4. Discussion
In the current study, we identified potentially similar miR sequences of human miRs and SARS-Cov-2 strains from different geographical regions. For this purpose, we selected different genome sequence studies released recently in PubMed® and GISAID databases, which included genome results for SARS-CoV-2 strains from four different geographical regions. We first aligned all the sequences with the mature hsa-miR database presented in miRBase. During this study, we made a cluster according to the co-existence of significantly aligned hsa-miRs with the SARS-CoV-2 genome and their biological significance in human cells. miRPath version 3 was used for the determination of the selected miRs’ potential biological effects via searching target gene-related results found in the KEGG and GO pathways. The mutational alterations were also analyzed for different miRs, which showed significant alignment scores with SARS-CoV-2 genomes from other geographical regions. Concomitantly, we also analyzed and compared Bioproject PRJNA615032 trancriptome data obtained from normal vs SARS-CoV-2-infected lung tissue biopsy samples. Data was submitted by tenOever Lab, Microbiology, Icahn School of Medicine at Mount Sinai on 24-Mar-2020. In addition, the gene expression differences between A549 and NHEB cell line data and the gene expression differences between normal and infected lung tissue samples were analyzed in the REACTOME database (https://reactome.org/) to propose the potentially affected signaling axis members. The clinical outcome of severe condition patients is discussed below, for selected miRs and their potential biological importance in host cells.

4.1. Biological Significance of Top Ranked miRs in Humans

4.1.1. miR-8066
Recently, the N nucleocapsid gene-related putative miR candidates were shown through in silico prediction tools [38]. In a similar manner, we determined miR-8066, a mature sequence found on SARS-CoV-2 genomes, in SARS-CoV-2 genome alignment analysis with human miRs. Additionally, miR-8067, which possesses a similar biological role to miR-8066, was identified as a stem loop region sequence in all SARS-CoV-2 genomes (Figure S1). Previous reports showed that both miRs are found in plasma samples of sepsis patients with severe clinical outcomes [39]. Similar miR family members such as miR-8054, miR-8057, miR-8061, and miR-8068, were also found in a non-survivor group of sepsis patients. It has been shown by in silico analysis that certain miRNAs have the core motifs of AUUGUUG, and that miR-8066 is one of these [40]. When miRs have this motif, it increases their likelihood to bind and activate Nf κB-mediated TLR-8 expression and induce cytokine synthesis [40]. miR-8066 was identified at high levels in tissue biopsies as well as in exosomes [41]. Therefore, SARS-CoV-2-mediated alterations of miRs may act as autocrine or paracrine agonists of host cells to trigger pro-inflammatory cytokines, due to their increased NfKB activity [42]. Similar findings were also suggested for cancer cells, which showed miR alterations in association with inflammation markers [43].
There is preliminary data about the existence of sequence similarity of human miRs in the spike region of the SARS-CoV-2 genome for both forward and reverse complementary strands [44]. Hsa-miR-8055, which was also previously shown to be a sepsis marker, was found to be involved in T-cell responses to specific antigens [45]. One of the differentially expressed circRNAs in children with fulminant myocarditis, Circ_0071542, was suggested to regulate the expression of MAPK, a well-characterized target in the development of disease through binding hsa-miR-8055 [45]. In our current study, we found that the miR-8066 sequence is present in four different SARS-CoV-2 genomes and shows strong association in KEGG pathways for TGF-beta signalling, mucin type O-glycan biosynthesis and cytokine-cytokine receptor interaction. These targeted pathways are known, critical mediators for the clinical outcomes for SARS-CoV-2-infected patients and give us insight about virus pathophysiology. It is well established that for enveloped viruses, such as SARS-CoV-2, N- or O-glycosylation of S protein determines the viral entry and membrane fusion with functional elicitation of host immune responses [46]. The high-resolution LC-MS/MS experiment was performed to detect site-specific quantitative N-linked and O-linked glycan profiling on SARS-CoV-2 subunit S1 and S2 proteins. The glycan profiling showed that two unexpected O-glycosylation sites on the receptor-binding domain (RBD) of subunit S1 increased the pathogenicity of SARS-CoV-2. Therefore, our understanding of complex sialylated N-glycans and sialylated mucin type O-glycans on the functional RBD domain