Bioinformatic analysis for potential biological processes and key targets of heart failure-related stroke

Bioinformatic analysis for potential biological processes and key targets of heart failure-related stroke 心力衰竭相关脑卒中的潜在生物学过程和关键靶点的生物学分析 1 2 3 first-author 1 2 3 1 2 1 2 1 2 1 2 3 corresp Zhaoyu LIU, liuzhy98@mail.sysu.edu.cn Zhaoyu LIU, liuzhy98@mail.sysu.edu.cn liuzhy98@mail.sysu.edu.cn https://orcid.org/0000-0002-2754-8661 1 2 corresp Jingfeng WANG, dr_wjf@vip.163.com Jingfeng WANG, dr_wjf@vip.163.com dr_wjf@vip.163.com https://orcid.org/0000-0002-9292-6884 Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou510120, China Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou510120, China Laboratory of Cardiac Electrophysiology and Arrhythmia in Guangdong Province, Guangzhou510120, China Laboratory of Cardiac Electrophysiology and Arrhythmia in Guangdong Province, Guangzhou510120, China Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou510120, China Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou510120, China This study aimed to uncover underlying mechanisms and promising intervention targets of heart failure (HF)-related stroke. HF-related dataset GSE42955 and stroke-related dataset GSE58294 were obtained from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis (WGCNA) was conducted to identify key modules and hub genes. Gene Ontology (GO) and pathway enrichment analyses were performed on genes in the key modules. Genes in HF- and stroke-related key modules were intersected to obtain common genes for HF-related stroke, which were further intersected with hub genes of stroke-related key modules to obtain key genes in HF-related stroke. Key genes were functionally annotated through GO in the Reactome and Cytoscape databases. Finally, key genes were validated in these two datasets and other datasets. HF- and stroke-related datasets each identified two key modules. Functional enrichment analysis indicated that protein ubiquitination, Wnt signaling, and exosomes were involved in both HF- and stroke-related key modules. Additionally, ten hub genes were identified in stroke-related key modules and 155 genes were identified as common genes in HF-related stroke. OTU deubiquitinase with linear linkage specificity ( OTULIN ) and nuclear factor interleukin 3-regulated ( NFIL3 ) were determined to be the key genes in HF-related stroke. Through functional annotation, OTULIN was involved in protein ubiquitination and Wnt signaling, and NFIL3 was involved in DNA binding and transcription. Importantly, OTULIN and NFIL3 were also validated to be differentially expressed in all HF and stroke groups. Protein ubiquitination, Wnt signaling, and exosomes were involved in HF-related stroke. OTULIN and NFIL3 may play a key role in HF-related stroke through regulating these processes, and thus serve as promising intervention targets. 目的 2 本研究通过加权基因共表达网络分析（Weighted gene co-expression network analysis，WGCNA）揭示心力衰竭（心衰）相关脑卒中的发病机制，为临床防治心衰相关脑卒中提供关键靶点。 创新点 2 本研究基于网络的研究策略，首次揭示了心衰相关脑卒中的潜在分子相互作用机制以及可能参与的生物学过程，并筛选出其中的关键基因。 方法 2 我们从基因表达综合数据库（Gene Expression Omnibus database，GEO）中分别获得心衰和脑卒中相关的芯片数据，并通过WGCNA鉴定各自的关键模块与关键基因。然后我们将二者的关键模块与关键基因取交集后，得到心衰相关脑卒中的关键基因，并利用Reactome和Cytoscape数据库对关键基因进行功能注释。最后在多个数据集中对心衰相关脑卒中的关键基因进行验证。 结果 2 我们在心衰和脑卒中相关芯片数据中分别鉴定出两个关键模块，随后功能富集分析发现两种疾病的关键模块中的基因均参与了蛋白质泛素化，Wnt信号通路和外泌体三个生物学过程。取交集后，我们共得到155个共同基因，他们可能参与了心衰相关脑卒中的发病机制。其中， OTULIN 和 NFIL3 被识别为心衰相关脑卒中的关键基因。功能注释分析显示， OTULIN 参与了蛋白质泛素化和Wnt信号通路， NFIL3 参与了转录和翻译过程。最后，在多个心衰和脑卒中的芯片数据中均证实 OTULIN 和 NFIL3 表达显著上调。 结论 2 蛋白质泛素化，Wnt信号通路和外泌体等生物学过程在心衰相关脑卒中的发病机制中发挥了重要作用。 OTULIN 和 NFIL3 通过调节这些通路因而可作为心衰相关脑卒中潜在的干预靶点。 Cardioembolic stroke Heart failure Bioinformatics Weighted gene co-expression network analysis (WGCNA) 心力衰竭 心源性卒中 生物信息学分析 加权基因共表达网络分析（WGCNA） 1 Introduction 1 Stroke is currently the second leading cause of death and a major cause of disability worldwide, costing approximately 3% to 4% of medical care expenditures in Western countries ( Struijs et al., 2006 Struijs et al., 2006 ; GBD 2015 Mortality and Causes of Death Collaborators, 2016 GBD 2015 Mortality and Causes of Death Collaborators, 2016 ; Ding et al., 2019 Ding et al., 2019 ). Cardioembolic stroke (CS), a subtype in which cardiogenic emboli cause cerebral thromboembolic ischemia, is estimated to account for 