Genome Identification of potential proteases for abdominal aortic aneurysm by weighted gene coexpression network analysis Journal: Genome Manuscript ID gen-2020-0041.R1 Manuscript Type: Article Date Submitted by the 28-Jun-2020 Author: Complete List of Authors: Zhang, Hui; Peking Union Medical College Hospital, Department of Vascular Surgery Yang, Dan; Chinese Academy of Medical Sciences and Peking Union Medical College, Department of Computational Biology and Bioinformatics,Draft Institute of Medicinal Plant Development Chen, Siliang; Peking Union Medical College Hospital, Department of Vascular Surgery Li, Fangda; Peking Union Medical College Hospital, Department of Vascular Surgery Cui, Liqiang; Peking Union Medical College Hospital, Department of Vascular Surgery Liu, Zhili; Peking Union Medical College Hospital, Department of Vascular Surgery Shao, Jiang; Peking Union Medical College Hospital, Department of Vascular Surgery Chen, Yuexin; Peking Union Medical College Hospital, Department of Vascular Surgery Liu, Bao; Peking Union Medical College Hospital, Department of Vascular Surgery Zheng, Yuehong; Peking Union Medical College Hospital, Department of Vascular Surgery Abdominal aortic aneurysm, next-generation sequencing, WGCNA, Keyword: proteases, matrix metalloproteinase Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 35 Genome 1 Identification of potential proteases for abdominal aortic aneurysm by weighted gene 2 coexpression network analysis 3 Short title: WGCNA identifies crucial proteases in AAA 4 5 Hui Zhang1, Dan Yang2, Siliang Chen1, Fangda Li1, Liqiang Cui1, Zhili Liu1, Jiang Shao1, Yuexin 6 Chen1, Bao Liu1, Yuehong Zheng1. 7 1Department of Vascular Surgery, Peking Union Medical College Hospital, Beijing 100730, PR 8 China; 2Department of Computational Biology and Bioinformatics, Institute of Medicinal Plant 9 Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 10 100730, PR China. 11 Correspondence: Draft 12 Yuehong Zheng, MD, Department of Vascular Surgery, Peking Union Medical College Hospital, 13 Shuaifuyuan #1, Dongcheng District, Beijing 100730, China. 14 Phone #: +8613811015811 15 Email: [email protected] 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 35 16 Abstract 17 Proteases are involved in the degradation of the extracellular matrix, which contributes to the 18 formation of abdominal aortic aneurysm (AAA). To identify new disease targets in addition to the 19 results of previous microarray studies, we performed next-generation sequencing (NGS) of the 20 whole transcriptome of Angiotensin II-treated Apo E-/- male mice (n=4) and control mice (n=4) to 21 obtain differentially expressed genes (DEGs). Identified DEGs of proteases were analyzed using 22 weighted gene coexpression network analysis (WGCNA). RT-qPCR was conducted to validate 23 the differential expression of selected hub genes. We found that 43 DEGs were correlated with 24 the expression of the protease profile, and most were clustered in immune response module. 25 Among 26 hub genes, we found that Mmp16 and Mmp17 were significantly downregulated in AAA 26 mice, while Ctsa, Ctsc and Ctsw were Draftupregulated. Our functional annotation analysis of genes 27 coexpressed with the five hub genes indicated that Ctsw and Mmp17 were involved in T cell 28 regulation and Cell adhesion molecule pathway, respectively, and that both were involved in 29 general regulation of the cell cycle and gene expression. Overall, our data suggest that these 30 ectopic genes are potentially crucial to AAA formation and may act as biomarkers for the diagnosis 31 of AAA. 32 Key words: Abdominal aortic aneurysm, next-generation sequencing, transcriptome, WGCNA, 33 extracellular matrix, proteases, matrix metalloproteinase, cathepsin. 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 35 Genome 34 Introduction 35 Abdominal aortic aneurysm (AAA) is defined as a 50% increase in the diameter of the infrarenal 36 aorta and is an age-related degenerative disease(B Timothy, Terrin, & Dalman, 2008). The only 37 treatment option for AAA is surgical repair(Schermerhorn et al., 2008). It is necessary to explore 38 suitable noninvasive pharmacological treatments for this disease from a health economics 39 perspective. The pathogenesis of AAA formation, development and rupture is not fully understood. 