Abstract

   Coronaviruses (CoVs) are single-stranded RNA viruses which following
   virus attachment and entry into the host cell, particularly type 2
   pneumocytes but also endothelial cells, release RNA into cytosol where
   it serves as a matrix for the host translation machinery to produce
   viral proteins. The viral RNA in cytoplasm can interact with host cell
   microRNAs which can degrade viral RNA and/or prevent viral replication.
   As such host cellular miRNAs represent key cellular mediators of
   antiviral defense. Polyphenols, plant food bioactives, exert antiviral
   properties, which is partially due to their capacity to modulate the
   expression of miRNAs. The objective of this work was to assess if
   polyphenols can play a role in prevention of SARS-CoV-2 associated
   complications by modulating the expression of host miRNAs. To test this
   hypothesis, we performed literature search to identify miRNAs that
   could bind SARS-CoV-2 RNA as well as miRNAs which expression can be
   modulated by polyphenols in lung, type 2 pneumocytes or endothelial
   cells. We identified over 600 miRNAs that have capacity to bind viral
   RNA and 125 miRNAs which expression can be modulated by polyphenols in
   the cells of interest. We identified that there are 17 miRNAs with both
   the capacity to bind viral RNA and which expression can be modulated by
   polyphenols. Some of these miRNAs have been identified as having
   antiviral properties or can target genes involved in regulation of
   processes of viral replication, apoptosis or viral infection. Taken
   together this analysis suggests that polyphenols could modulate
   expression of miRNAs in alveolar and endothelial cells and exert
   antiviral capacity.

   Keywords: polyphenols, microRNA, miRNA, nutriepigenomic, COVID-19,
   prevention

Introduction

   Faced with the enormous health and socioeconomic burden of the current
   COVID-19 pandemic, humanity makes every effort to develop effective
   measures to deal with the crisis in all segments of the society. While
   social distancing plays an important role, other lifestyle factors,
   including diet, may have potential to counteract viral susceptibility.
   In terms of nutrition, in addition to pivotal macro- and
   micro-nutrients, an adequate and continuous intake of various bioactive
   compounds, including polyphenols, has been pointed out as a
   contributing factor in the increased resistance to viral infections
   [[29]1]. Polyphenols are plant secondary metabolites that have various
   physiological functions in their dwelling plants. They are widespread
   in planta, with over 8000 different compounds described so far. Many of
   these compounds are found only in a limited number or in specific plant
   species [[30]2]. Only about 500 polyphenols are present in the human
   diet. They are classified as flavonoids, lignans, phenolic acids,
   stilbenes, non-phenolic metabolites and other polyphenols [[31]3].
   Polyphenols have been proven as molecules with strong in vitro free
   radical scavenging capacity. Within the cells however, they act
   predominantly by modulating the expression of numerous genes [[32]4],
   including those that are involved in the cellular antioxidant
   protection [[33]5]. Polyphenols or they metabolites bind to key
   proteins that are involved in cellular signaling [[34]6, [35]7], which
   is one of the mechanisms of their action in vivo. They also modulate
   the level of cellular microRNAs (miRNAs) [[36]8, [37]9], thus exerting
   a complex multi-layer regulation of cellular processes.

   Experimental studies have shown that polyphenols exert protective
   effects in respiratory infections caused by various pathogens
   [[38]10-[39]12], including viruses [[40]13, [41]14]. The antiviral
   activity of polyphenols has been investigated in vitro and in animal
   experimental studies. For example, curcumin has been shown to directly
   inactivate influenza A virus, block its adsorption and inhibit its
   proliferation in vitro [[42]15]. Curcumin increases the survival rate
   of mice infected with influenza A virus and decreases the production of
   cytokines by macrophages in vitro [[43]16]. These findings pinpoint
   curcumin as a potentially relevant plant bioactive in prophylaxis of
   the systemic cytokine storm [[44]17]. Similarly, hesperidin improves
   influenza A virus (H1N1) induced impairment of pulmonary function in
   rats by inhibiting cytokine production in pulmonary microvascular
   endothelial cells [[45]18]. Quercetin [[46]19] and flavanols [[47]20]
   also exert protective antiviral effects. Although limited,
   epidemiological studies confirm the protective effect of polyphenols in
   viral infections [[48]21]. Importantly, recent development of
   chemically modified epigallocatechin-3-O-gallate with improved
   antiviral efficacy against different types of viruses will
   significantly contribute to promotion of the use of polyphenols for
   prevention and treatment of viral infections [[49]22]. A combination of
   α-glucosyl hesperidin and epigallocatechin gallate was shown to have
   capacity to induce loss of rotavirus structural integrity as well as
   reduced infectivity (replication in cell culture) [[50]23]. Given the
   available data on the protective antiviral actions of polyphenols,
   there is an urgency to study their potential role in the context of the
   current COVID-19 pandemic, as prophylactic or therapeutic agents, or as
   treatment adjunct [[51]24-[52]27]. Quercetin is already included in the
   protocols for prophylaxis and early outpatient treatment for COVID-19
   ([53]https://covid19criticalcare.com). In order to give valuable
   recommendations for further research on this topic, it is necessary to
   get a deeper understanding of the interactions between the SARS-CoV-2
   virus and the polyphenol induced modulations within the host cells.
   Therefore, we hypothesize that polyphenols can play an important role
   in prevention of SARS-CoV-2 associated complications by modulating the
   expression of host miRNAs that play role in development of the viral
   infection.

