003cp\u003eNow, we aim to further explore the relationship between the target genes of miR-450a-2-3p and DEGs. The potential target genes of miR-450a-2-3p were predicted using TargetScan, a database for microRNA-target interactions, resulting in 4893 predicted target genes. Subsequently, the miR-450a-2-3p-related differentially expressed genes (MRDEGs) that overlapped with the miR-450a-2-3p-predicted genes and DEGs were identified. Finally, 31 meaningful MRDEGs were selected using two datasets, as indicated in the Venn plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Remarkably, approximately a quarter of the DEGs were predicted to be target genes of miR-450a-2-3p, highlighting its significant role in cardiac fibrosis. GO and pathway analyses were performed to determine the biological functions of the MRDEGs using the Metascape database. The MRDEGs were primarily associated with response to hormone, muscle tissue development, transmembrane receptor protein serine/threonine kinase signaling pathway, cell-cell adhesion, and other processes. Moreover, these MRDEGs were predominantly enriched in VEGFA VEGFR2 signaling and the Muscle contraction signaling pathway network map (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). These findings suggest that the anti-fibrotic effect of miR-450a-2-3p is likely achieved through these mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Increasing expression of miR-450a-2-3p and inhibiting ERK(1/2) reverse cardiac fibrosis in ISO induced mice\u003c/h2\u003e \u003cp\u003eAfter demonstrating that miR-450a-2-3p targets ERK(1/2) to mediate TGF-β-induced cardiac fibrosis in vitro, our objective was to assess whether miR-450a-2-3p could serve as a potential therapeutic target for reducing atrial fibrosis in vivo. The mice were randomly divided into four groups. The model group received intraperitoneal injections of isoproterenol (ISO), while the blank control group received an equal amount of normal saline. The experimental group and negative control group were injected with miR-450a-2-3p agomir and agomir NC via the tail vein, respectively. The heart weight to body weight ratio (HW/BW) was measured in each group, and the results showed no significant differences between the control group and the ISO-induced group. The miR-450a-2-3p agomir group and the ISO-induced group exhibited similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). HE staining results suggested that mice in the ISO group displayed cardiomyocyte hypertrophy, disordered arrangement, and a significant increase in myocardial interstitial cells compared to the control group. However, after injection of miR-450a-2-3p agomir, these pathological changes significantly improved (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Masson staining revealed collagen deposition and severe cardiac fibrosis in the ISO model group compared to the control group. However, mice injected with miR-450a-2-3p agomir showed a significant reduction in collagen deposition and collagen volume fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). These findings indicate the successful construction of a mouse myocardial fibrosis model at the tissue level, and miR-450a-2-3p can reverse the fibrosis process.\u003c/p\u003e \u003cp\u003eFurthermore, we investigated the regulatory effect of miR-450a-2-3p on fibrosis markers in mice with myocardial fibrosis. We performed RT-qPCR assays to detect the expression levels of miR-450a-2-3p in myocardial tissue between the control group and the ISO-induced group. Compared to the blank control group, the expression of miR-450a-2-3p in myocardial tissue of mice induced by ISO modeling was significantly reduced. In contrast, the expression levels of miR-450a-2-3p increased significantly in myocardial tissue injected with agomir, while the expression levels decreased significantly in myocardial tissue transfected with agomir NC. These results indicate the successful establishment of the model for the injection of miR-450a-2-3p agonists (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). At the mRNA level, overexpression of miR-450a-2-3p reduced ISO-induced upregulation of α-SMA, COL1, and COL3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G). Western blotting results also demonstrated that the expression of these collagen biomarkers at the protein level was significantly reduced after treatment with miR-450a-2-3p, consistent with the transcriptional level. However, although COL1 showed a downward trend in the miR-450a-2-3p treatment group compared to the modeling group, the results were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-K). These findings suggest that miR-450a-2-3p inhibits ISO-induced collagen formation in myocardial tissue.