Background

The single-stranded RNA pathogens known as coronaviruses (CoVs) are responsible for gastrointestinal, pulmonary, hepatic, and central nervous system disorders in both humans and animals [1]. Human cells may be infected by six distinct types of CoV, such as SARS-CoV, HCoVOC43, HKU-NL63, HCoV-HKU1, HCoV-229E, and MERS-CoV [2]. All coronaviruses have a similar genomic structure, with an open reading frame (ORF) 1a/b at the 5′ end that codes for 16 non-structural proteins (NSP1-NSP16) and four structural proteins that code by ORF at the 3′ end (the envelope (E), nucleocapsid (N), membrane (M) and spike (S) proteins). For the SARS-CoV-2 and SARS-CoV viruses to infect host cells, transmembrane serine protease 2 (TMPRSS2), the S protein must interact with the proteins of the extracellular membrane of the host [3, 4].

The novel coronavirus disease 2019 (COVID-19), caused by the new coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has speedily spread throughout the world after the first case was reported in late December 2019 in Wuhan, China. As of December 1, 2020, there will be 1,528,984 COVID-19-related mortality and 66,243,918 confirmed sick patients, according to the (WHO) [5]. A great deal of clinical and academic interest has been generated by the recognition of the biochemical and molecular mechanisms underlying SARS-CoV-2 infection and disease formation in human host cells in light of the global health crisis. Finding biomarkers that could be used as the disease's diagnostic and treatment targets is a crucial goal.

One of the indicators of the miRNA family may change the targets of genes related to immunity through networks of virus-host cell interactions [6]. Small noncoding RNA molecules, or miRNAs, are naturally occurring molecules that attach to specific mRNA targets through nucleotide similarity. By inhibiting translation or causing the target mRNAs to degrade, these chemicals contribute to the regulation of gene expression in a variety of organisms [7]. Viral infections and pathogenic processes, as well as chronic disorders, are all correlated with dysregulated miRNA expression [8, 9].

Cellular miRNAs can directly hinder viral replication within host cells, although the precise mechanisms underlying their interaction with viruses are still not entirely known. Several techniques have been proposed, including stopping viral replication and blocking viral genome translation, which can result in an anti-viral response by deregulating the synthesis of numerous endogenous miRNAs. miRNAs' anti-viral response may include modulating their mRNA targets: WNT, INF, MAPK, PIK3/AKT, and NOTCH are examples of signaling pathways that are involved in the viral infection by cellular response [10]. Other processes, such as cellular miRNA "evasion" and flaws in miRNA production, may, on the other hand, favor viral replication and virus survival in host cells [11].

In this study, we investigate the novel expression of miRNA 20a and miRNA 320 and their role as a biomarker in COVID-19 infection.

Methods

Patients and controls

A total of 145 COVID-19-positive patients without any chronic disease admitted to various hospitals in Baghdad/ Iraq underwent a case–control study from the period December 2021 to February 2022, and this research was approved by the College of Science/University of Baghdad's Research Ethics Committee (Ref.: CSES/0322/0071).

The patients' COVID-19 positivity was determined by a molecular test performed on nasopharyngeal swabs. They were examined 4–7 days after admission, and a chest computerized tomography (CT) scan was done to confirm the diagnosis. Patients included those who had a positive molecular test, and a COVID-19-positive CT scan were critically unwell in the intensive care unit, and had low oxygen saturation (SPO2 < 93). Healthy control samples of 145 healthy individuals were also included. They were blood donors (Iraqi National Blood Bank), and their serum profile for anti-infectious pathogens antibodies tested negative. They were also tested for anti-COVID-19 IgG and IgM antibodies using the COVID-19 IgG/IgM Rapid Test Cassette (Inzek B.V., The Netherlands), and their results were negative.

Blood sampling

Each participant's 5 milliliters of whole blood had to be drawn directly from the vein into an EDTA-containing tube, and the procedure had to be carried out in an aseptic way.

miRNA extraction and analysis

miRNA was isolated from the blood of both groups (patients and controls) according to [12] using the EasyPure® Blood Genomic miRNA Kit (Transgen, China) with catalogue number (ER601-01).

Primer preparation for miRNA-20a and miRNA-320 gene expression

The cDNA sequences of the housekeeping genes miRNA-U6 and miRNA-20a, as well as miRNA-320, were designed by the National Centre for Biotechnology Information (NCBI) Gene Bank database. After lyophilized primers were dissolved in nuclease-free water according to the manufacturer's instructions to form a stock solution with a concentration of 100 µl for each primer and kept at (-20°C). By mixing 10 µl of primer stock solution with 90 µl of nuclease-free water, a working solution was generated which was 10µM. The primers utilized in this investigation, as well as their related sequences, are shown in Table 1.