may help to evaluate better therapeutic or vaccine strategies [47]. Moreover, miR-8066 alters N-glycosylation patterns according to the microT-CDS pathway and may have biomarker potential for this mechanism. In addition, miR-8066 is found to be associated with one of the critical clinical problems of COVID-19, the cytokine storm, owing to its potential effect on the cytokine-cytokine receptor pathway. It is well established that miR-8066 affects PRLR, CXCL6, IL6, IL17 and ACVR1 target genes, which are crucial members of the cytokine regulatory network (Table 2). According to previous NGS platform results obtained from healthy versus SARS-CoV-2-infected lung tissue biopsies (Bioproject PRJNA615032), the most highly upregulated pathways are chemokine binding receptors (p value 7.94 × 10−4), FGFR activation pathways (p value 0.002) and its downstream pathway (p value 0.001), neutrophil degranulation (p value 0.003), and IL10 (p value 0.005), when compared with normal lung tissue biopsies (Table S2). On the contrary, according to the REACTOME pathway analysis of Bioproject PRJNA615032 data, neutrophil degradation was also downregulated in SARS-CoV-2-infected biopsy specimens (p value 1.74 × 10−6). Briefly, the downregulation of host cell responses’ related pathway members such as SRP-dependent co-translational protein targeting to membrane, L13a-mediated translational silencing of ceruloplasmin expression antigen processing, ribosomal organization, and the ERAD pathway, were observed in COVID-19-positive lung biopsy specimens. All of these preliminary gene expression differences, between normal and SARS-CoV-2-infected lung biopsies, are associated with detected miR pathways and highlight common clinical presentations of patients. Each of these biological mechanisms, according to potential disease progression-related alterations in the host genome, is highly associated with TGF-β, which is a cytokine affecting a number of host responses during infection [48]. TGF-β mediates the immune responses of host cells, as well as altering tissue remodelling by affecting cell survival, apoptosis and migration. It has been demonstrated that SARS-CoV N protein potentiate Smad-3 mediated TGF-β activation, and plasminogen activator inhibitor-1 (PAI-1), leading to severe pulmonary fibrosis and inactivation of pro-apoptotic genes by complex formation of Smad3 and Smad4 [48].

4.1.2. miR-5197
miR-5197-3p was identified as the most effective miR to interact with the guide RNA of SARS-CoV, MERS-CoV and COVID-19 [49]. According to a recent study, out of 2565 miRNAs, only three critical miRs, 5197-3p, 4778-3p and 6864-5p could interact with complete complementary miR (cc-miR) and possess a critical therapeutic potential due to their binding affinity on SARS-CoV-2 guide RNA. It was suggested that the generation of miR-5197-3p-based complete complementary miRNA may possess a significant therapeutic response, owing to its structural affinity to guide RNA of SARS-CoV-2, without any side effects on human genes [49]. Previous patents on hsa-miR-5197-5p indicated that any drug targeting this miR might be critical in the treatment of hepatitis B infections (WO2018193902A1). Similar to this finding, it was shown that miR-5197-3p might be used in vaccine strategies in the herpes simplex virus (HSV-1) (WO2013109604A1WO2013109604A1).
In another study, allelic variances of hsa-miR-5197 were found to be highly associated with non-small cell lung cancer (NSCLC) [50]. According to the Harvard and MD Anderson databases, miR-5197 shows significant differences between Caucasians and Chinese populations (data gained from a Han Chinese cohort from Nanjing, China) [50]. The rs2042253 polymorphism (T>C variation) of miR-5197 provided a protective effect on lung cancer survival. Moreover, this SNP is reported to have a high read frequency for paediatric acute lymphoblastic leukemia (ALL) in the UK cohort [51]. High throughput analysis of 408 lung cancer patients showed that miR-5197 polymorphism is highly correlated with chemotherapy-induced severe toxicity [52]. It has been accepted that nucleotide variations in pre-miRNAs may have contributed to the stability of stem-loop structure and may affect recognition sites for Drosha and Dicer cleavage [53]. In our current study, we did not find any mutations in miR-5197 sequences obtained from different SARS-CoV-2 genomes, however we found that mucin type O-glycan biosynthesis was related to both miRs 5197-3 and 8066. This KEGG pathway has been involved in the cell-to-cell transmission of human T cell leukemia virus type 1 (HTLV-1) [54] and plays roles in Ebola virus cell attachment [55]. HIV-1 infection of T cells has also been shown to result in altered glycosylation of cell surface glycoproteins [56].