\u003c/p\u003e \u003cp\u003eSimilarly, to confirm whether the in vivo improvement of miR-450a-2-3p on mouse myocardial fibrosis is achieved by inhibiting ERK(1/2), we observed changes in the nucleic acid and protein expression levels of ERK(1/2) under ISO and miR-450a-2-3p intervention. The results from both RT-qPCR and Western blotting assays demonstrated an increase in ERK(1/2) expression in the ISO-induced group, while miR-450a-2-3p inhibited ERK(1/2) expression, consistent with our previous cell experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL-O).\u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eAtrial fibrillation (AF) is frequently associated with cardiac fibrosis[3], however, the specific molecular mechanisms underlying AF remain unclear. The primary treatment methods for AF patients include drugs for rate control, oral anticoagulants for stroke prevention, antiarrhythmic drugs, and catheter ablation for conversion[9]. Despite significant advancements in recent decades, the fundamental mechanisms of AF have yet to be fully understood, and effective therapies for combatting AF are still lacking[10]. In this study, we merged two gene expression datasets from the GEO database and conducted an integrated analysis. We detected 127 DEGs and found that muscle tissue development, gland morphogenesis, and morphogenesis of an epithelial fold were significantly correlated with cardiac fibrosis. EndMT is a transcriptional program that downregulates the expression of endothelial genes and upregulates the expression of mesenchymal genes. Endocardial endothelial cells can transdifferentiate into fibroblasts via EndMT and contribute to extracellular matrix deposition[11\u0026ndash;14]. Pathway analysis showed that these 127 DEGs were mainly highly correlated with aldosterone synthesis and secretion and PI3K-Akt signaling. The renin-angiotensin-aldosterone system is known to play a crucial role in the structural and electrical remodeling of the heart[15]. Zhang et al. found that the principal signaling pathways of PI3K/AKT are mainly activated in various pathological states, such as fibrosis, apoptosis, and regeneration after myocardial infarction[16]. Upregulated PI3K and phosphorylation of AKT may be involved in the increased proliferation and migration of CFs[17].\u003c/p\u003e \u003cp\u003eIn this study, we found that miR-450a-2-3p regulates nearly a quarter of differentially expressed genes (DEGs). Further functional and pathway analysis of miR-450a-2-3p was conducted using Metascape. Notably, the transmembrane receptor protein serine/threonine kinase signaling pathway, VEGFA VEGFR2 signaling, and Muscle contraction signaling pathway were identified as significantly enriched pathways. ERK1/2, a serine/threonine kinase involved in the MAP kinase signal transduction pathway, has been reported to play a critical role in various biological processes, including myocardial fibrosis[18\u0026ndash;21]. Guo et al. demonstrated that H19 promoted cardiac fibroblast (CF) proliferation and collagen synthesis by suppressing the miR-29a-3p/miR-29b-3p-VEGFA/TGF-β axis[22]. Furthermore, studies have suggested that the relationship between tissue factor and VEGF and its receptor in atrial fibrillation may indicate a possible role for VEGF in the hypercoagulable state observed in AF[23]. It is evident that miR-450a-2-3p plays a crucial role in multiple functions and pathways related to cardiac fibrosis. Wu et al. reported reduced expression of miR-450a-2-3p in gastric cancer tissue, while the expression level of the oncogenic factor JHDM1D-AS1 was increased. Functionally, JHDM1D-AS1 depletion caused a significant reduction in cell proliferation and invasion both in vitro and in vivo, while the effects were nullified with the addition of miR-450a-2-3p inhibitor. Mechanistically, JHDM1D-AS1 promoted gastric cancer progression via the sponging of miR-450a-2-3p to increase PRAF2 expression[24]. Additionally, the expression of miR-450a-2-3p was also reduced in a study of tuberous sclerosis complex, indicating that miR-450a-2-3p may play a vital role in inhibiting cell proliferation[25]. However, the role of miR-450a-2-3p in myocardial fibrosis in patients with atrial fibrillation remains to be investigated.