Abstract
Background/Aim: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 infection, manifests a wide range of clinical symptoms ranging from mild to moderate and severe. Host-related factors influence the course of SARS-CoV-2 infection; for instance, the expression of host microRNAs (miRNAs) could influence the progression and complications of COVID-19. This study aimed to determine the expression pattern of endogenous miRNAs in 80 severe COVID-19 patients compared to a group of healthy individuals. Materials and Methods: The miRNA screening expression analysis was performed using TaqMan Low-Density Array, and the expression changes of miR-490-3p, miR-195-5p, miR-454-3p, and miR-431-5p were validated using RT-qPCR. In silico analysis was used to identify new targets and predict the pathways, biological processes, and interactions of the selected miRNAs. Results: The miR-490-3p, miR-195-5p, miR-454-3p, and miR-431-5p, were over-expressed in the total population of severe COVID-19 patients compared to the control group. miR-490-3p was found to be over-expressed in both female and male COVID-19 patients. Conclusion: Specific miRNAs might be a potential biomarker for predicting the clinical course of COVID-19.
Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 infection, has affected more than 0.77 billion people worldwide, including more than 6.9 million deaths. In Mexico, approximately 7.7 million cases and up to 330,000 deaths have been reported (1); therefore, COVID-19 surveillance remains a public health priority in Mexico for its prevention and control.
The clinical classification of COVID-19 varies widely, ranging from an asymptomatic infection to mild, moderate, severe, or critical disease. Most patients present with mild to moderate respiratory disease, experiencing cough, sore throat, fever, headache, myalgia, diarrhea, and mild pneumonia (2). In severe disease, dyspnea and hypoxia occur in addition to any combination of the symptoms of mild/moderate disease. The condition can progress to critical disease, characterized by acute respiratory distress syndrome (ARDS), shock, and multi-organ system dysfunction, ultimately terminating in death (3, 4). Patients with COVID-19 hypoxic respiratory failure have dysregulated immune response and cytokine secretion, including the release of pro-inflammatory cytokines and elevated concentrations of inflammatory markers (5).
Virus-host interactions induce the production of immunoregulatory factors against the SARS-CoV-2 virus, for instance, micro RNAs (miRNAs) that are small-sized, single-strand, non-coding RNA molecules that regulate viral gene expression (6). The expression pattern of host miRNAs is related to the severity of COVID-19, functioning as protective factors against the SARS-CoV-2 virus because specific endogenous miRNAs can inhibit or reduce transcription and translation of viral proteins (7, 8). In this sense, Haddad and Walid (9) proposed that certain human miRNAs (hsa-miR-497-5p, hsa-miR-21-3p, and hsa-miR-195-5p) may target genomic RNA variants located in the spike glycoprotein sequence of SARS-CoV-2, thereby affecting single-stranded RNA translation. However, the viral response also influences this regulation, since some viral microRNAs can degrade or perform in a sponge-like manner, trapping human endogenous microRNAs as miR-302c-5p. This could increase the expression of ACE2, thus inhibiting the antiviral response (10, 11).
Identifying potential biomarkers associated with the severity of COVID-19 is relevant, as these could be used in developing tests predicting disease severity. The objective of this work was to determine the pattern of expression of endogenous microRNAs and its relationship with the severity of COVID-19 in Mexican patients. To achieve this objective, we first analyzed global miRNA expression in both patients and controls. Then, we examined the expression of the most relevant miRNAs according to their expression pattern in a largest number of individuals.
Materials and Methods
Study type. The Health Institute of the Mexican State (ISEM) performed this observational descriptive study on healthcare. The hospital’s Bioethics Committee in Research approved the study (registry number 208C0101110500T-3157_2020-08). All participants consented to the data collection and signed the informed consent form in accordance with the Declaration of Helsinki.
Sample collection. From August 2020 to August 2021, the serum samples of 40 female and 40 male patients with severe COVID-19 and 80 healthy individuals as negative controls were collected. As previously described (12), the confirmation of the COVID-19 diagnosis was performed employing RT-qPCR.
RNA extraction. Nasopharyngeal swab samples in viral transport medium (VTM) were extracted using the Magna Pure 96 Instrument (Roche Diagnostics, Indianapolis, IN, USA) using the MagNA Pure LC RNA Isolation Kit - High Performance (Cat 03542394001, Roche). RNA HP Blood external lysis DNA and Viral NA Small Volume purification protocol was employed according to the manufacturer’s instructions.
