key: cord-1001068-l69vt2rb authors: Askari, Nahid; Hadizadeh, Morteza; Rashidifar, Maryam title: A new insight into sex-specific non-coding RNAs and networks in response to SARS-CoV-2 date: 2021-12-23 journal: Infect Genet Evol DOI: 10.1016/j.meegid.2021.105195 sha: c200fff910cee831b712537c05872f5bcd3ba0c6 doc_id: 1001068 cord_uid: l69vt2rb SARS-CoV-2 is the RNA virus responsible for COVID-19, the prognosis of which has been found to be slightly worse in men. The present study aimed to analyze the expression of different mRNAs and their regulatory molecules (miRNAs and lncRNAs) to consider the potential existence of sex-specific expression patterns and COVID-19 susceptibility using bioinformatics analysis. The binding sites of all human mature miRNA sequences on the SARS-CoV-2 genome nucleotide sequence were predicted by the miRanda tool. Sequencing data was excavated using the Galaxy web server from GSE157103, and the output of feature counts was analyzed using DEseq2 packages to obtain differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) and DEG annotation analyses were performed using the ToppGene and Metascape tools. Using the RNA Interactome Database, we predicted interactions between differentially expressed lncRNAs and differentially expressed mRNAs. Finally, their networks were constructed with top miRNAs. We identified 11 miRNAs with three to five binding sites on the SARS-COVID-2 genome reference. MiR-29c-3p, miR-21-3p, and miR-6838-5p occupied four binding sites, and miR-29a-3p had five binding sites on the SARS-CoV-2 genome. Moreover, miR-29a-3p, and miR-29c-3p were the top miRNAs targeting DEGs. The expression levels of miRNAs (125, 181b, 130a, 29a, b, c, 212, 181a, 133a) changed in males with COVID-19, in whom they regulated ACE2 expression and affected the immune response by affecting phagosomes, complement activation, and cell-matrix adhesion. Our results indicated that XIST lncRNA was up-regulated, and TTTY14, TTTY10, and ZFY-AS1 lncRN as were down-regulated in both ICU and non-ICU men with COVID-19. Dysregulation of noncoding-RNAs has critical effects on the pathophysiology of men with COVID-19, which is why they may be used as biomarkers and therapeutic agents. Overall, our results indicated that the miR-29 family target regulation patterns and might become promising biomarkers for severity and survival outcome in men with COVID-19. COVID-19 is caused by a new beta-coronavirus known as the severe acute Previous studies have reported that males are more susceptible to severe COVID- 19 and have a worse prognosis . During the COVID-19 pandemic, it is crucial to study the ncRNAs involved in the interaction between SARS-CoV2 and the host. Here we predicted specific male miRNAs that target the genome of SARS-CoV-2 and miRNAs that target specific differentially expressed genes (DEGs) in males. On the other hand, lncRNAs are non-coding transcripts of 200-100,000 nucleotides, most of which demonstrate cell-type-specific expression. In addition to gene imprinting and X chromosome inactivation, lncRNAs affect the regulation of many complex cellular and molecular processes during development. It was demonstrated that SARS-CoV-2 is present in the testis by detecting the high level of ACE2 expression in this organ. The lncRNAs and miRNAs in this organ affected ACE2 expression (Verma et al., 2020). Therefore, the present research aimed to identify lncRNAs and miRNAs in COVID-19-associated problems in different sexes using bioinformatics analysis. To the best of our knowledge, this is the first study of non-coding RNA expression profiling based on gender that has aimed to find different gender-associated miRNA and lncRNA patterns. Identifying target genes and pathways regulated by First, gene expression datasets of COVID-19 were searched using the keywords: To identify differentially expressed mRNAs (DEmRNAs), the expression dataset was normalized and analyzed by the DeSeq2 package in Bioconductor. It was then utilized to mine statistically significant DEGs based on the difference in their expression values between samples of the COVID_female_ICU vs. COVID_male_ICU, and COVID_female_NonICU vs. COVID_male_NonICU (4). DEGs with |log2FC| ≥ 1 and adjust P-value ≤ 0.05 were considered to be significantly differentially expressed. Moreover, we also identified differentially expressed lncRNAs (DElncRNAs). A useful approach for a biological understanding of gene expression data is gene set enrichment analysis (GSEA). We used the ToppGene database In this