SARS-CoV-2 and the host cell

   Coronaviruses (CoVs) are single-stranded RNA viruses that infect a wide
   variety of animals, and also humans. In parallel with the efforts for
   development of effective vaccines for COVID-19, research into the
   pathogenesis of SARS-CoV-2 infection has also focused on understanding
   the mechanisms involved in viral entry and replication [[54]28].
   Following virus attachment and entry into the host cell, the viral
   particle is uncoated, and its positive-sense single-stranded RNA genome
   is released into cytosol where it serves as a matrix for the host
   translation machinery to produce viral proteins. SARS-CoV-2 RNA encodes
   four main structural proteins: spike (S), envelope (E), membrane (M),
   and nucleocapsid (N) and several other accessory proteins, such as 3a,
   6, 7a, 7b, 8, and 10 [[55]29]. The spike protein enables the virus to
   bind to angiotensin-converting enzyme 2 (ACE2) on the host cell
   membrane, following which the viral genome makes its way inside the
   host cell [[56]28]. Importantly, the spike protein is the key target
   for development of SARS-CoV-2 vaccines [[57]30, [58]31]. Since
   SARS-CoV-2 utilizes human machinery to translate its RNA after the
   entry into the cell, it could possibly impact several RNA-binding
   proteins from the host to bind the viral genome resulting in altered
   post-transcriptional regulation.

   The clinical presentation of COVID-19 varies widely, from asymptomatic
   infection to a fulminant form with systemic cytokine storm. SARS-CoV-2
   infection that is localized mostly in the nose is usually asymptomatic.
   Progression of the infection to the lower respiratory tract is followed
   by onset of symptoms and worsening of the clinical presentation
   [[59]32]. In alveoli, SARS-CoV-2 initially attacks type 2 pneumocytes;
   subsequently, type 1 pneumocytes are also affected. Although they
   occupy only a small percentage of the inner alveolar surface, type 2
   pneumocytes are present in abundance, and play a critical role in
   alveolar function by generating the alveolar surfactant layer and
   acting as progenitor cells for type 1 pneumocytes [[60]33]. Type 2
   pneumocytes co-express ACE2, which is the primary cell surface receptor
   for SARS-CoV-2 and transmembrane serine protease 2 (TMPRSS2), a serine
   protease that is required to complete the entry process. Co-expression
   of ACE2 and TMPRSS2 is also found in the nasal secretory cells and
   absorptive enterocytes, which are also targeted by SARS-CoV-2 [[61]34].
   Other receptors and proteins have also been proposed to act as
   mediators of SARS-CoV-2 entry in the human cells, such as sialic acid
   receptor, CD147, or cathepsin B and L. All these molecules are also
   expressed in the endothelial cells [[62]35], suggesting their
   involvement in the major vascular derangements in COVID-19. Indeed,
   lung autopsy specimens of patients who died from respiratory failure
   caused by SARS-CoV-2 show severe endothelial damage with disruption of
   endothelial membranes and presence of SARS-CoV-2 within the endothelial
   cells, which is accompanied with widespread vascular thrombosis,
   microangiopathy and occlusion of alveolar capillaries [[63]36].
   Infection of endothelial cells and endotheliitis in COVID-19, however,
   are not limited to the lungs, and have been demonstrated in different
   organs, such as kidney, liver, heart, or small intestine [[64]37].
   Therefore, endothelial and alveolar cells are among the primary targets
   in developing of strategies to combat SARS-CoV-2, including studies on
   polyphenols as potentially protective factors.