RT-PCR real time. SARS-CoV-2 Virus Detection was performed by using One-Step Real-Time RT-PCR kit (RT-qPCR; Cat. MAD003941M, Vitro Master Diagnostica, Spain) as previously described (12). Briefly, the RT-qPCR reaction mixture (12 μl SARS-CoV-2 MMix and 8 μl of total extracted RNA) was subjected to a reverse transcription reaction (5 min at 25°C followed by 20 min at 50°C) followed by heating at 95°C for 5 min for enzyme deactivation. The PCR cycling program consisted of 45 cycles: 30 s at 95°C for denaturation, and 60 s at 60°C for annealing and extension, performed in the CFX96 Touch Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). Non-infectious synthetic DNA provided in the kit (PC SARS-CoV-2) was used as negative and positive controls.
RNA extraction. Serum samples were extracted on the QIAcube Connect (QIAGEN Hilden, Germany) employing the QIAamp Viral RNA Minikit 250 (Cat. Number 52906) according to the manufacturer’s protocol.
miRNA amplification. The global profiling for miRNA expression for 10 samples per group was performed using the TaqMan Array Human MicroRNA Panel v2.0 (Applied Biosystems, Foster City, CA, USA), which includes Cards A and B in a 384-well format (Cat.94566, Applied Biosystem) according to the manufacturer’s instructions. In brief, total RNA was first reverse transcribed with Megaplex RT primers Human A or B and the High-Capacity cDNA Reverse Transcription Kit (Cat 4368813, Applied Biosystems). The reactions were incubated for 5 min on ice; the RT protocol involved 40 cycles consisting of 2 s at 16°C, 1 min at 42°C, 1 s at 50°C, followed by 5 min at 85°C, and held indefinitely at 4°C.
The pre-amplificated products were amplified using sequence-specific primers included in TaqMan Low-Density Array (TLDA) plates (TaqMan® Array Human MicroRNA A+B Cards Set v3.0, No cat: 4444913, 8 pack). Briefly, 9 μl of pre-amplification products were added to 441 μl nuclease-free water and mixed with 450 μl TaqMan Universal Master Mix II, No UNG. The mixture was then dispensed into the 384 wells by centrifugation. The reactions were incubated in a 384-well plate at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and at 60°C for 1 min in Applied Biosystems 7900 HT Real-Time PCR system (Applied Biosystems).
miRNA screening analysis. The RAW data were collected and processed using the Plate Utility and Automation Controller software (Applied Biosystems). The expression level of each miRNA was determined using the -2ΔΔCt method.
Quantitative real-time-PCR analyses of microRNAs. cDNA was synthesized using Applied Biosystem’s AgPath-ID One-step enzyme RT protocol to validate miRNA expression changes detected in the TLDA. The expression of six microRNAs was analyzed using the following TaqMan Advanced MicroRNA Assays probes (Applied Biosystem): hsa-miR-490-3p (Assay ID: 478131_mir), hsa-miR-195-5p (Assay ID: 477957_mir), hsa-miR-454-3p (Assay: 478329_mir), hsa-miR-431-5p(Assay ID: 478889_mir), hsa-miR 625-3p (Assay: 479469_mir), has-miR- 519c-3p (Assay ID 479495_mir). The relative levels of microRNA expression were calculated and normalized using the −2^^CT method relative to the small nuclear RNA (snRNA) (13, 14). The data were normalized by employing U6-snRNA (Assay ID:001973) TaqMan assays, as they offer comparable reproducibility and performance to the miRNA TaqMan assays (15, 16).
Prediction of microRNA targets. miRNA target prediction was performed using the miRecords database (17). The pathways, biological processes, and interactions involving the studied miRNAs participate were predicted using ShinyGO v0. 75 (http://bioinformatics.sdstate.edu/go/).
Statistical analysis. microRNA expression was analyzed using the −2^^CT method, and the results were reported as mean±standard error (SEM). Statistically significant differences between the control and treated group were determined using a two-tailed Student’s t-test for normally distributed data and the Mann-Whitney U-test for non-normal distributions. p<0.05 was considered to indicate a statistically significant difference. The statistical analysis was performed in Prism Graph software (GraphPad Software, Boston, MA, USA).
Results
Demographic and clinical characteristics of the study population. The study population was divided into two groups: the healthy control and severe COVID-19 patients infected with the B.1 Variant of SARS-CoV-2, including 40 males and 40 females. The mean age of the control group was 51±7 years, compared to 51±8 years for the severe COVID-19 patients. Interestingly, the prevalence of obesity in COVID-19 patients (47.5%) and the BMI value (29±5.3 kg/m2) was higher compared to the control group (32.5% and 27.9±5.2 kg/m2). Particularly, this difference in obesity prevalence was statistically significant between control (27.5%) and COVID-19 (57.5%) male individuals (Table I). The prevalence of diabetes mellitus type 2 was very similar between both groups, with mean glucose values of 142 and 136 mg/dl in control and COVID-19 patients, respectively (Table I).