14% to 30% of ischemic stroke but has greater potential to lead to disability ( Adams et al., 1993 Adams et al., 1993 ; Murtagh and Smalling, 2006 Murtagh and Smalling, 2006 ). A growing number of cardiovascular diseases are recognized as causes of cardiogenic emboli, among which heart failure (HF) is responsible for 9% of all strokes ( Pullicino and Homma, 2010 Pullicino and Homma, 2010 ). HF is a severe clinical syndrome derived from the progression of various heart diseases, with reduced cardiac pumping and filling functions. Previous studies showed that stroke occurred in 4%‍–‍5% of patients with severe HF, compared with 0.5% in those without HF ( Mischie et al., 2013 Mischie et al., 2013 ). Moreover, HF patients bear more than three times the risk of stroke compared with the general population ( Kamel and Healey, 2017 Kamel and Healey, 2017 ). It is assumed that HF-related stroke may be related to the following pathophysiological mechanisms. First, the elevated or activated circulating markers like fibrin, pro-inflammatory cytokines, and platelets suggest a hypercoagulable state in HF patients ( Thomas et al., 2016 Thomas et al., 2016 ). Second, dyskinetic regions of the left ventricle where blood stasis happens create a hotbed of thrombosis, one of the potential sources of cardiogenic emboli ( Schoner et al., 2015 Schoner et al., 2015 ). Finally, atrial fibrillation (AF), which is well known as the chief precursor of CS, is a common complication of HF. HF accompanied by AF may present in approximately 2% of strokes ( Bogiatzi et al., 2014 Bogiatzi et al., 2014 ; Kang et al., 2017 Kang et al., 2017 ; Gao et al., 2019 Gao et al., 2019 ). However, the underlying molecular biological processes of these pathophysiological mechanisms remain unclear. Anticoagulation is the cornerstone of CS prevention and treatment ( Aguilar et al., 2007 Aguilar et al., 2007 ). However, in contrast to the efficacy of anticoagulation in AF-related stroke, this therapeutic approach failed to reveal an overall clinical benefit for HF-related stroke regardless of hypercoagulability in HF patients. Two randomized controlled trials recruited HF patients treated with anticoagulant therapy and showed that no improvement in stroke episodes or other outcomes had taken place in this population ( Cleland et al., 2004 Cleland et al., 2004 ; Cokkinos et al., 2006 Cokkinos et al., 2006 ). Also, the Warfarin and Antiplatelet Therapy in Chronic Heart Failure (WATCH) trial indicated that significantly increased bleeding risk counteracted the positive effects when HF patients received anticoagulants to prevent stroke ( Massie et al., 2009 Massie et al., 2009 ). Thus, anticoagulation is less effective in preventing stroke for HF patients, and new intervention targets need to be identified. Recent studies illustrated that genetic factors occupy an irreplaceable role in the pathogenesis of stroke ( Malik and Dichgans, 2018 Malik and Dichgans, 2018 ; Dichgans et al., 2019 Dichgans et al., 2019 ; Zhou LY et al., 2019 Zhou LY et al., 2019 ), and several gene expression profiling studies have been performed to reveal the underlying genes and biological processes during stroke occurrence and progression. Malik et al. (2018) Malik et al. (2018) identified 22 novel stroke risk loci and provided a framework for prioritization of stroke risk variants and genes. Zou et al. (2019) Zou et al. (2019) identified ZNF566 , PDZK1IP1 , ZFHX3 , and PITX2 genes as significant biomarkers for AF-related stroke. Zhu WH et al. (2019) Zhu WH et al. (2019) found that IL1α , IL1β , IL6 , IL8 , CXCL1 , CXCL2 , CXCL20 , CCL4 , ICAM1 , and PTGS2 had a protective effect against stroke in females. However, no gene expression profile studies have yet focused on HF-related stroke, and the underlying genes and biological processes of HF-related stroke can be clearly understood by revealing its gene expression variations and patterns. Weighted gene co-expression network analysis (WGCNA) is an advanced analytical method for discovering gene‍‒‍gene relationship and genetic network‒disease relationship, with the advantages of system-level insight and high sensitivity to genes with low abundance or small fold change ( Wang et al., 2020 Wang et al., 2020 ). Recently, WGCNA has been increasingly used to construct co-expressed gene networks and aid in identifying underlying biological processes and key genes in disease-related networks ( Raghow, 2016 Raghow, 2016 ). In this study, we explored common biological processes from the co-expression networks of HF and CS so as to uncover the underlying biological processes of HF-related stroke. Even further, we identified key genes from the common genes of HF and CS to pinpoint promising intervention targets for HF-related stroke. 