Interplay between host cell miRNAs and viral RNAs

   Replication cycle of SARS-CoV-2 potentially exposes its RNA to an
   antiviral cellular defense that relies on the host’s endogenous miRNAs.
   These miRNAs could directly degrade viral RNA and/or prevent viral
   protein translation, such as has been reported for the influenza virus
   [[65]38]. The role of host cellular miRNAs in negative regulation of
   virus replication is not surprising because siRNAs, which inhibit mRNA
   expression by comparable mechanism, have been shown to effectively
   impair virus replication. The first evidence that host cells might
   employ a miRNA in antiviral defense was provided by Lecellier et al.,
   showing that human miR-32 restricts the accumulation of primate foamy
   virus type 1 (PFV-1) in human cells by targeting viral RNA transcripts
   [[66]39]. Similarly, miR-24 and miR-93 can target vesicular stomatitis
   virus (VSV) L and P proteins [[67]40] and miR-29a targets human
   immunodeficiency virus (HIV) Nef proteins [[68]41] to inhibit
   replication. Therefore, the use of antisense oligonucleotides to block
   viral genome can be the most straightforward and easy to implement way
   towards development of new antiviral therapies. For example, in one
   study researchers developed four artificial miRNAs that were designed
   to target different regions of Chikungunya virus (CHIKV) genome. These
   miRNAs significantly inhibited CHIKV replication, up to 99.8% [[69]42].
   On the other hand, miR-122 upregulates the replication of the HCV RNA
   genome, having a key role in promoting viral RNA stability [[70]43].
   The miRNA-based therapy for HCV infection is performed using an antimiR
   which was designed to bind directly to the mature strand of the
   targeted miR-122 and thus to induce a functional blockade. This is a
   great example of therapeutic miRNA targeting with a significant
   reduction in HCV titers (> 300-fold), and without signs of a rebound
   even after discontinuation of the treatment [[71]44]. Viral RNAs can
   also serve as miRNA sponges and reduce cellular miRNA levels, which in
   consequence can modulate the host’s cellular processes to facilitate
   viral replication or to inhibit the host’s antiviral responses.
   Regarding SARS-CoV-2, it has been proposed that bats, considered as its
   host of origin, have tolerance to potentially deadly viruses because of
   the specific miRNA profile [[72]45].

miRNAs could explain diversity in response to SARS-CoV-2 infection

   Numerous reports have illustrated the large diversity in terms of
   responses and clinical outcomes to SARS-CoV-2 infection. One possible
   variable that has not been sufficiently addressed are the individual
   differences in patients’ miRNA profiles. A recent study has indeed
   suggested that SARS-CoV-2 virulence in aged patients may be due to a
   lower abundance of miRNAs, which may be a contributing factor in
   disease severity [[73]46]. Moreover, in the COVID-19 pandemic, a
   significant gender disparity was evidenced with the male lethality
   higher than the female one: in Italy 17.7% vs 10.7% [[74]47]. Sex
   chromosomes, in particular X chromosome, and sex hormones are key
   actors in these differences. Interestingly, a different expression in
   males and females of several miRNAs has been observed owing to sex
   hormones differences and/or localization on the X-chromosome, which is
   particularly enriched for miRNAs. Hence, the potential role of these
   gender-associated miRNAs in immunity regulation and in modulation of
   viral receptors and co-receptors should be considered as a crucial
   factor in the observed different pathogenicity and lethality of
   COVID-19 in men and women. These observations suggest that the
   individual epigenetic differences in the miRNA profiles during an
   infection could affect the effectiveness of the antiviral responses and
   the disease severity. Thus, modulating or manipulating endogenous
   miRNAs to improve immunity and clinical outcomes in SARS-CoV-2
   infection could be another avenue for further investigation [[75]47].

Identification of human miRNAs that could interact with SARS-CoV-2

   Bartoszewski et al. [[76]48] examined whether SARS-CoV-2 could be
   targeted by human endogenous miRNAs. The coronaviruses genomes were
   tested against the set of 896 confident mature human miRNA sequences
   that were obtained from the miRBase v2.21 using the RNA22 v2 microRNA
   target discovery tool web server [[77]49]. The authors observed that
   the pathogenic human coronaviruses could interact with host-specific
   miRNAs, disturb gene expression and consequently suppress immunity or
   prevent activation of unfolded protein response (UPR)-related
   apoptosis. The authors focused on the potential role of the 28 miRNAs
   that were uniquely specific for SARS-CoV-2. The majority of these
   miRNAs are well expressed in bronchial epithelial cells, and their
   dysregulation has been reported for various human lung pathologies that
   include chronic obstructive pulmonary disease, cystic fibrosis, and
   tuberculosis. Furthermore, many of these miRNAs have been proposed to
   act as tumor suppressors that target apoptosis-related pathways. Hence,
   SARS-CoV-2, by its potential to reduce host’s miRNA pool, may promote
   infected cell survival and thus continuity of its replication cycle.
   The authors concluded that the pathogenesis of human coronavirus could
   involve modulation of host miRNA levels by acting as miRNA sponges to
   facilitate viral replication and/or to avoid immune responses.

   Sacar Demirci and Adan [[78]50] performed a machine learning based
   miRNA prediction analysis for the SARS-CoV-2 genome and identified
   human miRNAs that appeared to be able to target viral genes encoding
   proteins that are involved in viral life cycle. Among 2,654 mature
   entries of Homo sapiens in miRBase, 479 of them could target SARS-CoV-2
   genes. The SARS-CoV-2 genes targeted by host-cellular miRNAs are mainly
   responsible for viral biogenesis, entrance, replication and infection.
   For instance, miR-203b-3p can target ORF1ab and ORF3a which result in
   the suppression of viral replication as already shown in influenza A
   virus [[79]51]. let-7c-5p can target ORF1ab in SARS-CoV-2 while it was
   found to be involved in H1N1 influenza A suppression by targeting its
   M1 protein [[80]52]. The presence of such miRNAs could be considered as
   a host’s innate antiviral defense mechanism.