Table 1 Primers of miRNA-20a and miRNA 320 used in quantitative RT– PCR
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Gene expression

cDNA synthesis from miRNA

To evaluate the synthesis of the cDNA, the EasyScript® One-Step gDNA removal with cDNA Synthesis SuperMix method was utilized (Transgen, China) which was employed to evaluate miRNA-20a and miRNA-320 expression. Reverse transcription reactions need to be put together in an environment free of RNase. All materials were thawed, the solutions were gently mixed, and the miRNA templates were added. 20µl of components reaction were used which contained 1µl of Anchored Oligo(dT)8 1 µl Primer(0.5 µg/µl), 1µl of Random Primer(0.1 µg/µl), 10 µl of 2 × EX Reaction Mix, 1 µl of Easy Script®RT/RI Enzyme Mix, 1 µl of gDNA Remover, 1 µl RNase-free Water and 5 µl of total miRNA The thermal cycler program of cDNA was composed of three steps: Step one was for a random primer with 25°C and 10 min, step 2 for anchored oligo(dt)8 with 42°C and 15min and step 3 for inactivating reverse transcriptase enzyme with 85 oC and 5 s. The synthesized cDNA was immediately utilized as a template for PCR or stored at -20°C for lengthy periods.

Quantitative real-time PCR (qRT–PCR):

MiRNA-20a and miRNA-320 expression levels were determined using qRT-PCR. qRT-PCR SYBR Green test (TransStart® Top Green qPCR SuperMix for gene expression) was performed to validate the target miRNA expression. This operation was done depending on the manufacturer's instructions (Transgene, China). The Cepheid Real-time PCR System (Smart Cycler Technologies) and qPCR Soft software were used to run qRT-PCR. By measuring the threshold cycle, the fold change and gene expression levels were calculated (Ct), and the threshold cycle (Ct) was measured to calculate the gene expression levels and fold change. Each reaction was carried out twice. The thermal profile was used to train the cycling process for optimal cycles. One cycle of initial denaturation at 94°C for 30 s, 40 cycles for three stages of denaturation (94 oC for 5 s), annealing (56 oC for both miRNA with 10 s), and extension (72 oC for 20 s). For this gene expression, a single melting curve cycle (65–95 oC) was used for 1 min. The real-time cycler software was used to calculate the threshold cycle (Ct) for each sample. Selected gene expression data were compared to housekeeping data. The Ct technique was used for data analysis, and the results were reported as folding changes in gene expression according to Aljoubory [13].

Statistical analysis

Statistical data for the Social Sciences (SPSS version 26.0 software) was used. The results are represented by either a number and a percentage, a mean and a standard deviation (SD). To determine the significance of the differences, an independent T-test and Mann–Whitney U test were utilized. A statistically significant value was defined as one with a P-value less than 0.05.

Results

All of the studied participants were of Iraqi ethnicity. The selected clinical characteristics of the included population were no variations in demographic characteristics between the COVID-19 cases and healthy controls. The COVID-19 group consisted of 145 patients (median age at diagnosis 40.56 ± 10.19 years), who were selected from different hospitals in Baghdad/ Iraq. No significant differences in age and gender distribution were seen between the studied groups. The control group was age-matched to the COVID-19 group (i.e., median age 38.78 ± 10.57 years) and comprised 145 healthy subjects as shown in Table 2.

Table 2 The distribution of the studied groups according to age and gender
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The current study examined the expression of miRNA in patients with COVID-19 since miRNAs have the potential to be biomarkers that alter post-transcriptional gene regulation. Subjects with COVID-19 had lower mean ΔCT values for miRNA-20a gene expression (4.02 ± 0.34) than controls (7.66 ± 1.11), and the observed difference was statistically significant (P-value was less than 0.0001) as shown in this investigation. This shows that the patients' miRNA-20a gene expression was considerably higher than that of the control group (Fig. 1, Table 3). The ΔCT values for miRNA-320 gene expression in COVID-19 patients (6.00 ± 0.37) were higher than that of controls (4.90 ± 0.60). Since the p-value was less than 0.0001, the observed difference was statistically significant. This shows that the miRNA-320 gene expression levels were considerably lower in the patients than in the controls (Fig. 1, Table 3).