Clinical and biochemical parameters of the groups.
Other biochemical parameters, such as urea, creatinine, LDH, cholesterol, HDL, LDL, and triglycerides, ranged within normal values, suggesting that renal function and lipid profile were normal in both groups. Similarly, the blood count did not show alterations in any groups (Table I).
miRNAs screening expression analysis. The miRNA screening expression analysis using TLDA showed that miR-195-5p, miR-454-3p, miR-490-3p, miR-511-5p, and miR-516-5p miRNAs were expressed exclusively in the COVID-19 severe group, whereas miR-431-5p, miR-519, and miR-625-5p miRNAs were detected only in the control group.
Analysis of miRNA expression. In expression analysis, a comprehensive literature was conducted for miRNAs identified as exclusive to control and COVID-19 patients, aiming to identify those more closely related to inflammation and viral infection. As a result of this analysis, the miR-490-3p, miR-195-5p, and miR-454-3p of the COVID-19 group and miR-431-5p of the control group were chosen for RT-qPCR validation.
Consistently, the total population analysis showed that the expression levels of miR-490-3p, miR-195-5p, and miR-454-3p were significantly higher in the COVID-19 patients compared to the control group. In contrast, a slight reduction was found in mir-431-5p expression between COVID-19 patients and the control group (Figure 1A). Interestingly, when expression analysis was performed by sex, both miR-490-3p and miR-195-5p exhibited a similar trend of increased expression in COVID-19 patients among males, consistent with the findings in the total study population. However, the expression of miR-454-3p and miR-431-5p was not changed between the control and COVID-19 groups (Figure 1B). In contrast, in females, miR-454-3p was found to be highly expressed in COVID-19 patients compared to the control group, whereas miR-195-5p expression was not significantly different between both groups. Consistent with the TLDA results in females, miR-490-3p was over-expressed in COVID-19 patients, while miR-431-5p was overexpressed in the control group (Figure 1C).
Relative expression of miRNAs -490, -195-5p, -454-3p, and -431-5p. A) expression of miRNAs in the total study population; B) comparison of COVID-19 and control groups in males; C) comparison of COVID-19 and control groups in females. Statistical significance was considered at p<0.05.
Prediction of miRNAs biological functions. Bioinformatic analysis showed that miR-490-3p and miR-454-3p target several genes related to the regulation of biosynthetic processes, such as those related to organic cyclic compounds (Figure 2A and B). In contrast, miR-195-5p primarily targets genes associated with the modification of proteins and macromolecules (Figure 2C).
Routes regulated by different miRNAs. A) miR-490-3p; B) miR-454-3p; and C) miR-195-5p.
Interestingly, in silico prediction of the biological participation of these miRNAs reveals that miR-490-3p participates in cardiac muscle adaptation and cardiac muscle hypertrophy in response to stress (Figure 3A); miR-454-3p participates in cell development, cell differentiation, and neurogenesis (Figure 3B); and miR-195-5 participates in the regulation of the catabolic process of organic substances, nervous system development, and protein localization (Figure 3C).
Prediction of biological process regulated by different miRNAs. A) miR-490-3p, B) miR-454-3p, and C) miR-195-5, regulates several signaling pathways.
Discussion
miRNAs regulate multiple biological processes involved in the progression and complications of COVID-19 (18), therefore, this work aimed to determine the expression pattern of endogenous miRNAs in severe COVID-19 patients.
The expression of host miRNAs could affect the SARS-CoV-2 life cycle, involving several signaling pathways that may promote SARS-CoV-2 infection or have an antiviral effect (19), for example, inhibiting viral replication and increasing the immune response. Our study showed that miR-490-3p, miR-195-5p, and miR-454-3p were over-expressed in a population of severe COVID-19 patients compared with the control group. Nersisyan et al. (20) reported that miR-195-5p, among others (miR-21-3p, miR-16-5p, miR-3065-5p, miR-424-5p, and miR-421), has multiple binding sites in SARS-CoV-2 RNA, thus regulating SARS-CoV-2 through direct binding to its genome. Consistent with our study, Kim et al. (21) reported that the up-regulation of miR-195-5p in hamster lung tissue infected with SARS-CoV-2 promotes cell cycle arrest and apoptosis. In contrast to our study, Li et al. (22) found that miR-454-3p was down-regulated in COVID-19 patients; this discrepancy may be attributed to differences in the sampling time. To our knowledge, there are no reports of miR-490-3p and miR-431-5p in COVID-19 patients; however, like miR-195-5p, miR-490-3p has antitumor properties and induces autophagy in myocardial cells (23, 24). Interestingly, a study by Huo et al. showed that knockdown of miR-431-5p delays angiotensin II-induced hypertension and reduces vascular damage in mice (25). In addition, angiotensin II increases thrombin formation, impairs fibrinolysis, and is strongly associated with lung injury in severe COVID-19 patients (26). Consistently, we detected a down-regulation of miR-431-5p in our population of severe COVID-19 patients that might be related to lung damage in these patients.