40 Aortic wall inflammation is considered one of the major causes of AAA development and 41 progression(Goldstone, Malone, & Moore, 1978). After infiltration of immune inflammatory cells, 42 a wide range of immunoreactive mediators are released, which leads to direct degradation of the 43 aortic wall. Similarly, the development of intraluminal thrombi in the aortic wall is a source of 44 immune-derived proteases that exacerbateDraft pathogenesis(Jean-Baptiste et al., 2011). Collectively, 45 these events result in significant changes in the medial layer, including degradation of 46 extracellular matrix (ECM) structural proteins such as elastin and collagen fibers, which ultimately 47 lead to aortic wall expansion and rupture(Jean-Baptiste et al., 2011). 48 Proteases are known to be involved in AAA by degradation of the ECM during tissue 49 remodeling(Haiying, Sasaki, Jin, Kuzuya, & Cheng, 2018). The role of ECM proteases and their 50 role in AAA development has been extensively studied because they may serve as suitable 51 candidates for pharmacological treatments and can be specifically targeted without impairing 52 overall immunity. Key proteases that play pathological roles in AAA include elastase, chymase, 53 tryptase, matrix metalloproteinases (MMPs), cathepsins, granzyme B, etc.(Alon, Lisa S, & David 54 J, 2012). Elastases induce aneurysms via cleavage of elastin and destruction of elastic lamellae, 55 as well as generating proinflammatory elastin fragments (elastokines)(Folkesson, Silveira, 56 Eriksson, & Swedenborg, 2011). Chymases and tryptases can activate MMPs and cathepsins, 57 resulting in elastic lamellae fragmentation. They also promote immune infiltration and smooth 58 muscle cell apoptosis(Jiusong et al., 2009; Zhang, Sun, Lindholt, Sukhova, & Shi, 2011). MMPs 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 35 59 are a large class of endopeptidases that are essential in physiological homeostasis and 60 remodeling processes in the ECM. Deciphering the specific contribution of ECM proteases may 61 provide valuable insights into the development of therapeutic approaches for AAA(Alon et al., 62 2012). There are supporting data that MMP-1, 2, 3, 9, 12, and 13 are increased in the aortic wall 63 of AAA(Rabkin, 2017). Cathepsins, or cysteine proteases, are a family of lysosomal proteases 64 that act effectively as elastolytic and collagenolytic enzymes and therefore play a significant role 65 in the degradation of the ECM(Schulte et al.; Sun et al.). Increased activity of cathepsins B, H, L, 66 and S has been detected in the aortic wall with AAA(Abisi et al., 2007). In addition, cathepsin C, 67 a protease responsible for activating zymogen granzyme, may also contribute to the development 68 of AAA by increasing the activation and release of granzyme and promoting the recruitment of 69 neutrophils to the affected aorta(Pagano et al., 2007). The data are weaker or insufficient for other 70 MMPs or cathepsins. Therefore, further Draftresearch is necessary to explore other possibly important 71 but neglected proteases. 72 The rapidly increasing availability of transcriptomics data generated by RNA sequencing (RNA- 73 seq) provides us with the opportunity to use this information to generate testable hypotheses to 74 understand the molecular mechanisms that control gene expression and biological processes. 75 With the latest developments in transcriptomics and next-generation sequencing technology, 76 coexpression networks constructed from RNA-seq data can also identify or infer the functional 77 status of genes from a systematic perspective(Sipko, Thomas, & Pedro, 2014). One way to infer 78 gene function and gene-disease associations from genome-wide gene expression is weighted 79 gene coexpression network analysis (WGCNA), which builds a gene network with a tendency to 80 coactivate across a group of samples and analyzes this network(van Dam, Võsa, van der Graaf, 81 Franke, & de Magalhães, 2018). The application of the above technology will help us discover 82 new AAA-related proteases and potential biomarkers associated with the diagnosis and treatment 83 of AAA. 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 35 Genome 84 In this study, we first identified novel genes that might underlie the aneurysm formation of AAA in 85 ApoE-/- mice via high-throughput or next-generation sequencing (NGS) of the transcriptome. Then 86 we explored the gene expression modules that correlate with protease activity in AAA progression 87 by WGCNA based on the RNA-seq data. Furthermore, we selected specific genes from protease 88 family for RT-qPCR analyses to examine whether the differences in gene expression associated 89 with aneurysm formation translated into similar changes in protein expression. 90 Materials and Methods 91 Animals and treatment 92 Male Apo E-/- mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were 93 bred as littermate controls and housedDraft in pathogen-free barrier cages. They were fed a normal 94 laboratory diet. Mice at 10-12 weeks