   Srivastava et al. [[81]53] used computational approach to investigate
   the potential binding sites of human miRNAs in SARS-CoV-2 genome using
   the Find Individual Motif Occurrences (FIMO), a motif-based sequence
   analysis tool [[82]54]. They identified 22 miRNAs that could
   potentially bind throughout the length of the SARS-CoV-2 viral genome.
   These results suggest that several important miRNAs are likely being
   titrated by SARS-CoV-2 genome that could result in dysregulation of
   post-transcriptional networks in the infected cells. Majority of the
   identified miRNAs were highly expressed in immune cells including
   CD8^+T cells, CD4^+T cells, NK cells, CD^+14 cells and mast cells,
   signifying that these miRNAs could contribute to the progression of the
   viral infection and host immune response. The results also indicate
   that the highly confident genes targeted by these sponged miRNAs were
   enriched for functional terms including ‘post transcriptional gene
   regulation’ and ‘cell to cell communication’ suggestive of a
   large-scale dysregulation across host’s tissues.

   Guterras et al. [[83]55] analyzed sixty SARS-CoV-2 genomes to identify
   regions that could work as miRNA sponges and potentially bind human
   miRNA. They searched for miRNAs that shared 100% identity of the 8mer
   seed region with SARS-CoV-2 genome regions, both positive- and
   negative-sense. Among 2.654 mature miRNA sequences in miRBase, they
   identified over 1000 miRNAs that showed 100% of similarity between
   their sequences and viral RNA. The search for miRNAs was then refined
   to identify those that interact with the SARS-CoV-2 genome with a
   perfect alignment of 11 nucleotides encompassing the seed region. Using
   this approach, 34 miRNAs for positive-sense and 45 miRNAs for
   negative-sense viral RNA were identified that can strongly bind to
   certain key SARS-CoV-2 genes. Literature review revealed the potential
   of these miRNAs in the immunopathogenesis and potential treatment
   possibilities of COVID-19. The disruption and dysfunction of these
   miRNAs may dysregulate the immune response and stimulate the release of
   inflammatory cytokines altering the cellular response to viral
   infection. These miRNAs have been identified in studies related to
   pulmonary and cardiac disorders, including lung cancer, asthma,
   pneumonia, or cardiac fibrosis, among others.

   Khan et al. [[84]56] also used bioinformatic analysis pipeline which
   allowed them to identify 106 host antiviral miRNAs against SARS-CoV-2.
   Whilst comparing these miRNAs with the antiviral miRNAs from VIRmiRNA
   [[85]57], they have found three (miR-17-5p, miR-20b-5p, and
   miR-323a-5p) host miRNAs against SARS-CoV-2 which have experimental
   evidence of having antiviral roles during infections. Next, they
   performed miRNA pathway enrichment analysis and found that several
   pathways might be modulated by the host miRNAs to suppress the entry of
   the virus, prevent the spread of the virions, and to minimize the
   systemic symptoms resulting from the infection. For example, host
   miRNAs might have a probable role in blocking the entry of the virus,
   as they are found to be targeting the pathways needed for viral entry,
   such as PDGF receptor-like signaling, Arf-6 signaling, PI3K-Akt
   signaling, EGFR signaling, signaling evens mediated by focal adhesion
   kinase, CDC42 signaling, the EphrinB-EPHB pathway, Cadherin signaling,
   or RTK signaling. They can also block some machinery like p38 MAPK
   signaling, FAK signaling, or PI3K-Akt signaling, which can be hijacked
   by viruses for their efficient replication, pre-mRNA processing, and
   translation. These host miRNAs might also try to reduce some
   host-induced inflammatory responses to prevent acute lung damage by
   targeting pathways such as VEGF signaling, Integrin signaling, TGF-beta
   signaling or TRAIL signaling. Some signaling pathways, such as CXCR4
   signaling, TGF-beta signaling, mTOR signaling, or PI3K-Akt signaling,
   can facilitate viral survival in infected cells by inhibiting
   apoptosis, autophagy, or early immune responses.

   Therefore, to develop effective therapeutics, there is a need to
   decipher the cellular targets in humans that interact with SARS-CoV-2
   and result in altered functional outcomes. All these bioinformatic
   analyses discovered that human miRNAs harbor binding sites across the
   SARS-CoV-2 genome that could act as post-transcriptional regulators.
   Therefore, miRNA regulation or manipulation could provide a novel basis
   for antiviral therapies. We and others have shown that miRNA expression
   profiles are often cell-type specific and differ between individuals,
   and importantly, can be affected by different factors such as nutrients
   present in our diets, including polyphenols.