Differential miRNA expression analysis was performed by sex (Figure 1B and C), showing different trends between males and females concerning the total study population. It has been reported that some miRNAs can be differentially expressed according to tissue and sex, showing inconsistent and even opposite expression between males and females, which could be an important factor affecting the screening of disease-associated miRNAs (27, 28). In our study, miR-490-3p was over-expressed in both female and male COVID-19 patients and thus may be a potential biomarker for severe COVID-19 for both sexes.
A previous report identified mitochondria and cellular respiration as primary targets of miR-195-5p deregulation, suggesting that this miRNA might impact the myocardium’s energy production during SARS-CoV-2 infection (29); however, our in silico analysis revealed that miR-195-5p regulates processes related to protein and macromolecule modification, participating in catabolic process, nervous system development. and protein localization. This suggests that in severe COVID-19 patients, miR-195-5p may contribute to metabolic dysregulation, potentially producing a neurological disruption in these patients (30, 31).
Another miRNA identified to be over-expressed in COVID-19 patients was miR-490-3p. In agreement with our data, a previous report described the exclusive up-regulation of miR-490-3p in COVID-19 patients with a fatal outcome (32). Hence, these data support the proposal of considering this miRNA as a possible biomarker of severity in SARS-CoV-2 infection. Functionally, the down-regulation of miR-490-3p promotes cancer cell characteristics, such as proliferation, migration, survival, and invasiveness, because this miRNA can regulate critical signaling pathways, such as MAPK, TGF-β, HIF-1A, Wnt, EGFR, and PI3K-AKT (33). Interestingly, in the context of Coronavirus disease, we identified that miR-490-3p targets several genes related to the regulation of biosynthetic processes and participates in cardiac muscle adaptation and hypertrophy in response to stress, suggesting that some canonical processes regulated by miR-490-3p, such as proliferation and cell survival might also be perturbed in cardiac muscle cells, possibly favoring cardiovascular complications of COVID-19 (34).
Similarly, it has been described that the over-expression of miRNA-454-3p promotes cell growth and apoptosis evasion in cervical cancer cells (35), and we detected a high expression of miRNA-454-3p in COVID-19 patients; however, our in silico analysis revealed that this miRNA participates in cell development, cell differentiation and neurogenesis; suggesting that the control of cell proliferation and apoptosis of nervous system cells might also be deregulated during SARS-CoV-2 infection. Interestingly, previous data reported the down-regulation of miRNA-454-3p in COVID-19 patients (22, 32); this discrepancy with our data may be attributed to the fact that these studies only included patients with mild to moderate symptoms; thus, the up-regulation of miRNA-454-3p might be exclusive to severe COVID-19 patients.
Conclusion
Our data suggest that the over-expression of miR-195-5p, miR-490-3p, and miR-454-3p might be potential biomarkers for the severity of COVID-19 in patients.
Acknowledgements
P.Z.-S. (43142), F.B.-G. (225525), M.M-C. (290662), L. C.-M. (509433), R.C.-V. (11369), and J.G.-M. (19815) are Fellows from the Sistema Nacional de Investigadoras e Investigadores, Mexico.
Footnotes
Authors’ Contributions
P.Z.-S., Investigation, Methodology, Supervision, original draft, M.M-C., Formal Analysis, original draft, review, A.P.G-G., Data Curation, Validation, J.M.H-H., Data Curation, Methodology, editing, L.J.C-M., Data curation, Formal analysis, J.G-M., Formal analysis, review and editing, R.M.C-V., Methodology, review and editing, F.B-G., Conceptualization, investigation, Methodology, software, supervision, original draft.
Funding
This study was supported by 20240656 IPN grant, with the collaboration of CONAHCYT grant number: CF-2023-I-323.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received September 17, 2024.
- Revision received October 8, 2024.
- Accepted October 11, 2024.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