\u003c/p\u003e \u003cp\u003eOur previous findings in cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs) demonstrated that miR-450a-2-3p can suppress cardiac fibrosis by downregulating ERK(1/2) signaling[8]. In CFs, overexpression of miR-450a-2-3p attenuated the upregulation of α-SMA, COL1, and COL3 induced by TGF-β1. The CCK8 assay revealed that TGF-β1 stimulation promoted CF proliferation, but this effect was reversed by treatment with the miR-450a-2-3p mimic. In HUVECs, TGF-β1 stimulation decreased the mRNA and protein expression levels of VE-CAD and PECAM-1/CD31, while increasing the expression levels of vimentin, COL1, and COL3. The miR-450a-2-3p mimic reversed these changes in gene expression levels. Scratch experiments demonstrated that TGF-β1 accelerated HUVEC migration, which was blocked by the miR-450a-2-3p mimic. Conversely, the miR-450a-2-3p inhibitor significantly facilitated HUVEC migration. Transfection with the miR-450a-2-3p mimic markedly reduced endogenous ERK(1/2) expression, while transfection with the miR-450a-2-3p inhibitor enhanced ERK(1/2) mRNA and protein expression levels in both CFs and HUVECs. Luciferase reporter gene assays confirmed that miR-450a-2-3p directly targeted ERK(1/2). Overexpression of ERK(1/2) abolished the inhibitory effect of miR-450a-2-3p on CFs. Furthermore, the scratch assay showed that the inhibitory effect of the miR-450a-2-3p mimic on HUVEC migration was significantly blocked by ERK(1/2).\u003c/p\u003e \u003cp\u003eThe aim of this study was to investigate the function and mechanism of miR-450a-2-3p in the pathogenesis of atrial fibrillation (AF) using a mouse model. Our data demonstrate that overexpression of miR-450a-2-3p, a downregulated miRNA in AF, can alleviate heart fibrosis, which may be achieved through the inhibition of ERK1/2 signaling. The first major finding of this study was the observation of reduced expression of miR-450a-2-3p in mice treated with isoproterenol (ISO). Subsequent experiments revealed that overexpression of miR-450a-2-3p in vivo resulted in the suppression of heart fibrosis, as evidenced by a reduction in the levels of fibrotic markers such as α-SMA, COL1, and COL3.\u003c/p\u003e \u003cp\u003eERK(1/2), also known as MAPK1, belongs to the MAPK family and is involved in various cellular processes, including proliferation, differentiation, transcription modulation, and development. Numerous studies have demonstrated the significance of ERK(1/2) in fibrogenesis. For example, Thum et al. discovered that miR-21 modulates the ERK(1/2) signaling pathway, influencing cardiac fibroblast growth and the secretion of cytokines associated with interstitial fibrosis[20]. M. Harada et al. found that TRPC3 regulates myocardial fibrosis proliferation by influencing Ca2\u0026thinsp;+\u0026thinsp;influx through the MAPK1/miRNA-26/NFAT pathway, thereby increasing TRPC3 expression in the myocardium[21]. Our study provides evidence for the in vivo regulation of miR-450a-2-3p on ERK(1/2).\u003c/p\u003e \u003cp\u003eHowever, it is important to acknowledge the limitations of the present study. Firstly, we did not extensively utilize bioinformatics tools or include additional datasets to explore potential therapeutic targets. Secondly, our study focused solely on investigating the role of one specific miRNA (miR-450a-2-3p), and future studies should aim to elucidate the functions of other potentially important miRNAs based on microarray data. Thirdly, while our animal model successfully replicated cardiac fibrosis in mice, it would be beneficial for future studies to investigate the role of miRNAs or genes in other animal models as well. Lastly, despite including a total of 85 participants, the input data might still be insufficient to identify and validate key genes involved in atrial fibrillation development. It is worth noting that these participants came from various regions with different diets, levels of physical activity, genetic variations, and susceptibility to cardiovascular diseases, all of which may have influenced the occurrence of atrial fibrillation.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eIn summary, our study identified 127 differentially expressed genes (DEGs) and 31 miRNA-regulated DEGs (MRDEGs) as potential key biomarkers for atrial fibrillation (AF) using bioinformatics methods. Furthermore, our in vivo experiments demonstrated that administration of miR-450a-2-3p agonists effectively prevented AF development in mice with isoproterenol (ISO)-induced cardiac fibrosis. These findings provide insights into the molecular mechanisms underlying AF pathogenesis by elucidating the role of miR-450a-2-3p targeting the ERK(1/2) pathway.