2 Materials and methods 1 2.1 Data sources 2 A flow chart for the present study is shown in Fig. 1 Fig. 1 . The HF-related microarray datasets (GSE42955 and GSE1145) and stroke-related datasets (GSE58294 and GSE22255) were obtained from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo https://www.ncbi.nlm.nih.gov/geo ). In dataset GSE42955, left ventricular (LV) samples from explanted hearts of HF patients (New York Heart Association (NYHA) functional classifications of III and IV) were regarded as HF group, and non-diseased donor hearts were regarded as control group. This dataset was verified using the GPL6244 platform (Affymetrix Human Gene 1.0 ST Array). In dataset GSE58294, whole blood from subjects with CS (stroke group) and healthy controls (control group) was collected. This dataset was verified using the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array). In dataset GSE1145, failing myocardial samples from explanted hearts of HF patients were regarded as HF group and non-diseased donor myocardial samples were used as control group. This dataset was verified using the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array). In dataset GSE22255, peripheral blood from subjects with stroke (stroke group) and healthy controls (control group) was collected. This dataset was verified using the GPL570 platform (Affymetrix Human Genome U133 Plus 2.0 Array). GSE1145 and GSE22255 were used as validation datasets. 2.2 Data preprocessing 2 The "Combat" function in the Surrogate Variable Analysis package (SVA, Version 3.13) was applied to remove batch effects among microarrays ( Leek et al., 2012 Leek et al., 2012 ). Subsequently, all of the raw data were analyzed using bioinformatics methods, including background correction, quantile normalization, and log 2 -transformation of the expression values ( Irizarry et al., 2003 Irizarry et al., 2003 ; Ritchie et al., 2015 Ritchie et al., 2015 ). The probes exhibiting the top 50% in high expression variance (and thus minimal loss of statistical information) were identified and retained with a genefilter package Version 1.64.0 ( Gentleman et al., 2021 Gentleman et al., 2021 ) for subsequent analysis. Finally, the probe identifications (IDs) in the gene expression matrix were reannotated with gene symbols. 2.3 Construction of co-expressed gene modules 2 WGCNA was executed as follows: (1) outlier samples were detected by hierarchical clustering analysis; (2) an appropriate "soft" threshold ( β ) was selected by the "pickSoftThreshold" algorithm to obtain a biologically significant scale-free network (scale independence of > 0.8); (3) the expression similarity between each gene pair m and n was defined as adjacency value: a mn =power( S mn , β )=| S mn | β ( S mn was defined as the absolute value of the correlation coefficient between the profiles of genes m and n); (4) the adjacency matrix was transformed into a topological overlap matrix (TOM) by the "TOMsimilarity" algorithm to counter the effects of spurious or missing connections between genes m and n ( Yip and Horvath, 2007 Yip and Horvath, 2007 ); (5) modules were detected by hierarchical clustering analysis of TOM and the Dynamic Branch Cut method. 2.4 Identification of disease-related key modules 2 The analysis was conducted as follows: (1) module eigengene value was calculated by "moduleEigengenes" algorithm, which summarizes the gene expression profiles of a given module; (2) HF- or stroke-related key modules were identified through hierarchical clustering and Spearman correlation analysis of a module's module eigengene value and a sample's clinical traits; (3) the contribution of key modules to sample traits was assessed by principal component analysis (PCA). 