MicroRNAs as biomarkers of coronavirus disease 2019 (COVID-19): a scoping review

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus which was identi�ed as the infectious agent responsible for coronavirus disease 2019 (COVID-19). MicroRNAs (miRNAs) are small non-coding RNAs that can be potential biomarkers of several diseases. The aim of this scoping review was to identify which miRNAs could be biomarkers of COVID-19 and their roles. Methods. A literature search was performed based on PubMed, PubMed Central, BVS/BIREME, Web of Science, Scopus, EBSCOhost, ProQuest, Embase, and Cochrane Library for studies published until January 28th, 2021. Animal and human studies that described miRNAs as biomarkers of SARS-CoV-2 infection/COVID-19 were included. Studies with a purely computational approach were excluded. Results. A total of 1,797 records were identi�ed, seven of which met the eligibility criteria. Six studies were conducted in humans (samples derived from blood) and one was performed in an animal model (lung tissue). The most important miRNAs identi�ed were miR-195-5p, miR-618, miR-146a-5p, miR-21-5p, miR-15b-5p, miR-142-3p, miR-155, miR-208a, miR-499, miR-103a-2-5p, miR-200c-3p, miR-2115-3p, and members of the let-7 family. Among these miRNAs, miR-146a-5p, miR-21-5p, miR-15b-5p, and members of the let-7 family may be important as they were deregulated in more than one study. Dysregulated miRNAs appear to play key roles in viral replication, proliferation of infected cells, immune response, in�ammation, or cardiovascular dysfunction. Conclusion. MiRNAs may be used as potential diagnostic or severity biomarkers, predictive biomarkers, biomarkers of T-cell immune response, and as therapeutic targets of COVID-19. Further studies are required to investigate and validate the role of miRNAs as biomarkers of COVID-19.


Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus that was identi ed as the infectious agent responsible for coronavirus disease 2019 (COVID-19) [1].After the initial outbreak in Wuhan (China), COVID-19 spread across the world, and was declared a pandemic in March 2020 [2].At the time of writing of this article (March 2021), more than 100 million cases of COVID-19 had been reported across 223 countries, with 2.7 million deaths [3].It is worth mentioning that there is no speci c treatment available for COVID-19 and that the management of the disease is empirical [4].To date, only three vaccines have had their emergency use approved by the Food and Drug Administration (FDA), the P zer-BioNTech, the Moderna, and the Janssen (Johnson & Johnson) COVID-19 vaccines [5,6].However, other vaccines have been approved in other countries.
Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) performed on nasopharyngeal or oropharyngeal swabs is the most widely used diagnostic method for SARS-CoV-2 infection [7].This technique has been criticized for being relatively invasive and is associated with an increased risk of cross-infection [8].Serological tests may also be used to diagnose COVID-19.These are based on the detection of SARS-CoV-2 speci c antibodies (IgM and/or IgG) in blood samples.The detection of IgM can be interpreted as an indicator of acute infection, whereas the detection of IgG represents previous infection/immunity.However, serological tests have low sensitivity when performed on the rst days after the onset of symptoms, have signi cant rates of false-negative results, and poor validation of results [9].Chest computed tomography is an alternative test for diagnosing and monitoring COVID-19 [10].Finally, some in ammatory (procalcitonin, C-reactive protein), hematologic (lymphocyte, thrombocytes), and biochemical (creatine kinase-MB, troponin I, D-dimer, aspartate amino-transferase, alanine amino-transferase, lactate dehydrogenese, gamma-glutamyltransferase) biomarkers have been associated with severe COVID-19 and might help in prognostic risk strati cation of patients with COVID-19 [11].
Therefore, identi cation of effective diagnostic biomarkers and predictors of severity for COVID-19 is becoming increasingly important so that patients can receive an accurate and targeted therapy.
MicroRNAs (miRNAs) are small non-coding ribonucleic acids that are approximately 22 nucleotides long, which enhance messenger RNA (mRNA) degradation and inhibit protein translation [12].In this way, they play essential regulatory roles in several biological processes and represent potential biomarkers of diseases to be explored [12,13].Moreover, it is known that virus infection changes host miRNA expression [14] and dysregulated miRNAs have already been studied as biomarkers of several infectious diseases [15].Thus, miRNAs can also be biomarkers of COVID-19 [16][17][18][19].The aim of this scoping review was to identify which miRNAs could be biomarkers of COVID-19 and their roles.