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Atrial fibrillation\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA \u0026nbsp; \u0026nbsp; Alpha-smooth muscle actin\u003c/p\u003e\n\u003cp\u003eCFs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Cardiac fibroblasts\u003c/p\u003e\n\u003cp\u003eCVF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Collagen volume fraction\u003c/p\u003e\n\u003cp\u003eERK1/2 \u0026nbsp; \u0026nbsp; \u0026nbsp;Extracellular regulated protein kinases1/2\u003c/p\u003e\n\u003cp\u003eHUVECs \u0026nbsp; \u0026nbsp;Human umbilical vein endothelial cells\u003c/p\u003e\n\u003cp\u003eISO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Isoproterenol\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMAPK \u0026nbsp; \u0026nbsp; Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003eMFB \u0026nbsp; \u0026nbsp; \u0026nbsp;Myofibroblast\u003c/p\u003e\n\u003cp\u003eRAAS \u0026nbsp; \u0026nbsp; \u0026nbsp;Renin-Angiotensin-Aldosterone system\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;1 \u0026nbsp; \u0026nbsp; Transforming growth factor-\u0026beta;1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLLS performed the experiments, conducted data analysis, and drafted the manuscript. LFY contributed to the conception and design of the study and critically revised the manuscript. The initial draft of the manuscript was written by LLS and all authors provided feedback on earlier versions. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (no. 82070352).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethic Committee of Xiangya Hospital Central South University (201803209). The research was conducted according to the World Medical Association Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data underlying this article will be shared on reasonable request to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChugh, S.S., et al., \u003cem\u003eWorldwide epidemiology of atrial fibrillation: a Global Burden of Disease 2010 Study.\u003c/em\u003e Circulation, 2014. \u003cstrong\u003e129\u003c/strong\u003e(8): p. 837-47.\u003c/li\u003e\n\u003cli\u003eCochet, H., et al., \u003cem\u003eAge, atrial fibrillation, and structural heart disease are the main determinants of left atrial fibrosis detected by delayed-enhanced magnetic resonance imaging in a general cardiology population.\u003c/em\u003e J Cardiovasc Electrophysiol, 2015. \u003cstrong\u003e26\u003c/strong\u003e(5): p. 484-92.\u003c/li\u003e\n\u003cli\u003eNattel, S., \u003cem\u003eMolecular and Cellular Mechanisms of Atrial Fibrosis in Atrial Fibrillation.\u003c/em\u003e JACC Clin Electrophysiol, 2017. \u003cstrong\u003e3\u003c/strong\u003e(5): p. 425-435.\u003c/li\u003e\n\u003cli\u003eSmaill, B.H., \u003cem\u003eFibrosis, myofibroblasts, and atrial fibrillation.\u003c/em\u003e Circ Arrhythm Electrophysiol, 2015. \u003cstrong\u003e8\u003c/strong\u003e(2): p. 256-7.\u003c/li\u003e\n\u003cli\u003eSnider, P., et al., \u003cem\u003eOrigin of cardiac fibroblasts and the role of periostin.\u003c/em\u003e Circ Res, 2009. \u003cstrong\u003e105\u003c/strong\u003e(10): p. 934-47.\u003c/li\u003e\n\u003cli\u003eZeisberg, E.M., et al., \u003cem\u003eEndothelial-to-mesenchymal transition contributes to cardiac fibrosis.\u003c/em\u003e Nat Med, 2007. \u003cstrong\u003e13\u003c/strong\u003e(8): p. 952-61.\u003c/li\u003e\n\u003cli\u003ePrunotto, M., et al., \u003cem\u003eStable incorporation of alpha-smooth muscle actin into stress fibers is dependent on specific tropomyosin isoforms.\u003c/em\u003e Cytoskeleton (Hoboken), 2015. \u003cstrong\u003e72\u003c/strong\u003e(6): p. 257-67.\u003c/li\u003e\n\u003cli\u003eLiu, L., F. 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Li, \u003cem\u003eCanonical transient receptor potential 3 channels in atrial fibrillation.\u003c/em\u003e Eur J Pharmacol, 2018. \u003cstrong\u003e837\u003c/strong\u003e: p. 1-7.\u003c/li\u003e\n\u003cli\u003eThum, T., et al., \u003cem\u003eMicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.\u003c/em\u003e Nature, 2008. \u003cstrong\u003e456\u003c/strong\u003e(7224): p. 980-4.\u003c/li\u003e\n\u003cli\u003eHarada, M., et al., \u003cem\u003eTransient receptor potential canonical-3 channel-dependent fibroblast regulation in atrial fibrillation.\u003c/em\u003e Circulation, 2012. \u003cstrong\u003e126\u003c/strong\u003e(17): p. 2051-64.\u003c/li\u003e\n\u003cli\u003eGuo, F., et al., \u003cem\u003eLncRNA H19 Drives Proliferation of Cardiac Fibroblasts and Collagen Production via Suppression of the miR-29a-3p/miR-29b-3p-VEGFA/TGF-beta Axis.\u003c/em\u003e Mol Cells, 2022. \u003cstrong\u003e45\u003c/strong\u003e(3): p. 122-133.\u003c/li\u003e\n\u003cli\u003eChung, N.A., et al., \u003cem\u003eIs the hypercoagulable state in atrial fibrillation mediated by vascular endothelial growth factor?\u003c/em\u003e Stroke, 2002. \u003cstrong\u003e33\u003c/strong\u003e(9): p. 2187-91.\u003c/li\u003e\n\u003cli\u003eWu, M., et al., \u003cem\u003eJHDM1D-AS1 aggravates the development of gastric cancer through miR-450a-2-3p-PRAF2 axis.\u003c/em\u003e Life Sci, 2021. \u003cstrong\u003e265\u003c/strong\u003e: p. 118805.\u003c/li\u003e\n\u003cli\u003eCai, Y., et al., \u003cem\u003e[MicroRNA differential expression profile in tuberous sclerosis complex cell line TSC2(-/-) MEFs and normal cell line TSC2(+/+) MEFs].