Antiviral properties of polyphenols

   Together with well described properties of polyphenols to prevent or
   delay development of noncommunicable diseases such as cardiovascular,
   metabolic or neurodegenerative disorders, studies, both in vitro and in
   vivo, have also revealed their antiviral properties either by entering
   into the defensive mechanism directly through interfering with the
   target viruses, or indirectly through activating the cells associated
   with the adaptive immune system [[86]58]. Several studies have
   described potential antiviral activity of plant food bioactives against
   herpes virus, human immunodeficiency virus, influenza virus or
   hepatitis C virus [[87]59]. In a recent review, it can be observed that
   different polyphenols, such as quercetin, quercetin 3-glucoside,
   resveratrol, rutin, kaempferol, catechin, epigallocatechin,
   epigallocatechin gallate, or ferulic acid derivatives can inhibit the
   replication of both influenza A and B viruses, suppress the influenza
   virus-induced cell death, or inhibit viral activity in the early stage
   of flu infection [[88]60]. These effects have been associated with the
   capacity of polyphenols to exert anti-inflammatory and immunomodulatory
   effects (such as modulation of the expression of cytokines including
   IL-6, IL-8 and IFN-β) or capacity to modulate molecular pathways
   associated with viral infection (such as Toll-like receptor 7 signaling
   pathway, cell cycle pathway or autophagy related pathways). Several
   studies have also observed potential anti-Zika virus activity of
   polyphenols, such as the citrus flavanones [[89]61, [90]62], flavonoids
   [[91]63] or quercetin-3-β-O-D-glucoside [[92]64]. Antiviral activity
   against Ebola virus has been reported for several polyphenols, such as
   quercetin [[93]65], quercetin 3-β-O-D-glucoside [[94]66] or
   epigallocatechin-3-gallate [[95]67]. Moreover, several studies have
   revealed potential antiviral effect of polyphenols against other
   coronaviruses such as Middle East respiratory syndrome-coronavirus
   (MERS-CoV). For example, experimental and computational study showed
   that flavonoids (herbacetin, isobavachalcone, quercetin 3-β-D-glucoside
   and helichrysetin) can block the enzymatic activity of MERS-CoV 3CL
   protease which regulates replication of the MERS-CoV [[96]68].

Polyphenols and miRNA expression in alveolar and endothelial cells

   Polyphenols exert beneficial effects on lung health, which has been
   demonstrated in several animal models. For example, prophylactic
   resveratrol inhalation partially preserves lung function in prematurely
   aging mice [[97]69], whereas oral treatment with curcumin and
   resveratrol, individually or in combination, has beneficial effects in
   lung carcinogenesis [[98]70]. It has also been demonstrated that
   quercetin can ameliorate lung inflammation in pulmonary artery
   hypertension, which is mediated by miR-204 modulation [[99]71].
   Interestingly, in vitro studies demonstrating beneficial effects of
   polyphenols in lung function, were performed mostly on type 2
   pneumocytes. In the majority of these studies the A549 cell line was
   used, a human lung adenocarcinoma cell line that is subsequently
   characterized as a representative to type 2 pneumocytes [[100]72].
   Using this cell line, for example, the anti-inflammatory and
   antioxidant effects of polyphenols have been demonstrated [[101]73,
   [102]74], as well as their involvement in reducing the toxicity of PM
   2.5 particles [[103]75].

   Even though studies demonstrated that polyphenols exert their health
   properties through modulation of expression of protein-coding genes and
   proteins, there are only few of them that investigated the effects of
   polyphenols on the modulation of miRNAs in type 2 pneumocytes. We were
   able to identify a total of five such studies [[104]76-[105]80], where
   the expression of miRNAs was analyzed by RT-PCR or microarray methods.
   The significantly modulated miRNAs are extracted in the [106]Table 1.
   On the other hand, the number of in vitro studies on polyphenol-induced
   miRNA modification in endothelial cells is higher, and we were able to
   identify a total of ten such studies ([107]Table 1), where the
   expression of miRNAs was also analyzed by RT-PCR or microarray methods
   [[108]6, [109]81-[110]89]. The most commonly used model of endothelial
   cells are human umbilical vein endothelial cells (HUVECs), but other
   models of endothelial cells are also used such as human microvascular
   endothelial cells (HMEC-1 cell line) [[111]81, [112]86, [113]90], or
   human retinal endothelial cells (HRECs) [[114]87, [115]88]. These
   experimental studies have demonstrated beneficial effects of
   polyphenols on the endothelial function, in the first place in the
   context of the complex pathophysiology of atherosclerosis, but diabetic
   retinopathy has also been studied. Still, given the findings from the
   scanning electron microscopy of lung tissue obtained during autopsy of
   patients who died from COVID-19, where the presence of the virus in the
   endothelial cells is clearly visible [[116]36], the above experimental
   data represent a valuable source of information that will help us to
   get a deeper insight into potential effects of dietary polyphenols on
   SARS-CoV-2 exposure.

Table 1.