2.5 GO and KEGG enrichment analyses of genes in disease-related key modules 2 Genes of HF-related key modules and stroke-related key modules were imported into the Metascape database ( https://metascape.org https://metascape.org ) to execute Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses ( Zhou YY et al., 2019 Zhou YY et al., 2019 ). GO describes the overrepresented biological functions among intramodular genes for three distinct aspects including molecular function, cellular component, and biological process ( Ashburner et al., 2000 Ashburner et al., 2000 ; The Gene Ontology Consortium, 2019 The Gene Ontology Consortium, 2019 ). KEGG describes the overrepresented pathways among intramodular genes ( Kanehisa and Sato, 2020 Kanehisa and Sato, 2020 ). A maximum P value of 0.01, minimum overlap of 3, and minimum enrichment of 1.5 were chosen as the threshold. 2.6 Identification of key genes in HF-related stroke 2 The common genes of HF-related stroke were obtained by taking the intersection of genes between HF-related key modules and stroke-related key modules. In addition, their protein–protein interaction (PPI) relationships were visualized with the STRING database ( https://string-db.org https://string-db.org ). In stroke-related key modules, gene significance and module membership of each intramodular gene were calculated by the "geneTraitSignificance" and "geneModuleMembership" algorithms to find stroke-related hub genes. Key genes of HF-related stroke were obtained by taking the intersection of the common genes of HF-related stroke and stroke-related hub genes. 2.7 GO and pathway annotation of key genes in HF-related stroke 2 GO annotates the key genes of HF-related stroke for three distinct aspects including molecular function, cellular component, and biological process. The Reactome database ( https://reactome.org https://reactome.org ) is an open-source, open-access, manually curated, and peer-reviewed pathway database ( Jassal et al., 2020 Jassal et al., 2020 ). Here, we used the Reactome database to annotate and visualize key genes-involved pathways. 2.8 Predicting regulatory targets of transcription factor 2 Transcription factor was introduced into the Cytoscape software Version 3.8.2 ( Shannon et al., 2003 Shannon et al., 2003 ) and the iRegulon plug-in Version 1.3 ( Janky et al., 2014 Janky et al., 2014 ) was used to predict the transcription factor targets. 2.9 Differential expression validation of key genes in HF- and stroke-related datasets 2 Differential expression analysis was performed with the limma package Version 3.38.3 ( Ritchie et al., 2015 Ritchie et al., 2015 ) on two HF-related datasets and two stroke-related datasets. P -value of < 0.05 was set as the threshold. 3 Results 1 3.1 Construction of co-expressed gene modules 2 To identify co-expressed gene modules in HF-related and stroke-related datasets, we executed WGCNA. As shown in Figs. 2a 2a and 2b 2b , samples from both datasets were clustered into two clusters, exactly corresponding to the disease group (HF or stroke) and control group, with no outliers detected. Then, based on scale independence of > 0.8, 8 and 6 were selected as the minimum β values for GSE42955 and GSE58294 respectively, to ensure biologically significant scale-free topology (Figs. 2c 2c and 2d 2d ). As shown in Figs. 2e 2e and 2f 2f , genes in GSE42955 were clustered into 17 modules and genes in GSE58294 were clustered into 20 modules by hierarchical clustering analysis and the Dynamic Branch Cut methods for the gene dendrograms. 3.2 Identification of disease-related key modules 2 To identify HF- and stroke-related key modules, module eigengene values of modules were correlated with a sample's clinical traits by applying an average linkage hierarchical clustering algorithm and the Spearman correlation coefficient. As shown in Figs. 3a 3a and 3c 3c , hierarchical cluster dendrograms and correlation heatmaps of module eigengene values and clinical traits indicated that the Maroon and Darkgrey modules were highly correlated with HF (Maroon: correlation coefficient=0.76, correlation P -value=4×10 -4 ; Darkgrey: correlation coefficient=0.76, correlation P -value=4×10 -4 ). Similarly, the Magenta and Darkolivegreen modules were highly correlated with stroke (Magenta: correlation coefficient=0.79, correlation P -value=3×10 -16 ; Darkolivegreen: correlation coefficient=0.76, correlation P -value=2×10 -14 ) (Figs. 3b 3b and 3d 3d ). Also, PCA results showed satisfactory distinguish ability for the Maroon and Darkgrey modules in response to HF, and for the Magenta and Darkolivegreen modules in response to stroke, indicating that genes in these four modules contributed to disease occurrence (Figs. 3e 3e and 3f 3f ). Therefore, we identified the Maroon and Darkgrey modules as HF-related key modules and the Magenta and Darkolivegreen modules as stroke-related key modules. 