1. Introduction
The emergence of SARS-coronavirus-2 (SARS-CoV-2), which causes coronavirus-mediated disease 2019, or COVID-19, has now spread pandemically, resulting in a serious global health crisis. Coronaviruses are positive-single stranded RNA (+ssRNA) zoonotic viruses with a ~30 kb genome (approx). The coronavirus subfamily is divided into four genera: α, β, γ, and δ, based on serotype and genome features. The genome of a typical CoV codes for at least 6 different open reading frames (ORFs), which have variations based on the CoV type 4. Some ORFs encode non-structural proteins while others code for structural proteins required for viral replication and pathogenesis. Structural proteins include the spike (S) glycoprotein, which has various roles in SARS-CoV based on sequence analysis and might share similar viral genomic and transcriptomic complexity. Other proteins include matrix (M) protein, small envelope (E) protein, and nucleocapsid (N) protein for virus entrance and spread [1]. SARS-CoV-2 infections mainly target the lungs with respect to other viral infections, which begin with upper respiratory tract symptoms. However, it is obvious that an important differentiating feature of the current SARS-CoV-2 infection is that it does not follow regular viral lower respiratory infection pathways. There are a wide variety of clinical symptoms presented by SARS-CoV-2 infection. Mainly (otherwise unexplained) fever and a failure to breathe fully are the most frequent symptoms, with non-productive cough, sneezing, sudden loss of smell and/or taste, pain in the chest cavity (mostly anteriorly), headache and prominent muscle ache, abdominal pain with diarrhoea, and neurological symptoms with facial nerve involvement [2]. Patients are generally referred to hospital when fever does not subside for 2 to 3 days, or respiratory effort gets harder. Clinical observation indicates that elderly patients present more unexplained fever for 3 days, in contrast to younger patients who present with difficulty in breathing without fever. In addition, the current lack of successful therapeutic intervention strategies to prevent the disease, or overcome serious mortality rates as a result of difficult lung pathophysiology mediated by SARS-CoV-2 infection, requires clarification of the molecular aetiology behind the mild or severe conditions of the disease. Increasing mortality rates are the major obstacle in the management of the disease in existing healthcare systems, which have insufficient numbers of intensive care units. Clinically, the initiation of acute respiratory distress syndrome (ARDS), which is the predominant severe pathology, leads to diffuse alveolar damage (DAD). In addition, the presence of a cytokine storm, the excessive and uncontrolled release of pro-inflammatory cytokines such as IL-6, TNFα, IL-1β, IL-8, and IL2R, is associated with ARDS, hypercoagulation and increased erythematosus sedimentation rate (ESR) [3]. SARS-CoV-2, as well as other viruses, leads to pathophysiological problems in the host cells, and alters the expression of a number of genes. Thus, clarification of molecular regulators on human cells is important to evaluate molecular mechanisms.
MicroRNAs (miRs) are non-coding RNAs of length approximately 20–22 nucleotides; they post-transcriptionally regulate gene expression by binding to the 3′-untranslated regions of target mRNAs, leading to degradation or translational inhibition. Each miR can target hundreds of mRNAs within a given cell type, and a single mRNA is often the target of multiple miRs, and thus over half of the human transcriptome is predicted to be under miR regulation, embedding this post-transcriptional control pathway within nearly every biological process [4,5]. Virally expressed miRs have recently been discovered, especially in viruses with DNA genomes. The best-known viral miRs are found mostly in herpesvirus families, where they enhance bilateral crosstalk between viral pathogenesis and host response mechanisms. Additionally, it has been shown that different virus families such as delta bovine leukaemia virus and foamy retroviruses could encode miRs [6]. As previously shown, viral miRs are critical in the immune evasion mechanisms, affecting host immunity-related gene regulation networks. This bilateral effect results in a rapid increase during the virus resistance against host defence mechanisms and leads to their survival in host cells [7]. The scope of miR generation from ssRNA viral genomes has been controversial, mainly due to the potentially deleterious effect of ssRNA viral genome cleavage into pre-/pri-miRs, making it unavailable for packaging into new virus particles. However, predictive studies on RNA viral genomes reveal RNA structures, which are conceivable Drosha and Dicer substrates. One of the well-described annotations was performed for HIV to define HIV-1 TAR RNA, a 59-nt long sequence that could generate a stem-loop structure similar to the pre-miR structure [8]. The identified numbers of viral miRs are not as many as for other organisms. One of the reasons for this is that viral miRs, due to their cytoplasmic location in host cells, render interaction with nuclear miR biogenesis elements of the host cells. Another hypothesis is that the cleavage of the viral RNA genome could generate miRs [9]. According to data obtained using in silico screening, there are fewer identified virus miRs compared with other species, and their functional roles in host cells are not well described. Therefore, greater effort is required to identify novel virally encoded miRs and to predict their host targets. Conversely, host cell miRs may alter the biological effect of the viruses [10]. Several interactions between viruses and the miRs in the host cells have been reported: the virus may either avoid being targeted by the cellular miRs [11]; block the cellular miRs to regulate key proteins in main signalling pathways [12,13]; synthesize their own viral miRs to create a more favourable cellular environment to survive in the host cells [14], or simply use the cellular miRs to their own advantage [15]. It should also be noted that host cell miR repertoires change dramatically in response to various diseases. Several underlying cardiovascular and lung conditions may significantly alter host miR expression, which would affect virus-host lung cell interactions, and may significantly determine the course of the disease.
The existence of sequences within the ssRNA viral genome with a high degree of sequence similarity to human (or mammalian) sequences is unlikely to be accidental. Hypothetically, some viral ssRNA molecules may be channelled into the miR processing pathways influencing the host cell, whilst other ssRNAs are packaged, combinations of which may increase overall viral activity. The general prediction mechanism of putative miRs is based on the determination of seed region specificity. The seed sequence, which is the critical part of the target prediction, is essential for the binding of the miR to the target mRNA. The seed sequence or seed region is referred to as an evolutionary conserved heptameric sequence, which is mostly situated at positions 2–7 from the miR 5’-end. Therefore, point mutations at seed regions are critical to evaluate the target specificity and functional consequences of the potential miR and target mRNA. The complex interaction between the viruses and the host miRs mostly become more advantageous for the viruses as they enable them to avoid the immune system response and allow them to employ the host’s miRs [16]. Recently it has been suggested that miRs play a role in the host’s defence system against viral infections such as HIV-1, HSV, HCV, dengue and influenza [17,18,19,20,21]. Therefore, targeting specific miRs could prove to be a novel strategy for treatment. The best-known anti-miR treatment for viral infection is targeting miR-122 to cure HCV [18]. It has also been reported that anti-miR-based HCV therapy is genotype independent, which makes it safe, effective and well tolerated by patients [22].
In this study, our first aim was to identify human miRs that show sequence similarities to the SARS-CoV-2 genome, and their conservation ratio in SARS-CoV-2 isolates obtained from different geographical regions. Following determination of significantly similar miR sequences, we evaluated their potential effect on host cells through analysis of their target genes and related KEGG and GO pathways using bioinformatics tools. In the final part of the study, the miR-mediated alterations of different pathways were compared to public transcriptome data obtained from SARS-CoV-2-infected cell and tissue biopsy samples. To this end, the study aims to clarify the role of potential miR-mimic sequences in the SARS-CoV-2 genome with their host target genes, which may propose a new perspective for antiviral strategies.