\u003c/em\u003e Beijing Da Xue Xue Bao Yi Xue Ban, 2017. \u003cstrong\u003e49\u003c/strong\u003e(4): p. 580-584.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genes-and-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gnnu","sideBox":"Learn more about [Genes \u0026 Nutrition](http://genesandnutrition.biomedcentral.com)","snPcode":"12263","submissionUrl":"https://submission.nature.com/new-submission/12263/3","title":"Genes \u0026 Nutrition","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"atrial fibrillation, cardiac fibrosis, microRNA, mice","lastPublishedDoi":"10.21203/rs.3.rs-3930815/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3930815/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eCardiac fibrosis is a significant contributor to atrial fibrillation (AF). Our aim is to identify biomarkers for AF using bioinformatic methods and explore the regulatory mechanism of miR-450a-2-3p in cardiac fibrosis in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTwo datasets, GSE115574 and GSE79768, were obtained from the Gene Expression Omnibus (GEO) database and subsequently merged for further analysis. Differential gene expression analysis was performed to identify genes that were differentially expressed (DEGs) and genes that were differentially expressed in relation to miR-450a-2-3p (MRDEGs). To investigate the underlying mechanism of cardiac fibrosis, a mouse model was established by treating mice with isoproterenol (ISO) and miR-450a-2-3p agomir.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eA total of 127 DEGs and 31 MRDEGs were identified and subjected to functional enrichment analysis using Gene Ontology (GO) and pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) to determine the functions and pathways involved in atrial fibrillation (AF). In animal experiments, histological staining using H\u0026amp;E and Masson's trichrome, as well as quantification of collagen volume fraction (CVF), were performed. The increased expression of α-smooth muscle actin (α-SMA), collagen type I (col1), collagen type III (col3), and extracellular signal-regulated kinase (ERK) 1/2 at both the mRNA and protein levels indicated the presence of significant myocardial fibrosis in mice induced with isoproterenol (ISO). However, after the overexpression of miR-450a-2-3p agomir through caudal vein injection, there was a notable improvement in the cardiac morphology of the treated group. The expression levels of α-SMA, col1, col3, and ERK1/2 also significantly decreased, as confirmed by Western blot and RT-qPCR analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur study elucidates the mechanistic connection between ISO-induced myocardial fibrosis and the miR-450a-2-3p/ERK (1/2) signaling pathway, highlighting their role in the development of cardiac fibrosis. Modulating miR-450a-2-3p expression and inhibiting ERK (1/2) activation represent promising approaches for therapeutic intervention in atrial fibrillation.\u003c/p\u003e","manuscriptTitle":"miR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-19 19:23:59","doi":"10.21203/rs.3.rs-3930815/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-03-10T14:38:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-04T11:15:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0b7b476e-8fb2-40f7-a726-3aa4e9c2c1cd","date":"2024-02-26T09:49:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-25T15:15:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-25T15:10:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-16T05:12:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genes \u0026 Nutrition","date":"2024-02-05T11:46:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genes-and-nutrition","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gnnu","sideBox":"Learn more about [Genes \u0026 Nutrition](http://genesandnutrition.biomedcentral.com)","snPcode":"12263","submissionUrl":"https://submission.nature.com/new-submission/12263/3","title":"Genes \u0026 Nutrition","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2bbd6262-7c5c-424b-b915-d8681880824c","owner":[],"postedDate":"February 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:08:55+00:00","versionOfRecord":{"articleIdentity":"rs-3930815","link":"https://doi.org/10.1186/s12263-024-00753-6","journal":{"identity":"genes-and-nutrition","isVorOnly":false,"title":"Genes \u0026 Nutrition"},"publishedOn":"2024-08-19 15:57:35","publishedOnDateReadable":"August 19th, 2024"},"versionCreatedAt":"2024-02-19 19:23:59","video":"","vorDoi":"10.1186/s12263-024-00753-6","vorDoiUrl":"https://doi.org/10.1186/s12263-024-00753-6","workflowStages":[]},"version":"v1","identity":"rs-3930815","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3930815","identity":"rs-3930815","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}