3.3 Functional enrichment analysis of genes in disease-related key modules 2 To reveal the underlying molecular biological processes in disease-related key modules, and more importantly, to find the common processes that link HF and CS, we performed GO and KEGG enrichment analyses. The top 20 GO and KEGG terms in HF-related and stroke-related key modules are shown in Fig. 4 Fig. 4 . In the HF-related key modules, the GO and KEGG terms were enriched in muscle system process, Wnt signaling, ubiquitin transferase activity, circadian rhythm, cell adhesion molecule binding, Fanconi anemia pathway, fibronectin binding, focal adhesion, extracellular matrix (ECM)‍-recepter interaction, transforming growth factor-‍β (TGF-‍β) activity, regulation of lipid localization, maintenance of location, response to nutrient availability, vacuolar lumen, cell migration, and exosomes. In stroke-related key modules, the GO and KEGG terms were enriched in complement and coagulation, Wnt signaling, telomere maintenance, myeloid leukocyte activation, cell body, transport vesicle, aging, Fc receptor signaling pathway, DNA-templated transcription, immune system process, chemical synaptic transmission, oxytocin signaling pathway, proteasome-mediated ubiquitination, synapse organization, exosomes, cellular component disassembly, and response to wounding. All the enriched GO and KEGG terms in HF-related and stroke-related key modules were shown in Table S1. Notably, genes in both HF-related key modules and stroke-related key modules were enriched in protein ubiquitination, Wnt signaling, and exosomes, which could prove to be biological processes for HF-related stroke. 3.4 Identification of key genes in HF-related stroke 2 To identify key genes in HF-related stroke, the centrally located intramodular hub genes in stroke-related key modules were identified at first. Module membership summarized the intramodular centrality of each gene. Hence, the top five genes in terms of module membership were selected as hub genes in each stroke-related key module, and they were nuclear factor interleukin 3-regulated ( NFIL3 ), RTN3 , AKIRIN2 , SNRPA1 , YIPF1 , PSMB5 , TIMM8A , PPP6C , OTU deubiquitinase with linear linkage specificity ( OTULIN ), and RAP1A . Additionally, gene significance measure summarized the clinical significance of each gene. As shown in Fig. 5 Fig. 5 a, the high correlation between gene significance and module membership implied that hub genes in the Magenta and Darkolivegreen modules were also highly correlated with CS. Next, the common genes of HF-related and stroke-related modules were obtained by intersection. As shown in Fig. 5 Fig. 5 b, a total of 155 common genes were obtained and their PPI were revealed. After further intersecting these common genes with hub genes of stroke-related key modules, OTULIN and NFIL3 were obtained, indicating that the two genes played a more critical role in the pathogenesis of HF-related stroke than other common genes. Therefore, OTULIN and NFIL3 were identified as key genes of HF-related stroke. 3.5 Functional annotation on key genes of HF-related stroke 2 To reveal the functions of OTULIN and NFIL3 in HF-related stroke, we used GO and pathway annotation. As shown in Fig. 6 Fig. 6 , NFIL3 mainly functioned as a transcription factor in the nucleus. Notably, OTULIN was involved in protein ubiquitination and Wnt signaling in the cytoplasm. Since protein ubiquitination and Wnt signaling were identified above as potential biological processes in HF-related stroke, OTULIN might act in HF-related stroke by participating in these two processes. To further understanding how NFIL3 acts in HF-related stroke, we used the iRegulon plug-in (Version 1.3) in Cytoscape (Version 3.8.2) to predict its regulatory targets, and we observed that DDIT3 , one of the common genes in HF-related stroke, was a potential target of NFIL3 ( Fig. 7 Fig. 7 a). GO annotation on DDIT3 also revealed that NFIL3 was involved in regulation of protein ubiquitination and the Wnt signaling pathway ( Fig. 7 Fig. 7 b). Thus, we believe that NFIL3 might control protein ubiquitination and Wnt signaling by regulating DDIT3 transcription in HF-related stroke. 