In our previously reported cohort, miRNA-3115 and miRNA-7151 were expressed in non-severe group exclusively. In addition, patients who expressed miRNA-3115 and miRNA-7151 were characterized by a lower inflammatory response, and these miRNAs were present more prevalently in those who did not develop a subsequent worsening disease as compared to those who worsened.

To date, no data are available on miRNA-3115 and miRNA-7151 effects in COVID-19 and our data seem to potentially support, for the first-time, a possible protective role of these miRNAs in COVID-19 patients. The pathophysiological roles of miRNA-3115 and miRNA-7151 are currently poorly understood. Interestingly, using a widely accepted database for prediction of miRNAs functional targets, we have evaluated candidate transcripts with very high scores ≥80 (range 0–100) observing that miRNA-3115 is potentially related to a negative modulation of G-protein-coupled receptor 17 (GPR17) and miRNA-7151 to an inhibitory effect on cathepsins B/L (CTSB/L) and Kringle Containing Transmembrane Protein 1 (KREMEN1) [9]. Interestingly, GPR17 is a seven-transmembrane domains receptor with a tissue broad distribution playing different roles in cells remodeling and repairing [10]. Recently, the inhibition of GPR17 in mouse models using Cangrelor, an antagonist anti-platelet drug, was associated with a significant decrease in the inflammatory response injury and pulmonary fibrosis during sepsis [11]. Also, CTSB/L is an endosomal protease involved in cleaving and activating SARS-CoV-2 spike-protein, a required step for viral entry in host-cells [12, 13]. In vitro studies have demonstrated that blocking CTSB/L effectively reduces and prevents viral entry [12, 13]. Finally, KREMEN1 is a transmembrane protein expressed in various tissues and, recently, was also demonstrated to act as an ACE2-alternative viral entry receptor for SARS-CoV-2 and its in vitro blockage was associated with a substantial reduction of viral infectivity [14, 15]. These evidences may support the hypothesis that miRNA-3115 and miRNA-7151 expression could represent a protective factor against worse COVID-19 through the inhibition of the pathways mentioned above.

Besides miRNAs effects, in our cohort, vitamin D deficiency was previously demonstrated to predict the occurrence of severe outcomes prospectively and independently [6]. Several pathophysiological hypotheses were proposed to explain the negative influences of vitamin D deficiency in COVID-19 [1, 16,17,18,19], however, no data are currently available regarding vitamin D and miRNAs relationship in this clinical setting. Despite the non-statistically significant differences in either rates of hypovitaminosis D or 25(OH) vitamin D levels among patients with and without miRNAs expression, possibly also due to the limited number of study subjects, we found an association between vitamin D deficiency and lack of miRNA-7151 expression in COVID-19 patients with worse outcomes which may support the hypothesis that the co-existence of these two conditions may have a strong negative prognostic role. It remains to be investigated in larger cohorts if lack of vitamin D and miRNA-7151 can act synergistically and independently, or with a cause-effect relationship or even if supplementation of vitamin D may have more effective protective role in those with vitamin D deficiency and lack of protective miRNA expression [20].

First limitation of our study is the low number of patients included. This is mainly related to the stringent inclusion and exclusion criteria adopted. Another important limitation is the lack of evaluation of potential pathophysiological mechanisms underlying the associations between miRNAs expression, vitamin D deficiency, and COVID-19 severity.

However, besides these limitations, this is the first study that specifically evaluated vitamin D and miRNAs relationships in influencing COVID-19 outcome. Moreover, we firstly reported on the expression of two novel miRNAs as potentially useful protective biomarkers of COVID-19 severity and the prospective design used in the study allowed us to hypothesize a combined predictive role of low vitamin D levels and miRNA expression.

In conclusion, the expression of two novel miRNAs was reported for the first-time to be associated with a less severe COVID-19 form and to prospectively predict the occurrence of disease outcome. Moreover, the association observed between vitamin D levels and miRNA expression may potentially expand our knowledge about vitamin D role in acute COVID-19, but further investigations should be also performed in Long COVID scenario and in response to anti-COVID-19 vaccination, settings in which vitamin D levels were recently proposed to have a potential role [21,22,23].