Methods
This review was conducted following the recommendations of the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement for Scoping Reviews (PRISMA-ScR) [20], and the review protocol was registered in the Open Science Framework (https://doi.org/10.17605/OSF.IO/M5VJ6).

Search strategy.
A comprehensive literature search published until January 28 th , 2021 was performed in the PubMed, PubMed Central, BVS/BIREME, Web of Science, Scopus, EBSCOhost, ProQuest, Embase, and Cochrane Library databases to identify relevant studies.The search strategy included combinations of terms related to COVID-19 and miRNAs.The full strategy search for all databases can be found in the Appendix S1.No language restrictions were applied.
2.2.Study selection.Cell, animal, and human studies that described miRNAs as biomarkers of SARS-CoV-2 infection/COVID-19 were included.Studies with a purely computational approach were excluded.Preprints, books and book chapters, editorials, comments, conference proceedings or abstracts, and literature reviews and guidelines were also excluded.The studies retrieved from the databases were allocated to the Rayyan QCRI program [21] to exclude duplicate les (phase 1), analyze the titles and abstracts of the articles (phase 2), and analyze complete articles whose abstracts were previously selected (phase 3).Two reviewers (MBV and ASN) independently reviewed the titles and abstracts of all studies identi ed by the searches, and discussed and addressed any discrepancies arising, with a third reviewer (PM).In addition, references cited in all included articles were reviewed to identify any studies that might have been missed.

Data extraction and analysis.
For each included study, information such as author, date of publication or online availability, country, publication type, population, samples, methods used to identify miRNAs, time when miRNAs were analyzed, miRNAs differently expressed in SARS-CoV-2 infected animals/patients with COVID-19, pathophysiological implications of deregulated miRNAs, and the role of miRNAs as biomarkers of SARS-CoV-2 infection/COVID-19 were extracted.Two authors (MBV and ASN) independently completed the data extraction using a preformatted spreadsheet in Microsoft Excel.Disagreements were resolved with a third author (PM).The results of this scoping review are presented as a narrative synthesis.After following the PRISMA-ScR guidelines [20], no quality assessment was performed because scoping reviews aim to identify all the available evidence and highlight their main characteristics, regardless of the quality of such evidence.