   Polyphenol-modulated miRNAs in pneumocytes, lung and endothelial cells.
   Cells/Tissue Polyphenol Concentration Significantly modulated miRNAs
   Ref
   A549 cells Curcumin 10 - 20 μM miR-206 [117]76
   A549 cells Curcumin 10 μM hsa-miR-330-5p; hsa-miR-331-5p; hsa-miR-1276;
   hsa-miR-544a; hsa-miR-29c-5p; hsa-miR-335-5p; hsa-miR-296-3p;
   hsa-miR-34a-5p; hsa-miR-26a-1-3p; hsa-miR-190a; hsa-miR-362-3p;
   hsa-let-7f-2-3p; hsa-miR-302b-3p; hsa-miR-338-3p; hsa-miR-455-3p;
   hsa-miR-29c-3p; hsa-miR-154-3p; hsa-miR-21-3p; hsa-miR-377-5p;
   hsa-miR-34c-5p; hsa-miR-1257; hsa-miR-744-3p; hsa-miR-502-5p;
   hsa-miR-33a-3p; hsa-miR-424-3p; hsa-miR-92a-1-5p; hsa-miR-10b-3p;
   hsa-miR-769-3p; hsa-miR-1179; hsa-miR-516a-3p; hsa-miR-148a-5p;
   hsa-miR-604; hsa-miR-499a-5p; hsa-miR-1262; hsa-let-7a-3p;
   hsa-miR-25-5p [118]77
   A549 cells Curcumin 50 - 100 μM miR-98 [119]78
   A549 cells Curcumin 20 - 40 μM miR-21 [120]79
   A549 cells Curcumin 15 μM miR-320; miR-26a; let-7i; miR-130a; miR-16;
   miR-125b; miR-23a; miR-27b; miR-155; miR-625; miR-576-3p; miR-186*;
   miR-9*; let-7e [121]80
   Rat lung Quercetin Intra-peritoneal daily injections of 30 mg/kg
   Quercetin for 3 weeks. miR-204 [122]71
   Human microvascular endothelial cells (HMEC-1) Curcumin 1 - 5 μM
   miR-126 [123]81
   HUVECs Resveratrol 50 μM miR-221; miR-222 [124]82
   HUVECs Curcumin 10 μM miR-93 [125]83
   HUVECs Purified epicatechin metabolites: 3′-O-methyl(-)-epicatechin
   (3′MEC), 4′-O-methyl(-)-epicatechin-7-β-D-glucuronide (4′MEC7G) and
   (-)-epicatechin-4′-sulfate (EC4′S) Mixture of 3′MEC, 4′MEC7G and EC4′S,
   1 μM each hsa-let-7a; hsa-let-7f; hsa-miR-10a; hsa-miR-10b;
   hsa-miR-1290; hsa-miR-130b; hsa-miR-134; hsa-miR-181a*; hsa-miR-221*;
   hsa-miR-224; hsa-miR-30a*; hsa-miR-30c; hsa-miR-30e*; hsa-miR-320a;
   hsa-miR-320c; hsa-miR-320d; hsa-miR-361-5p; hsa-miR-365; hsa-miR-543;
   hsa-miR-769-5p [126]84
   HUVECs Mixture A or/and Mixture B One mixture contained compounds
   present in the circulation 1-5 h after consumption of anthocyanin-rich
   sources, named mix A. It was composed of cyanidin-3-arabinoside,
   cyanidin-3-galactoside, cyanidin-3-glucoside, delphinidin-3-glucoside,
   peonidin-3-glucoside and 4-hydroxybenzaldehyde. Once dissolved in the
   culture medium, the final concentrations were 0.1 μM for anthocyanins
   and 0.5 μM for 4-hydroxybenzaldehyde. The other mixture contained
   metabolites present in the circulation for around 15 h after the
   consumption, named mix B. It was composed of hippuric acid, vanillic
   acid, ferulic acid and protocatechuic acid at final concentration in
   the culture medium of 2 μM, 2 μM, 1 μM and 0.2 μM, respectively. Mix
   A/(+) TNF: hsa-let-7f; hsa-miR-126*; hsa-miR-1260; hsa-miR-1268;
   hsa-miR-130a; hsa-miR-181b; hsa-miR-1915; hsa-miR-26a; hsa-miR-30b;
   hsa-miR-361-5p; hsa-miR-374a; hsa-miR-376c; hsa-miR-455-3p; hsa-miR-99b
   Mix B/(+) TNF: hsa-let-7a; hsa-let-7f; hsa-miR-1246; hsa-miR-125b;
   hsa-miR-126*; hsa-miR-1260; hsa-miR-1275; hsa-miR-196b; hsa-miR-216a;
   hsa-miR-23a; hsa-miR-374a; hsa-miR-720; hsa-miR-923 Mix A+B/(+) TNF:
   hsa-let-7c; hsa-let-7f; hsa-miR-1207-5p; hsa-miR-1246; hsa-miR-125a-5p;
   hsa-miR-125b; hsa-miR-1305; hsa-miR-181b; hsa-miR-1915; hsa-miR-361-5p;
   hsa-miR-572; hsa-miR-575; hsa-miR-638 [127]6
   HUVECs Curcumin 40 μM hsa-miR-4298; hsa-miR-23a; hsa-miR-423-3p;
   hsa-miR-671-5p; hsa-miR-21; hsa-miR-29b; hsa-miR-27a; hsa-miR-491-5p;
   hsa-miR-1307; hsa-miR-181a; hsa-miR-92a; hsa-miR-339-5p; hsa-miR-25;
   hsa-miR-27b; hsa-miR-941; hsa-miR-222; hsa-miR-138; hsa-miR-149;
   hsa-miR-1226; hsa-miR-3198; hsa-miR-1973; hsa-miR-665; hsa-miR-3127;
   hsa-miR-196b; hsa-miR-30e; hsa-miR-1246; hsa-let-7g; hsa-miR-1979;
   hsa-miR-1275; hsa-miR-1975 [128]85
   Human microvascular endothelial cells (HMEC-1) Quercetin 30 μM miR-216a
   [129]86
   Human retinal endothelial cells (HRECs) Resveratrol 50 μM miR-15a
   [130]87
   HUVECs and HRECs HT-3O sulfate (HT-3Os), the major plasma metabolite of
   Hydroxytyrosol (HT) 10 μM let-7 [131]88
   HUVECs (CRL-1730) Resveratrol 10 - 50 μM miR-126 [132]89
   [133]Open in a new tab