Fig. 1
figure 1

Relative gene expression level of: A Relative expression levels of miRNA-20a were increased in patients with COVID-19 compared with controls. B Relative expression levels of miRNA-320 were decreased in patients with COVID-19 compared with controls,**** < 0.0001, ΔCT = Delta cycle threshold

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Table 3 Expression level (ΔCT) of miRNA-20a and miRNA-320 in COVID-19 patients and control
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So the fold change values for miRNA-20a gene expression in cases with COVID-19 (10.65) were higher than that of the controls (1.03) while the fold change values for miRNA-20a gene expression in cases with COVID-19 (0.48) were lower than that of the controls (1.08). This indicates that the miRNA-20a and miRNA-320 gene expression in the patients might be used as a biomarker for COVID-19 patients (Table 4).

Table 4 Expression fold (2−∆∆Ct) of miRNA-20a and miRNA-320 in COVID-19 patients
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Furthermore, receiver operating characteristic (ROC) curve analysis was used to identify miRNA-20a and miRNA-320 in blood as a biomarker for distinguishing COVID-19 patients from healthy controls. The AUC for miRNA-20a in coronavirus disease 2019 (COVID-19) patients was 1.00 (0.974 to 1.000), with optimal sensitivity and specificity values of 100% and 95%, respectively, to the cutoff value of 14.55. (Fig. 2A). The AUC for miRNA-320 in blood expression was 1.00 (0.974 to 1.000) for patients with COVID-19 compared to the control group, with optimal sensitivity and specificity values of 100% and 91%, respectively, to the cutoff value of 24.55 (Fig. 2B). As a result, the expression of miRNA-20a and miRNA-320 in blood samples has sufficient sensitivity and specificity to distinguish samples without coronavirus disease 2019 (COVID-19) from samples with COVID-19. Receiver operating characteristic (ROC) curve analysis revealed that miRNA-20a and miRNA-320 had significant sensitivity and specificity, indicating that they are suitable biomarkers for COVID-19 disease (Fig. 2).

Fig. 2
figure 2

ROC curve analysis of miRNA-20 and miRNA-320 in CΟVΙD-19 cases, miRNA-20a and miRNA-320 showed good discriminating efficiency A ROC curve of miRNA-20a (control vs. COVID-19) the AUC was 1.00 (0.974 to 1.000), the sensitivity and specificity values of 100% and 95%, respectively, the cutoff value of 14.55. B ROC curve: miRNA-320 (control vs COVID-19), the AUC was 1.00 (0.974 to 1.000), sensitivity and specificity values of 100% and 91%, respectively, the cutoff value of 24.55 AUC: area under the curve, CI: confidence interval, OR = odd ratio

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Discussion

miRNAs have been identified as potential targets that could aid in the comprehension of COVID-19 infection mechanisms and the development of novel antiviral drugs [14]. In this study, the expression levels of miRNA-20a and miRNA-320 in COVID-19 patients were compared to those in healthy controls. Significantly elevated levels of miRNA-20a were observed in COVID-19 patients compared to controls. ROC curve analysis verified this result's accuracy with a P-value of 0.0001, which agreed with Liu et al. who reported that miRNA20a was up-regulated in pediatric pneumonia and human lung adenocarcinoma A549 cells [15]. Similar findings were found in investigations on other chest diseases. Xu et al. also discovered that non-small cell lung cancer (NSCLC) patients had higher levels of miRNA-20a than healthy controls [16]. However other studies showed different investigation to the findings of Giannella et al. 2022 and Li et al. 2020, it has been observed that the expression of miRNA-20a is notably reduced in individuals diagnosed with COVID-19 in comparison to the control groups [17, 18]. MIR20, miRNA-20, MIRH1, MIRN20,MIR17HG, MIRN20A, MIRHG1, miRNA20A, C13orf25, miRNA20A, and hsa-miRNA-20a or hsa-miRNA-20 are all names for human miRNA-20a. It's found on chromosome 13q31.3.MicroRNA-20a gene expression was significantly greater in COVID-19 patients compared to controls. Another research discovered that overexpression of miRNA-20a decreases the production of mediators of inflammation such as IL-2, IL-6, and IL-8 [19]. This miRNA was shown to be downregulated in multiple sclerosis (MS) patients compared with the control group [20]. However, because of the significant difference in miRNA20a gene expression between patients and controls, it might be used as a biological marker to identify COVID-19 patients. Previous research has shown that miRNA-20a is associated with cell proliferation and progression through the cell cycle by modulating the expression of transcription factor E2F1. Fan et al. [21] also reported that miRNA-20a overexpression was able to promote cell proliferation and invasion by inhibiting the expression of targeted amyloid precursor protein in ovarian cancer cells. In addition, miRNA-20 has been shown to play an essential role in inflammatory responses. Philippe et al. [22] reported that miRNA-20 inhibited the production of inflammatory cytokines such as IL-6, C-X-C motif chemokine ligand 10, IL-1, and TNF- in LPS-activated fibroblast-like synoviocytes. In contrast, it has been reported that miRNA-20a promotes macrophage inflammatory responses by modulating the expression of signal-regulatory protein [23].