study, GSE157103 (GPL24676) was used; the RNA-seq dataset contained 126 samples (each sample had two repetitions). Differential expression analysis of genes was firstly carried out to identify DEmRNAs and DElncRNAs using (Supplementary 1). We also determined DElncRNAs in this stage (Table 1) . For identification of the binding sites of human miRNAs on the coronavirus 2 reference genome, 2565 mature miRNAs were collected, 444 miRNAs were detected among all the 2654 human mature miRNAs at various locations with direct binding sites throughout the SARS-CoV-2 reference genome (Supplementary 2). We searched for the perfect matching interactions at the seed region and sorted out 160 miRNAs. Fifteen miRNAs with more than three binding sites were detected among them (Table 2 ). Our findings suggest that the miR-29 family (miR-29a, miR-29b, and miR-29c) had the maximum binding sites (11 sites). We also discovered miRNAs with more than three binding sites on the SARS-CoV-2 genome sequence ( Figure. and is a potential target for antiviral therapy. Enrichment analysis of differentially expressed genes has been shown in figure 2. Pathways and functional gene ontology analysis of DEGs was conducted. Production of molecular mediator of immune response, Phagosome, complement activation, cell-matrix adhesion and macrophage chemotaxis were most prevalent in DEGs related to COVID_female_ICU vs COVID_male_ICU. In addition, immunoglobulin production, ventricular cardiac muscle cell action potential, lipoxin metabolic process, regulation of adaptive immune response and Transcriptional misregulation in cancer were more significant in DEGs related to COVID_female_NonICU vs COVID_male_NonICU ( Figure 2 ). Moreover, we recognized miRNAs that target DEGs by ToppGene, among all miRNAs, hsa-miR-29a-3p and hsa-miR-29c-3p in addition to having five and four binding site respectively in SARS-CoV-2 genome, regulated some DEGs (Table 3 ). At last the DElncRNAs that had interaction with hsa-miR-29a-3p, hsa-miR-29c-3p and their targets was identified (supplementary 3). lncRNAs-miRNAs-mRNAs networks have been shown in figure 3. COVID-19 is an infectious disease, which results in slightly more severe outcomes in men. This study aimed to analyze the correlation between characteristics of ICU Our findings showed that miR-761 had three seed binding sites on the SARS-COVID-2 genome, and it may cause the shortness of breath and severe problems that occur after COVID-19 infection in the lung bronchi. MiR-6715b was reported as a biomarker to predict cancer stages (Xu et al., 2020) . Our results showed that miR-6715b participates in the progression of COVID-19 in males. It was reported the conserved sequence for the evolutionary miR-15/107 family, including miRNA-6838-5p . In the present study, we observed that miR-6838-5p was significantly correlated in men with COVID-19. This J o u r n a l P r e -p r o o f miRNA can be a signature and has the potential to be used as a factor in the treatment of COVID-19 patients. Besides, Angiotensin-converting enzyme 2 (ACE2), which helps SARS-CoV-2 inter into the host cells, is expressed in the testis. Additionally, miRNAs in this organ affect ACE2 expression (Olaniyan et al., 2020) . The results of the present study indicated that the antiviral therapies and diagnostics can be developed differentially on the basis of gender, but it is obvious that, many variables, such as hormone-specific reactions and the activation of Xlinked genes may influence the innate and adaptive immune response to viral infection and contribute to the discrepancy in COVID-19 outcomes. Our results indicated changes in the expression of miRNAs (125, 181b, 130a, 29a Besides, the testis has a crucial role in the immunological response, and there is a high level of ACE2 expression in the testis (Olaniyan et al., 2020) . Therefore, coronaviruses may localize in the testis, and thus, men with COVID-19 show worse symptoms than women. The immune system affects testis development and acts during spermatogenesis. lncRNA XIST has the potential of interacting with some proteins and miRNAs such as miR-29a (Brown et al., 1991) . On the other hand, miR-29a has five seed binding sites on the SARS-COVID-2 genome reference. Thus some serious problems in men with COVID-19 may be related to the interaction between lncRNA XIST and miR-29a. Our findings showed a complex mapping of disease-related factors and ncRNAs significantly affecting the pathways (Table 2) . 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