Angiotensin converting enzyme 2 in coronavirus disease (COVID-19):Evidence from bioinformatics analysis

Recently, the outbreak of coronavirus disease 2019 (COVID-19) is threatening human health globally. There is a dire need to find potential therapeutic agents. Angiotensin converting enzyme 2 (ACE2), as an entry receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is considered as potential therapeutic target in COVID-19 pandemic. Here, our bioinformatics analysis revealed that the biological function of ACE2 was correlated with regulation of blood pressure and mediation of SARS-CoV-2 entry into host cells. Ten ACE2 cooperative proteins were identified by using STRING with a high score. ACE2 expressed highly in the small intestine, testis, and kidney. The level of ACE2 expression in tumor tissues varies in different types of cancers compared with that in normal tissues. It was worth noting that the expression level of ACE2 in the tumor has no effect on patient survival. MiRNA hsa-miR942-5p, and three transcription factors (TFs) including Signal transducer and activator of transcription 4 (STAT4), Estrogen related receptor α (ESRRA), and Signal transducer and activator of transcription 3 (STAT3) were selected as novel ACE2 regulators. Moreover, nine Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 26 April 2020 doi:10.20944/preprints202004.0466.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. potential therapeutic drugs were predicted by two online databases. Thus, our research may expand the overall view of ACE2 in COVID-19 treatment.


Introduction
The spread of COVID-19 is progressing rapidly. The high lethality of COVID-19, and its enormous economic and social impact, makes it paramount for us to battle this virus. Numerous therapeutic strategies have been applied to treat or block the spread of COVID-19.1 So far, there have been no specific drugs or methods identified to prevent the spread of the virus or to treat patients.2 Intensive research on the coronavirus is needed to elucidate its pathogenic mechanisms.
SARS-CoV-2 is a member of the β-coronavirus family, which includes the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). 3 In 2003, ACE2 in host cells was confirmed as the entry receptor of SARS-CoV4. Based on the similarity of the spike proteins between SARS-CoV and SARS-CoV-2, SARS-CoV-2 is also expected to bind to ACE2 as its cellular entry receptor.5 Therefore, ACE2-expressing cells and tissues potentially act as targets of the novel coronavirus.
Furthermore, the binding affinity between ACE2 and SARS-CoV-2 is approximately 10-to 20-fold higher than that between ACE2 and SARS-CoV,6 which might contribute to its quick transmission.
ACE2, a homolog of angiotensin-converting enzyme (ACE), is a transmembrane protein including an extracellular domain, transmembrane region, and intracellular component.7 The best-known function of ACE2 is to catalyze angiotensin II to angiotensin 1-7, which dilates the arterial vessels, counteracting the adverse action of the renin-angiotensin system.8 It was reported previously that ACE2 has a protective role in severe acute lung injury and reduces the vascular permeability and lung edema induced by acid aspiration.9 Moreover, loss of ACE2 increases urinary albumin excretion and is associated with glomerular lesions and more severe renal injury.10 ACE2 regulates innate immunity and microbial ecology in the intestine, and a deficiency of ACE2 increases the susceptibility to intestinal inflammation induced by epithelial damage.11 Therefore, ACE2 might become a potential target molecule for COVID-19 from the pharmacological perspective. 12 Presently, many supportive care plans and non-specific drugs are recommended to improve the symptoms of infected patients.13 However, few drugs or methods have been developed targeting the ACE2 receptor on host cells. Integrative bioinformatics methods for analyzing public data have been extensively applied to unravel reliable and precise clues for further research.14 By re-analyzing the public online data of ACE2, our study intends to offer a comprehensive atlas of ACE2 expression in the different tissues and diseases and shed a favorable light on potential treatments for COVID-19.
Currently, our research data provides the following suggestions for prevention and treatment of COVID-19. First, we can partially explain the symptoms of COVID-19 and the high comorbidities in hospitalized patients. Also, it was shown in our study that the expression level of ACE2 has no effect on cancer patients' survival. However, we suggest that more attention should be paid to individuals with cancer and impaired immune systems during the outbreak of COVID-19. Second, miRNA hsa-miR-942-5p, and the transcription factors STAT4, ESRRA, and STAT3, could be useful regulators for ACE2 expression. Also, nine drugs were identified as potential therapeutic medicine for COVID-19.

ACE2 expression analysis
To explore the biological function of ACE2, gene ontology (GO) enrichment was performed by DAVID version 6.8. Proteins with a high combined score with ACE2 identified with the STRING online database were selected to search for proteins associated with ACE2.
The protein-protein interaction (PPI) network and co-expression analysis of ACE2 and associated proteins were constructed by the STRING online database. The GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis generated by STRING were visualized by the ggplot2 package in R (3.6.2) software.

Tissues ACE2 expression analysis
The Human Protein Atlas (https://www.proteinatlas.org/), a human protein database, was used to map the expression of ACE2 in normal tissues. Gene-Cloud of Biotechnology Information (GCBI, https://www.gcbi.com.cn), a genetic and data combination platform, was used to analyze the expression of ACE2 in different diseases. Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia2.cancer-pku.cn), a gene expression analysis web server, was used to perform the survival analysis and determine the prognostic value of ACE2 in cancer patients.