O-glycans are also linked to avian influenza [57]. Mucosal O-linked glycans are furthermore associated with herpes simplex virus 1 infection [58]. Mucin type O-glycan biosynthesis miR pathways have been identified in avian oncogenic retrovirus (Avian leukosis virus subgroup J (ALV-J)) [59]. This KEGG pathway is therefore related both to human as well as veterinary viral infections and further studies will be needed to establish the function of miR-5197 during SARS-CoV-2 infection.

4.1.3. miR-3611
In our current study, we found that miR-3611 is a positive hit for stem loop region and mature miRNA prediction. Because of its potential presence in SARS-CoV-2-mediated cellular responses, we have analysed data from the lung tissue biopsy database (PRJNA615032 Bioproject) to understand its function. A previous study reported altered miRNA expression in chronic obstructive pulmonary disease (COPD). COPD is an airway disorder and respiratory disease that is associated with persistent inflammation [60]. A study on miR expression analysis, conducted on COPD and healthy volunteers, showed significant down-regulation of miR-3611 expression in COPD patients [61]. The long non-coding RNA, H19, has been linked to many carcinomas, including lung cancer [62]. It has been shown that miR-3611 is significantly down-regulated in a H19 knockdown lung cancer cell line (SPC-A1), which indicated overexpression of this miR in ‘normal’ H19 intact lung cancer cell lines [63]. In our study, we identified that miR-3611 shows high similarity with all four main SARS-CoV-2 genomes and we did not detect any mutations between genomes. However, miR-3611 was strongly associated with KEGG pathways for metabolism of xenobiotics by cytochrome P450, morphine addiction and GABAergic synapse. Moreover, morphine addiction was, besides linkage with miR-3611, also linked to miR-5197-3. This KEGG pathway has been related to enhanced HIV-1 infection [30] and morphine treatment has been shown to promote HIV-1 replication in macrophages via inhibition of the TLR9 pathway [64]. Furthermore, an increased rate of HIV-1/HTLV-I infection has been observed due to morphine in injection drug users [65]. Morphine is also associated with enhanced hepatitis C virus (HCV) replicon expression [66,67]. Opioids have furthermore been shown to enhance simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys [68], while reduced clearance of pulmonary influenza virus infection was observed in morphine-treated Lewis rats [69]. Interestingly, opioids such as morphine are also used as indirect antitussives to suppress cough, which is commonly associated with respiratory viral infections [70], including SARS-CoV-2. Indeed, morphine was used in recent cases in China during sedation and analgesia for endotracheal intubation, to avoid patients’ cough and agitation during the procedure [39]. In light of the association of morphine with promotion of viral replication, it may have effects in SARS-CoV-2 that need to be further investigated.

4.1.4. miR-3934-3p
miRNA expression profiles have been used to classify cancers into various subtypes. miR3934 is found upregulated in colon cancer and was suggested as a biomarker for lung cancer as its expression correlated with survival rate and prognosis of NSCLC [71,72]. Moreover, it has been reported that miR-3934-5p expression significantly increases in NSCLC cell line A549 [73]. It is also a SNP linked to TGF-β signalling and has been identified as downregulated in rectal carcinoma mucosa, compared with normal mucosa [74].