   These studies have identified 125 different miRNAs differentially
   expressed by polyphenols in alveolar or endothelial cells. Few of the
   miRNAs have been identified in more than 1 study, for example,
   modulation of expression of hsa-let-7f and hsa-miR-126 have been
   observed in 4 different studies, hsa-miR-1246, hsa-miR-125b,
   hsa-miR-23a and hsa-miR-361-5p in 3 studies. Moreover, among these
   miRNAs, some have been identified as presenting antiviral activities.
   We used VIRmiRNA [[134]57] to obtain the list of miRNAs with antiviral
   properties, that included 148 different miRNAs. Comparison with 125
   miRNAs identified as modulated by polyphenols in alveolar and
   endothelial cells, has demonstrated that 30 are in common (hsa-let-7a,
   hsa-let-7c, hsa-let-7f, hsa-let-7g, hsa-miR-10a, hsa-miR-10b,
   hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-126, hsa-miR-130a, hsa-miR-138,
   hsa-miR-149, hsa-miR-155, hsa-miR-15a, hsa-miR-16, hsa-miR-181a,
   hsa-miR-21, hsa-miR-221, hsa-miR-222, hsa-miR-224, hsa-miR-25-5p,
   hsa-miR-26a, hsa-miR-27b, hsa-miR-29b, hsa-miR-30b, hsa-miR-30c,
   hsa-miR-491-5p, hsa-miR-92a, hsa-miR-93, hsa-miR-98) ([135]Fig. 1).
   Among these common miRNAs is miR-221 which has been identified as
   presenting antiviral ability against respiratory syncytial virus in
   bronchial epithelial cells [[136]91]. Regarding hsa-miR-125b, its
   antiviral activities were described for human papillomavirus and
   respiratory syndrome [[137]92, [138]93]. Taken together this analysis
   suggests that polyphenols could modulate expression of miRNAs in
   alveolar and endothelial cells and exert antiviral capacity.

Figure 1.

   Figure 1.
   [139]Open in a new tab

   Venn diagram showing the overlapping between microRNAs with antiviral
   properties and microRNAs which expression can be modulated by
   polyphenols in type 2 pneumocytes and endothelial cells.

Modulation of miRNAs by polyphenols related to SARS-CoV-2

   Our next step was to compare the list of human miRNAs that may interact
   with SARS-CoV-2 genome with the miRNAs which expression can be affected
   by polyphenols in alveolar and/or endothelial cells. We identified 644
   miRNAs from the literature that can interact with SARS-CoV-2 genome
   (based on the publications of Bartoszewski et al. [[140]48], Sacar
   Demirci and Adan [[141]50], Srivastava et al. [[142]53], Guterres et
   al. [[143]55], and Khan et al. [[144]56]) and 125 miRNAs which
   expression can be modulated by polyphenols. Comparison of the 2 lists
   revealed 17 miRNAs in common: hsa-let-7a-3p, hsa-miR-25-5p,
   hsa-miR-1246, hsa-miR-125a-5p, hsa-miR-1262, hsa-miR-1290,
   hsa-miR-148a-5p, hsa-miR-154-3p, hsa-miR-21-3p, hsa-miR-320c,
   hsa-miR-335-5p, hsa-miR-34a-5p, hsa-miR-377-5p, hsa-miR-455-3p,
   hsa-miR-499a-5p, hsa-miR-544a and hsa-miR-744-3p. This analysis
   suggests that polyphenols could modulate miRNA expression of host cells
   and potentially improve outcome upon SARS-CoV-2 exposure.

