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.
1
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.
2
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.
3
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.
4
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.
4
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.
5
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.
6
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.
7
On the other hand, they were downmodulated in prostate cancer cells under androgenic
effect.
8
Furthermore, miR‐98‐5p, an oestrogen‐responsive miRNA undergoing ERα‐positive modulation,
7
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.
9
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.
10
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.
6
To further increase the complexity of this circuitry, miR‐222 is downmodulated by
androgen.
11
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.
12
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.
13
,
14
Accordingly, prediction studies suggest an efficient cleavage of the SARS‐CoV‐2 but
not SARS‐CoV S protein by Furin.
15
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,
16
might represent a potential therapeutic target in COVID‐19.
15
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.
17
Downregulated levels of Furin have been recently detected in Chinese patients with
hypertension, one of the main risk factors for COVID‐19 lethality.
18
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.
19
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.
20
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.
21
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%.
22
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
John Wiley & Sons, Ltd
This article is being made freely available through PubMed Central as part of the
COVID-19 public health emergency response. It can be used for unrestricted research
re-use and analysis in any form or by any means with acknowledgement of the original
source, for the duration of the public health emergency.
CONFLICT OF INTEREST
We have no conflicts of interest to disclose.