In addition, it was shown that miR-3934-3p downregulated TGFBR1 and SMAD3. In a similar vein, HSV-1 viral infection led to a significant down-regulation of these targets [74]. Moreover, the activation of the TGF-β/Smad pathway is critical for lung fibrosis, which was previously shown in SARS-CoV-related cases. Dysregulation of ACE2 may influence the toll-receptor signalling pathway, via IL6, and affect downstream immune responses. Irrespective of SARS-CoV-2 or pneumonia in TCGA-LUAD, the altered immunoreaction was the primary cause (lung adenocarcinoma; SARS-CoV-2; ACE2; miR-125b-5p; IL6). TGF-β and cigarette smoke have been shown to suppress miR-141-5p to promote CCR5 expression on primary bronchial epithelial cells, which results in increased viral entry and infection by R5-tropic HIV [75]. Given that TGF-β signalling is upregulated by trans-activator (Tat) protein, cigarette smoke and in chronic lung diseases, it has been determined the effects of persistent TGF-β signalling on HIV infection in primary bronchial epithelium re-differentiated ex vivo [75]. In our current study, miR-3934-3p was found to be associated with KEGG pathways for glycosaminoglycan biosynthesis heparan sulfate/heparin, mucin type O-glycan biosynthesis and vitamin digestion and absorption. The relevance of these pathways is as follows:
“Glycosaminoglycan biosynthesis—heparan sulfate/heparin” was here related to miRs 3934-3. Heparan sulfate proteoglycans have previously been identified to provide the binding sites for SARS-CoV-2 invasion at the early attachment phase [76]. Furthermore, human coronavirus NL63 has been shown to utilise heparan sulfate proteoglycans for target cell attachment [77]. This KEGG pathway has been related to Ebola virus, where heparan sulfate has been identified as an important mediator in polarised epithelial cells [78,79]. In hepatitis C viral infection, the virus hijacks this pathway via interaction with apolipoprotein E for cell entry [63], while heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection [70]. Endogenous HERV-K furthermore binds to heparin for cell entry [80], and, while heparin has been found to further Zika virus infection, it acts as an antiviral against Dengue replication [81]. Heparin sulphate is identified as an inhibitory regulator of porcine epidemic diarrhoea virus infection [82] and acts as an attachment factor for rabies virus entry and infection [83], as well as an enhancer of Nipah and Hendra virus infections, which are highly pathogenic, zoonotic paramyxoviruses [84]. This highlights the importance of this KEGG pathway both in human, zoonotic and veterinary viral infections.
Another important pathway, vitamin digestion and absorption, was found to be related to miR-3934-3. This KEGG pathway has been highlighted amongst others as a link between dysbiosis of the gut microbiome and chronic diseases [85]. Furthermore, intestinal triglyceride-rich lipoproteins have been related to vitamin metabolism in relation to coronary artery disease as well as viral infections [86]. In AIDS, malabsorption of vitamin 12 has been related to gastric secretory failure, including chronic diarrhoea, due to advanced HIV infection [87,88]. Additionally, vitamin A deficiency has been associated with more progressive HIV disease [89]. Veterinary viral infections, such as infectious bronchitis virus (IBV) and reovirus (RV) in chickens, have been identified to affect vitamin A metabolism due to epithelial damage [90]. Interestingly, a link between vitamin D deficiency and SARS-CoV-2 infection severity was recently reported [91]. Also, vitamin B3 was found to be highly effective to help lung tissue damage repair [92] and it was suggested to be given to COVID-19 patients as soon as the CT lung abnormalities were detected [93].

4.1.5. miR-1307-3p
miR-1307 has previously been shown to be one of the lung-tissue-associated miRs [94] and was reported as an especially important target in lung development in newborns. Expression of mir-1307-3p has furthermore been linked to acid metabolism in response to aspirin in human cardiac and peri-cardiac fat-derived mesenchymal stem cells (MSCs) [95]. Moreover, it was shown that several miRs, including miR-1307-3p, are involved in TGF-β and semaphorin signalling, as well as inflammatory responses. miRs play an important role, especially during lung morphogenesis in the early stages of development. Interestingly, miR-1307 has been associated with the severity of pulmonary hypertension in systemic scleroderma [96]. Persistent problems with SARS-CoV-2-infected patients, such as oxygen dependency, urgent need for mechanical ventilation, persistent wheezing, and increased risk for pulmonary infections correlate with involvement of miR-1307-3p, as indicated above, due to its role in pulmonary hypertension and chronic lung diseases. The TGF-β superfamily plays critical roles in pre- and postnatal lung development, importantly shaping alveolarisation and controlling the extracellular matrix composition and tissue homeostasis, among other functions. TGF-β signalling is therefore strongly linked to both pulmonary and cardiovascular diseases [97,98,99].