Results
3.1.Search results.The electronic search found 1,797 potentially relevant studies.After removing duplicates and reviewing the titles and abstracts, 29 articles were selected for full-text reading.In addition, no relevant studies were identi ed by searching the reference lists of the selected studies.After careful full-text reading, only seven studies [22][23][24][25][26][27][28] met the inclusion criteria and thus were included in the review.A owchart of the literature search is shown in Figure 1.The references for the excluded studies, along with the reasons for their exclusion, are available in the Appendix S2.
From our perspective, the COVID-19 pandemic changed the pro le of published articles; currently, more articles are being published as rapid or short communications with low details.We also noted that many articles only commented on previously published articles, extrapolating the results for SARS-CoV-2 infection, or were about the elucidation of mechanisms, suggesting some important role in SARS-CoV-2 infection.In addition, we found many studies that used only bioinformatics data.Although these characteristics are justi ed by the urgency demanded by the pandemic and the need for knowledge about this new virus, such articles were excluded from our analysis.Surprisingly, the seven studies included in this scoping review [22][23][24][25][26][27][28] were research articles, and six of them were conducted in humans [23][24][25][26][27][28]; studies on miRNAs as biomarkers have most often been conducted in cells and animal models.Most of the studies were conducted in China [23][24][25][26], since this country was the rst to be affected by the SARS-CoV-2 [1].
Though it is important to standardize the time of sample collection for analysis after infection or symptom onset, we observed that there was no standard time for the analysis of miRNAs between studies.Therefore, it was not possible to compare this important parameter.Further research studies with standardized times need to be developed, including more prospective longitudinal studies, to verify how these miRNAs behave over infection time.Moreover, in most human studies [23][24][25][26], miRNAs described as potential biomarkers were detected by sequencing, and they need to be validated through RT-PCR in a higher number of patients.
Three studies indicated that miR-21-5p was important for COVID-19 [24,27,28].Similar to miR-146a-5p, miR-21-5p regulates in ammation and is a marker of the proin ammatory state [27].In two studies, it was shown that miR-21-5p was downregulated in patients with severe COVID-19 [24,27], whereas it appeared as upregulated in a different study [28].However, miR-21-5p appeared to be more speci c to severe COVID-19 since it was signi cantly downregulated in patients with severe disease compared to those with moderate disease [24].Moreover, this miRNA appeared to be more speci c of SARS-CoV-2 infection since it was signi cantly more deregulated in patients with COVID-19 than in patients with in uenza-induced ARDS [28].In a study that evaluated miRNAs as markers of cardiovascular damage in patients with COVID-19, miR-21-5p was associated with cardiac brosis and might be a predictor of chronic myocardial damage and in ammation in COVID-19 survivors [28].
Two studies found an association between miR-15b-5p and SARS-CoV-2 infection/COVID-19 [22,24].Controversially, one study showed that it was downregulated in the lung tissue infected by SARS-CoV-2 [22], whereas others showed that it was upregulated in patients with COVID-19 [24].A study that used machine learning to predict the likelihood of miRNAs targeting the SARS-CoV-2 genome found a score of 99 in this assessment for miR-15b-5p [32].Besides directly targeting the viral genome, miR-15b-5p may also regulate the host immune response [33,34].Thus, miR-15b-5p is associated with viral replication and proliferation of infected cells [22,24], and a decrease in the levels of this miRNA may favor viral replication and host pathogenesis [35].Kim et al. [22] indicated that this miRNA is a potential diagnostic biomarker for SARS-CoV-2 infection.At the same time, Tang et al. [24] suggested a key role of miR-15b-5p in COVID-19 pathogenesis and patient deterioration, and that it might also be a speci c biomarker of severe COVID-19.
Two studies found that members of the let-7 family were deregulated in patients with COVID-19 [25,26].Both showed the importance of let-7 in T-cell activation [25,26].In fact, let-7 miRNAs play a key role in the activation of the immune system and the in ammation response by targeting IL-6 gene and reducing its expression [19,36].Downregulation of let-7 family members leads to an intense in ammatory process.Thus, these miRNAs may also be effective predictive biomarkers of clinical outcome [26].
Therefore, miR-146a-5p, miR-21-5p, miR-15b-5p, and members of the let-7 family may serve as important biomarkers of COVID-19.However, more studies including non-COVID-19 patients with pneumonia or ARDS as positive controls for moderate and severe groups as well as asymptomatic COVID-19 patients are required to validate the speci city of these miRNAs as biomarkers [24].
We have included a small number of studies in this scoping review despite the COVID-19 emerging in December 2019 (over a year ago) [1].In other words, there is a lack of studies evaluating miRNAs as biomarkers of COVID-19.Therefore, we encourage that further primary studies are conducted, mainly including populations from North and South America, where COVID-19 has had a high incidence.This opens a great opportunity to develop new research.
This study has some limitations: some studies may have been missed due to not being indexed in the databases searched.In addition, studies of interest available after the established search period have not been included.Moreover, this review did not analyze the quality of the studies, considering the inherent characteristics of the scoping reviews.

Table 1 :
Characteristics of the studies included in this scoping review.