3.6 Differential expression validation in HF- and stroke-related datasets 2 To validate the correlation between key genes and HF-related stroke in terms of fold change in expression, we performed differential expression analysis on the two datasets analyzed above (GSE42955 and GSE58294), and also on another HF-related dataset (GSE1145) and stroke-related dataset (GSE22255). As shown in Fig. 8 Fig. 8 a, with the threshold of P < 0.05, 55 differentially expressed common genes were identified in GSE42955, 43 differentially expressed common genes were identified in GSE58294, 85 differentially expressed common genes were identified in GSE1145, and 60 differentially expressed common genes were identified in GSE22255. By further intersecting the differentially expressed common genes of the four datasets, we found that NFIL3 and OTULIN were among the significantly differentially expressed genes in all HF and stroke groups, in line with the above results showing that OTULIN and NFIL3 played key roles in HF-related stroke ( Fig. 8 Fig. 8 b). 4 Discussion 1 In this study, WGCNA was performed to identify two HF-related and two stroke-related key modules. Functional enrichment analysis of these key modules identified the underlying biological processes in HF-related stroke, including protein ubiquitination, Wnt signaling, and extracellular exosome. In addition, 155 genes were identified as common genes in both HF- and stroke-related key modules, and are thus the top possibilities for genes that play a biological role in HF-related stroke. Most importantly, among these common genes, OTULIN and NFIL3 were identified as the hub genes of stroke-related key modules, indicating their key roles in HF-related stroke. Functional annotation revealed that key genes acted in HF-related stroke by participating in or regulating the biological processes identified above. Finally, NFIL3 and OTULIN were validated to be up-regulated in HF and stroke groups, supporting the importance of OTULIN and NFIL3 in HF-related stroke. Variations in gene expression and their patterns provide novel insight to the mechanism of HF-related stroke and assist in finding intervention targets. Jickling et al. (2010) Jickling et al. (2010) found expression differences in HF-specific genes such as MAPK1 , GNAQ , and MDM2 in peripheral blood of CS patients, and thus these genes might act in processes such as thrombopoietin signaling, nuclear factor-‍κB (NF-‍κB) activation, lymphocyte development, and inflammatory disorder. García-Berrocoso et al. (2020) García-Berrocoso et al. (2020) described several dysregulated genes (e.‍g., CREM , CYBB , and PTEN ) repeatedly identified in CS, which were associated with cardiomyopathy processes like cardiomyocyte apoptosis, ventricular abnormalities, and fibrosis. The current study is the first to perform gene co-expression pattern analysis (WGCNA) for CS. We applied WGCNA, a network-focused rather than individual gene/protein-focused analytic strategy, to evaluate expression similarity among genes under disease conditions and measure correlation between highly co-expressed gene sets (modules) and disease, which is a more effective technique for detecting gene expression with low abundance and variation compared to conventional differential expression analysis, as well as being less prone to information loss ( Horvath and Dong, 2008 Horvath and Dong, 2008 ; Pei et al., 2017 Pei et al., 2017 ). Quite a few studies have demonstrated its popularity in systematically identifying critical genes and relevant mechanisms of HF or stroke ( Liu et al., 2019 Liu et al., 2019 ; Niu et al., 2019 Niu et al., 2019 ; Pu et al., 2020 Pu et al., 2020 ; Wang et al., 2020 Wang et al., 2020 ). This study discovered three key biological processes in HF-related stroke by function enrichment