   Among the miRNAs in common is hsa-miR-1246. It has been observed that
   this miRNA has homologous sequence to ACE2, which plays role in
   integration with the virus and mediates viral entry. One recent study
   reported higher expression of ACE2 in the small airway epithelium of
   smokers compared to nonsmokers [[145]94]. The same study observed that
   the expression of miR-1246 is downregulated in the cells of smokers
   compared with nonsmokers, suggesting that miR-1246 plays a role in the
   regulation of ACE2, thus shedding light at the pathogenesis of COVID-19
   and risk factors in the population. On the other hand, it was reported
   that curcumin can increase the expression of this miRNAs in endothelial
   cells [[146]85]. This could suggest that consumption of curcumin would
   increase the expression of hsa-miR-1246, lower ACE2 levels and
   consequently the SARS-CoV-2 infection. It was also reported that human
   alveolar basal epithelial cells infected with influenza A viruses can
   modulate the expression of 20 miRNA, including up-regulation of
   miR-1290 [[147]95]. When miR-1290 antagonist, LNA-1290 is administrated
   to the cells, there is significant reduction of viral protein levels
   and viral titers [[148]96]. Interestingly, we have described that
   flavanols can decrease the expression of this miRNA in endothelial
   cells [[149]84]. Several studies also suggested the role of miR-21-3p
   in viral infection. For example, in A549 cells, miR-21-3p promoted
   replication of influenza A virus [[150]97]. Infection of A549 cells
   with influenza A virus (H1N1 or H5N1) in the presence of miR-21-3p
   inhibitor significantly decreased the viral load in the cells probably
   through regulation of HDAC8. On the other hand, in A549 cells exposed
   to curcumin, a significant down-regulation in the expression of this
   gene has been identified [[151]77]. Potential role in viral and/or
   antiviral properties have been described for few other common miRNAs,
   such as let-7a-3p [[152]98], miR-125a-5p [[153]99] or miR-455-3p
   [[154]100].

   Together with literature analysis, we also performed bioinformatic
   analysis to identify potential target genes of these 17 common miRNAs.
   Using MirWalk database ([155]http://mirwalk.umm.uni-heidelberg.de)
   [[156]101] we identified 1734 potential targets with minimum free
   energy <-20kcal/mol indicating a stronger predicted pairing between the
   miRNA and the target mRNA. With the aim to identify potential pathways
   in which these target genes are involved in we performed pathway
   analysis using MirWalk database as well as GeneTrail2 database.
   Interestingly, both databases showed that the potential target genes
   are involved in different cellular pathways, including those that have
   been previously identified as involved in regulation of SARS-CoV-2
   replication [[157]56]. These pathways include EGFR signaling, focal
   adhesion kinase, integrin signaling, mTOR signaling, p38 MAPK
   signaling, PDGF receptor-like signaling, PI3K-Akt signaling and VEGF
   signaling, described to regulate entry of the virus, its efficient
   replication, inflammatory responses or viral survival in infected cells
   ([158]Fig. 2). Moreover, comparison of the target genes with genomic
   data present in databases using Enrichr tool
   ([159]https://maayanlab.cloud/Enrichr/) [[160]102] revealed significant
   homology with gene expression of healthy lung biopsy vs. SARS-CoV-2
   infected lung, SARS-CoV-2 infection in lung tissue or A549 infected
   with SARS-CoV-2. Therefore, from the data on the effect of polyphenols
   on expression of miRNAs which potentially can target the SARS-CoV-2
   genome as well as their potential targets, it could be suggested that
   these bioactives modulate expression of miRNAs in the host cells in
   such a way to prevent viral entry, its replication and survival, thus
   preventing or lowering the risk of development of complications in
   COVID-19.

Figure 2.

   Figure 2.
   [161]Open in a new tab

   Network between 17 miRNAs, common between cellular miRNAs than can
   interact with SARS-CoV-2 RNA and miRNAs modulated by polyphenols, and
   their potential target genes. Blue circles represent miRNA, red circles
   are their potential targets. In yellow are target genes involved in
   pathways regulating virus entry, replication and viral infections.

Conclusion

   The role of host miRNAs in viral infection, both pro-viral and
   antiviral properties, have been little described, particularly for
   SARS-CoV-2. Nevertheless, the existing knowledge described in this
   paper can provide solid bases to support the hypothesis that
   polyphenols can have protective properties against SARS-CoV-2 by
   modulating the expression of miRNAs of the host cells, hypothesis that
   can be corroborated with few studies that suggested that these
   bioactives can interact with ACE2 or other proteins involved in
   COVID-19 ([162]Fig. 3). It is therefore urgent to develop studies to
   assess their capacity to prevent COVID-19 complications, such as in
   vitro studies with alveolar and endothelial cells, animal studies with
   SARS-CoV-2 infection along with diets supplemented with polyphenols. It
   is primordially important also to evaluate correlation between intake
   of polyphenols by the population and the incidence of COVID-19. Such
   study will provide direct demonstration of the impact of polyphenols on
   prevention of SARS-CoV-2 infection and development of complications.

Figure 3.

   [163]Figure 3.
   [164]Open in a new tab

   Potential mechanism of action of polyphenols on SARS-CoV-2 replication
   via modulating host cell miRNA expression (from [165]Biorender.com).

Footnotes

   Conflict of interest

   The authors do not have conflict of interest.

References