Prediction of transcription factors for ACE2 and its associated genes
To understand the transcriptional mechanism involved in the modulation of ACE2 and its associated genes, the transcription factors were predicted by the iRegulon app in Cytoscape. In this study, the criteria for prediction of TFs was the following: the minimum identity between orthologous genes was 0.05, the maximum false discovery rate (FDR) on motif similarity was equal to 0.001, and targets > 5 and the NES > 4.5 were set as the threshold. The network was established with transcription factors-genes and visualized with Cytoscape.

Drug-target interactions
ACE2 served as the potential target for searching for drugs through DrugBank

ACE2 expression analysis
Our data indicated that the GO analysis of ACE2, which is mainly enriched in angiotensin maturation and regulation of systemic arterial blood pressure by renin-angiotensin, and at the same time we can see that ACE2 has a role in inflammatory responses and mediates entry of the virus into host cells ( Table 1). The cellular component enrichment showed that the protein ACE2 was composed of three parts: an extracellular region, components of the membrane, and plasma components. Its molecular function is mainly enriched in virus receptor activity, glycoprotein binding, endopeptidase activity, and carboxypeptidase activity. Furthermore, KEGG pathway enrichment suggested that the protein ACE2 is involved in the pathway of the renin-angiotensin system, and the protein digestion and absorption pathways.
Ten proteins (Type-1 angiotensin II receptor (AGTR1), Type-2 angiotensin II receptor plasma membrane part. By examining the KEGG pathway enrichment, we found that these proteins are mainly enriched in the renin-angiotensin system and protein digestion and absorption, which is similar to the ACE2 protein pathway enrichment.

Tissues ACE2 expression analysis
The expression levels of ACE2 protein and mRNA in normal tissues are shown in Figure   3A and B. Our data indicate that ACE2 is highly expressed in the small intestine, kidney, and testis, which indicates that there are high levels of ACE2 in the digestive system, urinary system, and genital system. GCBI online databases were searched to explore the expression of ACE2 in different diseases. As shown in Figure 3C, research related to ACE2 is mainly related to hypertension, severe acute respiratory syndrome, diabetes mellitus, kidney diseases, and cardiovascular diseases.
The gene expression differences between tumor and normal control tissue indicated that among these 33 cancers, some cancers like adrenocortical carcinoma (ACC), kidney renal papillary cell carcinoma (KIRP), and kidney renal clear cell carcinoma (KIRC) express higher levels of ACE2 than the normal tissues ( Fig 3D). Other cancers such as stomach adenocarcinoma (STAD), sarcoma (SARC), and kidney chromophobe (KICH) express lower levels of ACE2 than normal tissues. To understand the role of ACE2 in cancers, we explored the overall survival analysis through the GEPIA online database as shown in Fig 4A~J. The results showed that there are 4740 ACE2 higher expression samples and 4738 lower expression samples among 33 cancers.
There is no obvious difference in overall survival rate between the two groups ( Fig 4A). Further analysis showed that the expression level of ACE2 had no effect on patients' survival in higher level cancers like ACC and KIRP (Fig 4B and C) or in the lower level cancers like KICH, SARC, and STAD ( Fig 4H~J). Among the 33 types of tumors, the expression of ACE2 had an obvious effect only for four cancers, KIRC, brain Lower Grade Glioma (LGG), liver hepatocellular carcinoma (LIHC), and ovarian serous cystadenocarcinoma (OV). It is worth mentioning that high ACE2 increased the overall survival of patients with KIRC, LIHC, and OV, but decreased the overall survival rate of patients with LGG.

Prediction of miRNAs
The miRNAs of ACE2 that could be predicted in all three databases were identified as potential miRNAs. We found 60 miRNAs were predicted by DIANA, 180 miRNAs were predicted by miRwalk, 13 miRNAs were predicted by starBase, and 9 miRNAs were predicted by two databases. Only one miRNA, hsa-miR-942-5p, was predicted by all three databases as indicated in Fig 5B.

Prediction of transcription factors
As shown in Fig 5A, three transcription factors, STAT4, ESRRA, and STAT3, were predicted by iRegulon plugin in Cytoscape. STAT4 is linked to 6 genes, ESRRA is linked to 7 genes, and STAT3 is linked to 8 genes. ACE2 is targeted by all three transcription factors.

Drug-target interactions
Using ACE2 to explore drug protein interactions, nine promising drugs were compiled and selected from two databases ( Table 2). The drugs approved by the FDA are lisinopril, ramipril, chloroquine, and hydroxychloroquine. Lisinopril and ramipril are angiotensin converting enzyme inhibitors (ACEIs) and block the contact of angiotensin II with angiotensin converting enzyme by dilating constricted blood vessels 17 . Chloroquine and its derivative hydroxychloroquine are used to treat malaria, and are receiving increased attention due to their anti-inflammatory, immunomodulating, and antitumoral effects 18 .

Discussion
To explore the role of ACE2 in COVID-19, this research is, for the first time, using a bioinformatics method to explore the biological function of ACE2, reveal the expression of

Conclusion
Currently, COVID-19 is spreading rapidly and has thrown the whole world into a panic.
Intense investigation into the molecular mechanisms of the virus receptor ACE2 is essential for its prevention, diagnosis, and therapy. In this study, we identified the biological function of ACE2, partially explained the clinical manifestations and comorbidities of COVID-19, and predicted miRNA hsa-miR-942-5p, transcription factors STAT4, ESRRA, STAT3, and nine targeted drugs aimed at ACE2 by using bioinformatics methods. Further in-depth experiments are needed to confirm the rational therapy for treating COVID-19.

KEGG Pathway
Renin-angiotensin system, protein digestion and absorption