4.1.6. miR-3691-3p
It has been shown that oxidative stress, which has detrimental effects on intercellular communication, plays an important role in many lung pathologies such as acute lung injury and COPD. A proteomic profile of exosomes from human bronchial epithelial cells, under normoxia and hypoxia, has reported reduced expression of miR-3691-3p in hypoxia [100]. Interestingly, miR3691-3p targets several cell signalling pathways, such as TGF-β signalling, FGF2 and also VCAM1, which is relevant for lung injury and repair. In the current study, we have also identified that miR3691-3p exists in four of the genomes, which we have extensively studied here, and therefore it seems to be a conserved miR.

4.1.7. miR1468-5p
Sudden cardiac death is a major problem amongst the unexplained deaths in COVID-19, and it has been identified that many of those patients suffered from primary myocardial fibrosis (PMF), without any known aetiology. Recently, higher expression of miR-1468-3p was identified as a disease-associated and age-dependent cardiac biomarker, as it promotes cardiac fibrosis and cell senescence, although no difference was noted in the mature form of miR-1468 between healthy and COVID-19-diseased cardiac tissue [101]. TGF-β1 plays a key role in fibrosis-related pathologies including cardiac fibrosis, and, furthermore, miR-1468 activates non-canonical TGF-β1 and MAPKs signalling pathways [101]. Moreover, miR-1468-5p expression has been found to be upregulated in regulatory T cells, which have a significant role in autoimmune disorders, transplant rejection, allergic diseases, and asthma [102]. miR-1468-5p has previously been associated with glioma, where it inhibits growth and cell cycle progression by targeting ribonucleotide reductase large subunit M1 (RRM1), based on a study on patients from the Chinese Glioma Genome Atlas [103]. miR-1468-5 is also linked to progressing hepatocellular carcinoma [104]. Interestingly, in Alzheimer’s disease, miR-1468-5p has been identified to be at lower abundance compared with healthy controls [105]. It has furthermore been identified as a biomarker in late seizure in patients with spontaneous intracerebral haemorrhage [49]. The link between miR-1468-5 in viral infection and other comorbidities will need to be further investigated.
miR-129-2-3p, here identified as a common mutated miR, has previously been identified as a regulator in human cancer development and progression [106]. It has been identified as a diagnostic and prognostic biomarker for renal cell carcinoma [107] and a suppressor of serous ovarian cancer [104]. Its upregulation suppresses breast cancer cell proliferation and induces its apoptosis, while downregulation, via hypermethylation, increases breast cancer progression due to BCL2L2 overexpression [107]. It furthermore attenuates cell migration and invasion in renal cell carcinoma by affecting the downregulation of various metastasis-related genes [108]. miR129-2 has been linked to a range of haematological malignancies, including lymphoma [109]. It is also linked to lung adenocarcinoma including regulation of cell proliferation [110], as well as to hepatocellular carcinoma [111]. Interestingly, miR-129-2-3p has been found to be upregulated in human papilloma virus-positive (HPV) head and neck squamous cell carcinoma [112] and in HPV transfected keratinocyte cells [113]. In the light of increasing understanding of the link between SARS-CoV-2 and comorbidities, underlying changes in miR-129-2-3p expression may be of considerable importance.
A number of KEGG pathways have been strongly linked to the main miRs identified as being related to sequences within the SARS-CoV-2 genomes. These include: “Mucin type O-glycan biosynthesis”, “TGF-β signalling pathway”, “Morphine addiction”, “Metabolism of xenobiotics by cytochrome P450”, “Other types of O-glycan biosynthesis”, “Vitamin digestion and absorption”, “Glycosaminoglycan biosynthesis—heparan sulfate/heparin”, “GABAergic synapse”, “Cytokine-cytokine receptor interaction”, “Signalling pathways regulating pluripotency of stem cells”, “Amphetamine addiction”, “Axon guidance”, “Hippo signalling pathway”, “Prolactin signalling pathway”, “mRNA surveillance pathway”, “Glycosphingolipid biosynthesis—lacto and neolacto series”, “Bile secretion”, “Circadian entrainment”, “N-glycan biosynthesis”, “Mismatch repair”, “Drug metabolism—cytochrome P450”, “Glutamatergic synapse”, “Glycosaminoglycan degradation”, “Antigen processing and presentation”. The relevance of several of these KEGG pathways has been discussed above in direct relation to the various miRs and provides a novel insight into the putative interplay of these pathways and the microRNAs identified in COVID-19, and may also help in furthering understanding of the interplay of miRs, viral infections and comorbidities.