microRNAs as new possible actors in gender disparities of Covid-19 pandemic.

Based on clinical and epidemiological investigations conducted so far, gender disparity exists in outcomes regarding Coronavirus disease 2019 (COVID‐19) affected patients as men show more serious forms and higher lethality than women. At present, mechanisms underlying the observed difference are not clear enough.

The SARS‐CoV‐2 virus, responsible for COVID‐19, belongs to Coronavirus family and has high similarity with virus causing SARS‐CoV and MERS. Hence, thanks to knowledge derived from previous studies performed on these coronaviruses, scientists have been able to understand different aspects of SARS‐CoV‐2 virulence. Nonetheless, besides several similarities connecting these homologous viruses, a key point is the gender‐related outcome discrepancies emerging from epidemiological data.

Coronaviruses are enveloped viruses with a positive single‐strand RNA genome, responsible for enteric, respiratory and central nervous system diseases with different grades of severity in a variety of animals and humans. They are able to infect target cells through their surface protein Spike (S) able to bind cellular receptors and subsequently fuse the viral envelope with the host cell membranes. Protein sequence analysis revealed 76% protein identity between SARS‐CoV and SARS‐CoV‐2 S protein, thus supporting shared host cellular receptor ACE2.

The interaction of ACE2, a metallopeptidase whose cleavage might promote viral uptake, with the Spike protein results in SARS‐CoV‐2 target cell invasion. To complete the infection process, SARS‐CoV‐2 needs host co‐receptors able to cleavage viral and cellular proteins. Among them, TMPRSS2, ADAM17 and Furin have been described to be involved in this process.

One of the most significant explanations for gender differences in COVID‐19 lethality is the sex hormone–based modulation of cellular receptor and co‐receptors used by SARS‐CoV‐2 to enter in human host cells. Indeed, the X‐linked ACE2 gene expression is regulated by oestrogen, whereas TMPRSS2‐coding gene is characterized by an androgen responsive promoter.

Beyond the direct effects on the aforementioned coding genes, an important role can be played by microRNAs. Several of these small RNAs, acting as post‐transcriptional modulators of gene expression, are regulated by sex hormones. In addition, the X‐chromosome is enriched for microRNAs.

In order to investigate the mechanisms underlying gender differences in COVID‐19 susceptibility and outcome, we focused our attention on miRNAs implicated in the modulation of co‐receptors acting with ACE2 to favour coronavirus infection.

TMPRSS2 is a serine protease encoded by an androgen responsive gene. As such, men are expected to show a higher expression of TMPRSS2 with respect to women, although some controversial results were reported. In fact, mRNA expression of TMPRSS2 in the lung tissue showed no difference between men and women. For this reason, beyond the direct TMPRSS2 gene regulation by androgens, we considered the possible miRNA‐based post‐transcriptional regulation of TMPRSS2. According to the TargetScan predictive online tool, among miRNAs putatively targeting TMPRSS2, we were attracted by let‐7a‐g/i, and miR‐98‐5p, another member of let7 family, which might be modulated in a gender‐specific manner. Let‐7a‐g/i are located in the intragenic region of an estradiol‐modulated gene and resulted upregulated by oestrogen/ERα activation. Let‐7a expression resulted positively regulated by oestrogen and progesterone treatment in ovarian cancer cells. Also, in breast cancer cells (MCF7 cell line) 17β‐estradiol (E2) was able to induce the expression of Let‐7 family members. On the other hand, they were downmodulated in prostate cancer cells under androgenic effect. Furthermore, miR‐98‐5p, an oestrogen‐responsive miRNA undergoing ERα‐positive modulation, can bind and repress IL‐6 gene expression in turn influencing some proinflammatory cytokines, such as TNF‐α, IL‐1β and IL‐10. Therefore, oestrogens seem to induce expression of miRNAs capable to target and inhibit TMPRSS2 mRNA translation, probably reducing its necessary availability for SARS‐CoV‐2 infection. Noteworthy, among their numerous roles, let‐7 miRNAs were reported to be closely associated with immunity, regulating cytokine expression during pathogen infection. To deepen further our analysis, we have to consider another type of regulation of gene expression related to long non‐coding RNA (LncRNA), which are non‐coding RNAs capable to act as sponges for miRNAs and coding mRNAs. We have identified the lncRNA H19, harbouring canonical and non‐canonical binding sites for the let‐7 family of miRNAs. In vitro and in vivo analysis demonstrated that H19 could modulate let‐7 availability. Among many oncogenic roles, H19 is able to modulate the expression of IL‐6 via sponging the let‐7 family members in turn possibly inducing TMPRSS2 expression by sequestering its target miRNAs. We could hypothesize that H19 action may be likely responsible for a greater virus ability to infect cancer cells.