analysis of HF-related and stroke-related key modules, including protein ubiquitination, Wnt signaling, and extracellular exosome. This finding indicated that the coagulation-anticoagulation pathway might not be a crucial mechanism for HF-related stroke, explaining the low effectiveness of anticoagulation therapy in stroke prevention for HF patients without AF. A previous study has shown that ubiquitin ligase blunts cardiomyocyte hypertrophy, the "knocking out" of which exacerbates cardiomyocyte hypertrophy in HF models ( Barac et al., 2017 Barac et al., 2017 ). In an ischemic stroke model, on the other hand, the ubiquitin E3 ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) aggravated ischemic stroke by ubiquitinating and activating Ras-related C3 botulinum toxin substrate 1 (Rac1) ( Li et al., 2017 Li et al., 2017 ). However, its effect on disease may be inversely depending on the target of ubiquitination because ubiquitination plays a central role in diverse cellular functions, so comprehensive and in-depth research is needed when studying its role in HF-related stroke ( Wojcik and di Napoli, 2004 Wojcik and di Napoli, 2004 ; Barac et al., 2017 Barac et al., 2017 ). The Wnt signaling pathway has been proven to participate in cardiac maturation and it is generally believed that cardiac hypertrophy involves reactivation of fetal programs, meaning that Wnt signaling may be reactivated in HF ( Blankesteijn et al., 2008 Blankesteijn et al., 2008 ). van de Schans et al. (2007) observed that knocking out a key member of Wnt signaling to block signal transduction inhibited the development of cardiac hypertrophy. In a mouse model of ischemic stroke, Wei et al. (2018) Wei et al. (2018) found a decrease in Wnt-3a (one of the canonical Wnt signaling ligands) and its downstream second messenger β-catenin, while Wnt-3a supplementation could alleviate stroke damage by its neuroprotective and regenerative actions. Changes in exosomes have been extensively reported in HF, and as one form of cell‒cell communication, exosomes exert various effects on cardiac hypertrophy and fibrosis depending on source cells, contents, and target cells ( Bang et al., 2014 Bang et al., 2014 ; Yang, 2018 Yang, 2018 ; Ranjan et al., 2019 Ranjan et al., 2019 ). Exosomes may function throughout time-series processes from stroke occurrence to neurorestorative events after stroke. Stroke onset involves processes such as exosome-mediated thrombosis and neuroinflammation ( Chen and Chopp, 2018 Chen and Chopp, 2018 ). The neuroprotective effects of exosomes after stroke include pro-neurogenesis, pro-angiogenesis, and neuronal plasticity ( Zhang and Chopp, 2016 Zhang and Chopp, 2016 ). We identified 155 common genes, which might act in HF-related stroke. Among these common genes, OTULIN and NFIL3 were identified as the hub genes of CS, indicating that they played a critical role in HF-related stroke. OTULIN encodes enzymes and specifically recognizes and removes Met1-linked ubiquitin chains from protein substrates, giving it biological significance in angiogenesis, craniofacial and neural development, and regulation of Wnt signaling as well as the NF-‍κΒ pathway ( Rivkin et al., 2013 Rivkin et al., 2013 ; Stangl et al., 2019 Stangl et al., 2019 ). Lentivirus-mediated overexpression of OTULIN attenuates microglia activation, proinflammatory cytokines release, and neuroinflammation by depressing the NF-κB pathway in ischemic stroke models ( Xu et al., 2018 Xu et al., 2018 ). OTULIN has not been reported to function in HF, but it is closely related to the immuno-inflammatory pathway and the Wnt signaling pathway, as we found by GO annotation, and both these pathways are known to be involved in HF. NFIL3 is well known as a basic leucine zipper transcription regulator, which activates interleukin 3-induced signaling pathways in T cells and hematopoietic cells, and also works in diverse cell processes such as development and survival of immune cells and circadian rhythm control ( Tamai et al., 2014 Tamai et al., 2014 ; Velmurugan et al., 2018 Velmurugan et al., 2018 ). Studies have not confirmed its role in HF or stroke. However, a review was devoted to elucidating the potential of NFIL3 as a new therapeutic target for HF due to its impact on several receptor-mediated