The current work describes the function of miRNA-20a in COVID-19 and offers details that can be applied to future studies on the biomarker function of miRNA-20a.

In COVID-19 patients, miRNA-320 levels were significantly lower than in healthy controls. ROC curve analysis confirmed the veracity of this result with a P value of 0.0001, confirming Duecker et al.'s finding that downregulation of miRNAs-320 has been documented in patients exhibiting acute mild symptoms, and this downregulation continues to decrease progressively in patients with severe progression of the disease [24].

miRNA-320 was shown to be downregulated in patients who had COVID-19 symptoms. Deep vein thrombosis (DVT) patients have already been studied using the miRNA-320 [25,26,27]. DVT is a serious consequence that affects COVID-19 patients as well, thus it's critical to identify high-risk COVID-19 patients so that thrombosis prophylaxis can be started right away [28]. Jiang et al. discovered that miRNA-320a and miRNA-320b substantially increase concentrations of D-dimer in patients with deep vein thrombosis (DVT) but not in those with post-thrombotic syndrome contradicts our results [29]. A preventive mechanism against abrupt thrombosis in COVID-19 patients may have caused a decrease in miRNA-320 expression, which could account for these disparities. MiRNA-320 downregulation protects against cardiac ischemia–reperfusion injury by promoting Nrf2 expression, according to Zhu et al.[25].

Downregulation of miRNA-320 has been related to Nrf2's control of oxidative stress and inflammation [26]. Along with miRNA-320, many miRNAs were discovered to be dysregulated in the patient groups. The two microRNAs with the greatest levels, miRNA-374a-3p and miRNA-15a-3p, have been associated with acute nephritis and neuroinflammation, respectively. A further molecular diagnostic miRNA biomarker for liver fibrosis in people with chronic hepatitis B is miRNA-4721, which has been proposed. The potential diagnostic biomarker for biliary atresia, Hsa-miRNA-4429, has been postulated [30,31,32,33,34].

Even though little to no study has been conducted on these miRNAs, it is vital to keep them in mind as possible indications of the devastating COVID-19 illness. Earlier COVID-19 research focused on miRNAs such as 126-5p, miR-27, miR-21-5p, miR-146, and miR-142 [35, 36]. Garg et al. [36] discovered dysregulated levels of miR-21-5p and miR-126-5p, two miRNAs linked to inflammatory processes, in the circulating microRNAs of seriously ill COVID-19 patients. When De Gonzalo-Calvo et al. [37], compared the profiles of circulating miRNA of patients who did not require critical care to those admitted to the intensive care unit, they discovered an elevation of miRNA-27a-3p, miRNA-148a-5p, and miRNA-27b-3p that distinguished ICU from non-ICU patients[37]. Those who investigated cellular miRNAs during the acute and post-acute phases of COVID-19 discovered that severe patients had higher levels of miRNA-29a-3p and miRNA-146a-3p [38]. The value of miRNAs as circulating diagnostic biomarkers was determined using the AUC of the ROC curves. According to Hosmer and Lemeshow, "0.5 = no discrimination; 0.5–0.7 = poor discrimination; 0.7–0.8 = acceptable discrimination; 0.8–0.9 = excellent discrimination; and > 0.9 = outstanding discrimination"[39]. The current research discovered that miRNA-20a in blood samples has excellent discrimination for use as a diagnostic biomarker, Other studies with the same goal found similar results with another miRNA based on the AUC of the ROC curve in the healthy group to COVID-19 patients. With AUCs of 0.959 (95% CI = 0.917–1.000, p 0.0001), Markou et al. determined that miRNA-210 was a useful biomarker for distinguishing patients from healthy individuals using ROC curve analysis [40]. The findings of the numerous studies frequently differ, sometimes noticeably, in terms of the sample preparation, materials testing, and molecular approach, as well as the severity of the patients, evaluated.

Conclusion

The results of our investigation indicate a higher frequency of expression for miRNA-20a, whereas miRNA-320 exhibited a lower frequency of expression.This finding implies that miRNA-20a and miRNA-320 have the potential to function as early indicators for the identification of individuals with COVID-19. ROC analysis was employed to assess the specificity and sensitivity of these miRNAs in COVID-19 patients.