ADAM17 is a disintegrin and metalloproteinase domain 17 responsible for the release of ectodomains of a different variety of membrane‐anchored cytokines, receptors, ligands and enzymes, such as ACE2. ADAM17 has been already identified as a co‐receptor for SARS‐CoV infection (SARS), even if there are some controversies regarding its role. Heurich and co‐authors suggested a role for ADAM17 in the protection from viral infection, whereas others studies suggested that ADAM17 action was necessary for cellular viral entry. Hence, further studies are needed to better dissect the role of ADAM17 in SARS‐CoV‐2 infection. Interestingly, ADAM17 plays a decisive role in inflammation since it is involved in the activation of proinflammatory cytokines and cytokine receptors, including TNFα and IL‐6R. It is well known that part of the severe consequences of COVID‐19 infection depends on the cytokine release syndromes, or “cytokine storm” where IL‐6 is the main proinflammatory factor capable of causing significant damage to organ function together with IL‐7, IL‐16 and IL‐18. Further exploring possible mechanisms associated with gender differences in COVID‐19 outcomes, we identified miRNAs putatively implicated in ADAM17 regulation. Among them, the most noteworthy is the X‐linked miR‐222 belonging to the miR‐221&222 cluster. This couple of miRNAs presents an Estrogen Receptor Element (ERE) binding site in its promoter region, suggesting a role for oestrogen in their upregulation. Moreover, according to a negative feedback loop, miR‐221&222 inhibit ERα mRNA translation by direct binding to its 3’UTR, being in turn repressed by ERα. To further increase the complexity of this circuitry, miR‐222 is downmodulated by androgen. Another important miRNA that targets ADAM17 mRNA is miR‐145. This miRNA resulted to be upmodulated by Vitamin D, which has been recently suggested as a protective factor in SARS‐CoV‐2 infection and severe outcomes.

Coronaviruses were reported to use different cellular entry mechanisms in terms of membrane fusion activities after receptor binding. Another cofactor possibly favouring SARS‐CoV‐2 infection is the proteolytic enzyme Furin. SARS‐CoV‐2 S protein contains four redundant Furin cut sites (PRRA motif) that are absent in the SARS‐CoV sequence. , Accordingly, prediction studies suggest an efficient cleavage of the SARS‐CoV‐2 but not SARS‐CoV S protein by Furin. Indeed, Furin plays an important functional role being able to cut the S protein at the S1/S2 site, a cleavage essential for the S‐protein–mediated cell‐cell fusion and entry into human lung cells. In this view, Furin, known to act as an oncogene and involved in several metabolic diseases, might represent a potential therapeutic target in COVID‐19. The Furin enzyme is ubiquitously expressed in human tissues, including lungs, liver and small intestine, which are second target organs well documented in COVID‐19 patients. It is a membrane‐binding molecule, but an active secreted isoform also exists, potentially facilitating the cleavage of the SARS‐CoV‐2 S protein in the cellular neighborhood. Downregulated levels of Furin have been recently detected in Chinese patients with hypertension, one of the main risk factors for COVID‐19 lethality. No evidence demonstrated a clear gender difference in Furin expression so far. Nevertheless, its transcript might be directly modulated by X‐linked and/or sex‐hormone sensitive miRNAs, eg miR‐20b, miR‐19a and miR‐19b‐3p, miR‐106a. miR‐20b, induced by E2 treatment, is involved in a negative autoregulatory feedback loop with ERα. Indeed, miR‐20b is able to target and inhibit the 3′UTR of ERα, thereby making difficult to assess its possible modulation to different oestrogen concentration. The others putative miRNAs targeting Furin are miR‐19a and miR19b‐3p and miR‐106a, which are regulated by E2‐mediated upregulation via secondary transcription factors.

Interestingly, in silico analyses showed that the oestrogen‐modulated miRNAs miR106a, miR‐20b and miR‐19a/b regulate also toll‐like receptor (TLR) 7 (TargetScan, v. 7.2) thus leading to speculate about a possible circuitry involving oestrogens, miRNAs and immune responses.

Finally, to corroborate further the significant involvement of miRNAs in SARS‐CoV‐2 infection, we can also consider the influence of gender‐related life styles on miRNAs dysregulated expression.

For example, deregulation of miR‐145, let‐7 and miR‐222 have been described in lung cancer as consequence of cigarette smoking. Such considerations may be related to higher expression of SARS‐CoV‐2‐host cellular receptor and co‐receptor (TMPRSS2) observed in smokers compared to non‐smokers. Although the number of smoking women is steadily increasing, in Europe they are still half respect to male smokers, thus supporting another possible gender‐related difference associated with Covid‐19 severity and lethality.

For COVID‐19 pandemic, a significant gender disparity was evidenced being the male lethality higher than the female one: in Italy 17.7% vs 10.7%. Sex chromosomes, in particular X chromosome, and sex hormones are key actors in these differences. In this picture, a powerful regulatory intermediation is represented by miRNAs as these small regulatory RNAs are able to modulate approximately 50% of protein coding genes. Interestingly, a different expression in males and females of several miRNAs has been observed owing to sex hormones modulation 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 (see Table 1).

Table 1

miRNAs putatively involved in gender differences underlying SARS‐CoV‐2 virulence

miRNAs	Regulation	Putative target	References
hsa‐let‐7a‐g/i	Up‐modulated by Estrogen/ERα activation and progesterone. Down‐modulated by androgen.	TMPRSS2	6, 8
hsa‐miR‐98‐5p	X‐linked miRNA. Up‐modulated by ERα.	TMPRSS2	7
hsa‐miR‐145	Up‐modulated by Vitamin D	ADAM17	12
hsa‐miR‐222	X‐linked miRNA involved in a negative feedback loop with ERα. Down‐modulated by androgen.	ADAM17	6
hsa‐miR‐19a/b‐3p	Up‐modulated by 17β‐Estradiol	Furin	20
hsa‐miR‐20b	Up‐modulated by 17β‐Estradiol	Furin	19
hsa‐miR‐106a	Up‐modulated by 17β‐Estradiol	Furin	20

CONFLICT OF INTEREST

We have no conflicts of interest to disclose.