processes which participate in pathogenesis of HF: calcium signaling, autocrine signaling, and insulin-like growth factor II signaling ( Velmurugan et al., 2018 Velmurugan et al., 2018 ). In addition, NFIL3 appears to be neuroprotective owing to its pro-survival and anti-apoptotic effects on neurons ( Tamai et al., 2014 Tamai et al., 2014 ). Interestingly, we predicted that DDIT3 was a potential regulatory target of NFIL3 , because it is reported to be involved in the regulation of protein ubiquitination and Wnt signaling. Therefore, based on these functions of OTULIN and NFIL3 , we speculated that OTULIN was likely to act in HF-related stroke as an executor by participating in protein ubiquitination and Wnt signaling, while NFIL3 might control protein ubiquitination and Wnt signaling as a regulator by regulating DDIT3 transcription in HF-related stroke. HF is a clinically, genetically, and pathophysiologically homogenous clinical entity. HF can be roughly classified into heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) ( Simmonds et al., 2020 Simmonds et al., 2020 ). Although it had been found that there was no evident correlation between left ventricular ejection fraction (LVEF) and the risk of stroke in HF patients, HFrEF and HFpEF showed significant differences in genetics, pathophysiology, and outcomes ( Kotecha et al., 2015 Kotecha et al., 2015 ; Chung et al., 2020 Chung et al., 2020 ). In HFpEF, genes enriched in mitochondrial adenosine triphosphate synthesis/electron transport are upregulated, which however are downregulated in HFrEF. HFpEF-specific altered genes are enriched in endoplasmic reticulum stress, autophagy, and angiogenesis ( Hahn et al., 2021 Hahn et al., 2021 ). In addition, HFrEF and HFpEF present with differences in inflammation and endothelial function, cardiomyocyte death and hypertrophy, alterations in the giant spring titin, and fibrosis ( Simmonds et al., 2020 Simmonds et al., 2020 ). Moreover, incidences of death (all-cause, cardiovascular, and sudden), intracerebral hemorrhage, and HF hospitalization increase progressively in HFrEF patients compared with HFpEF patients ( di Tullio et al., 2016 di Tullio et al., 2016 ). The patients included in our study were diagnosed as HFrEF with systolic dysfunction (LVEF of < 40%); hence, our findings should be interpreted carefully and whether they can be applied to HFpEF warrants further investigation. 5 Conclusions 1 Based on our genetic findings, protein ubiquitination, Wnt signaling, and exosomes may be involved in HF-related stroke. A total of 155 common genes are likely to act in the mechanism of HF-related stroke, among which OTULIN and NFIL3 play a more critical role in HF-related stroke by participating in or regulating protein ubiquitination and Wnt signaling; they thus have the potential to become promising intervention targets. References Adams HP , Bendixen BH , Kappelle LJ , et al. , 1993 . Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment . 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Bioinformatic gene analysis for potential biomarkers and therapeutic targets of atrial fibrillation-related stroke . J Transl Med , 17 : 45 . https://doi.org/10.1186/s12967-019-1790-x https://doi.org/10.1186/s12967-019-1790-x Bioinformatic gene analysis for potential biomarkers and therapeutic targets of atrial fibrillation-related stroke Zhaoyu LIU and Jingfeng WANG directed the project. Haifeng ZHANG designed experiments. Chiyu LIU and Sixu CHEN performed experiments and data analysis. Chiyu LIU drafted the manuscript. Yangxin CHEN, Qingyuan GAO, and Zhiteng CHEN perfected the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the National Natural Science Foundation of China (No. 81900387), the Guangdong Basic and Applied Basic Research Fund (No. 2019A1515011806), the Fundamental Research Funds for the Central Universities (No. 19ykpy97), and the Science and Technology Program of Guangzhou City of China (No. 201803040010). 10.1631/jzus.B2000544 1673-1581（2021）09-0718-15 2020-09-11 2021-02-08 2021-09-15 2022-05-09 2021-09-06 © Zhejiang University Press 2021 Chiyu LIU, Sixu CHEN, Haifeng ZHANG, Yangxin CHEN, Qingyuan GAO, Zhiteng CHEN, Zhaoyu LIU and Jingfeng WANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 9 22