Clinical significance and mechanistic study of SOX2-OT and miR-27b-3p expression in children with viral myocarditis

Abstract

Background

Viral myocarditis (VMC) primarily affects children and adolescents, with a certain risk of mortality. Its clinical manifestations are complex and variable, making early diagnosis difficult. Therefore, there is an urgent need to identify effective biomarkers.

Methods

This study collected clinical data from 110 children with VMC and 100 healthy children. The expression levels of SOX2-OT and miR-27b-3p in cells and serum were assessed by RT-qPCR. The diagnostic and prognostic values of these markers were evaluated through ROC curve analysis and logistic regression. ELISA was used to assess the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β. Commercial kits quantified serum LDH, CK-MB, and cTnI levels. The interaction between SOX2-OT and miR-27b-3p was validated by dual-luciferase reporter and RIP assays.

Results

Serum SOX2-OT was elevated and miR-27b-3p was decreased in VMC patients, and the combination of the two showed excellent diagnostic performance. Serum SOX2-OT and miR-27b-3p were strongly correlated with disease severity. In patients with poorer prognosis, SOX2-OT was significantly higher, and miR-27b-3p lower; their combined assessment also predicted prognosis effectively. Mechanistically, SOX2-OT acted as a “sponge” to inhibit miR-27b-3p, promoting inflammation and cardiomyocyte injury in vitro.

Conclusions

Elevated serum SOX2-OT and decreased miR-27b-3p serve as potential biomarkers for early diagnosis and prognosis in VMC. Their combined detection greatly improves accuracy. The SOX2-OT/miR-27b-3p axis plays a key role in VMC pathogenesis by modulating inflammation and myocardial injury, providing a promising molecular target for future therapeutic interventions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12887-026-06692-y.

Background

Myocarditis is a common inflammatory disease characterized by localized or diffuse inflammation of the myocardium, frequently occurring in children and young adults [1]. It can be caused by various factors, including infections, immune responses, and toxins [2]. In recent years, the incidence of viral myocarditis (Viral Myocarditis, VMC) caused by viral infections has been steadily increasing [3]. The clinical manifestations are diverse, ranging from mild symptoms such as tachycardia or palpitations to severe heart failure, arrhythmias, and even sudden death, seriously threatening the life of affected children [4]. Endomyocardial biopsy (EMB), considered the “gold standard” for diagnosing myocarditis, is invasive and destructive, and is generally not accepted by patients’ families [5]. Therefore, the diagnosis of viral myocarditis in children relies on a combination of clinical manifestations and auxiliary tests. However, due to the variable presentation, lack of specific biomarkers, and difficulties in communication with young patients, diagnosis remains challenging. There is a need for more advanced detection techniques and definitive diagnostic indicators to enable early and accurate diagnosis, thereby improving prognosis.

Non-coding RNA (ncRNA) has increasingly become a research hotspot, with its regulatory role in cardiovascular diseases gaining greater attention [6, 7]. Particularly, long non-coding RNAs (lncRNAs) with their rich regulatory functions are closely linked to gene expression changes in cardiac tissues during disease processes [8]. Although research on lncRNAs in VMC is still limited, several have been implicated, such as MEG3, which participates in macrophage M1/M2 polarization imbalance [9] and coxsackievirus group B type 3 (CVB3) replication [10], and NEAT1, which shows potential as a diagnostic marker for VMC [11]. It has been found that SOX2-OT (SRY-box 2 overlapping transcript), a significant lncRNA, is involved in cardiomyocyte development, injury repair, and disease progression [12, 13]. For example, Liang et al. demonstrated that silencing SOX2-OT by regulating miR-2355-3p could inhibit NLRP3 expression and alleviate arrhythmias associated with heart failure [14]. SOX2-OT also regulates myocardial fibrosis and inflammation responses in heart failure via multiple mechanisms [15, 16]. Additionally, SOX2-OT can mediate mitochondrial dysfunction in infectious myocarditis [17]. However, there have been no reports regarding the relationship between SOX2-OT and VMC.

On the other hand, microRNAs (miRNAs), due to their short length and high efficiency in targeting regulation, are key modulators in cardiovascular diseases [18]. In this study, we specifically examined the role of miR-27b-3p in cardiovascular pathophysiology. Previous research has confirmed that miR-27b-3p participates in key processes such as endothelial inflammation, vascular smooth muscle cell phenotypic switching, and cardiac fibrosis [19, 20]. This miRNA exhibits protective or detrimental effects across multiple cardiovascular diseases, including atherosclerosis and myocardial injury, by regulating numerous target genes [21]. Particularly relevant to the VMC pathogenesis, the regulatory network of miR-27b-3p involves interactions with TGF-β signaling pathways, apoptotic pathways, and inflammatory mediators [22]. These established functions in relevant cardiac pathologies, combined with bioinformatics predictions of its potential to bind to the SOX2-OT, provide rationale for selecting miR-27b-3p as a candidate for VMC research. However, despite these compelling links, its expression patterns and clinical significance, particularly in VMC, remain unexplored.

Based on the above, this study included 110 children with VMC and 100 healthy controls, measuring the levels of SOX2-OT and miR-27b-3p to evaluate their clinical significance in pediatric viral myocarditis. Additionally, an in vitro VMC model was constructed to explore the potential mechanisms by which these molecules influence VMC.

Methods

Study participants

This study enrolled 110 children with VMC who received treatment at Pu’er People’s Hospital from January 2021 to January 2024. Inclusion criteria: (1) Children meeting the diagnostic standards for VMC as per the American Heart Association guidelines; (2) Elevated levels of cardiac troponin I (cTnI) and creatine kinase-MB (CK-MB), with abnormal routine electrocardiograms; (3) Children with febrile infections prior to hospital admission; (4) Complete medical records; (5) High compliance. Exclusion criteria: (1) Children with functional disorders of the lungs, kidneys, liver, brain, or blood system; (2) Children with malignant tumors; (3) Children with coronary artery disease, congenital heart defects, or other cardiovascular conditions.

As controls, 100 healthy children were also recruited. All subjects’ baseline clinical data were collected, including age, gender, disease duration, levels of myocardial markers (cTnI, CK-MB), and cardiac function indices (EF, FS). During the study, peripheral blood samples were collected from participants using EDTA anticoagulant tubes, separated by centrifugation, and stored at -80 °C for subsequent RNA extraction. This study was performed in line with the principles of the Declaration of Helsinki. All procedures were approved by the Pu’er People’s Hospital’s Ethics Committee (No. 2020-0048), and written informed consent was obtained from the guardians of all children.

All patients received standard treatment for VMC, including antiviral medications and intravenous immunoglobulin. Follow-up was conducted for 12 months to assess prognosis. Poor prognosis was defined as recurrence of myocarditis, arrhythmias, or heart failure during hospitalization, need for heart transplantation, or death.

Cell culture and virus

H9c2 cells (American Type Culture Collection, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin, maintained at 37 °C in a 5% CO₂ humidified atmosphere. Cells were infected with 0.6 mL of CVB3 (titer of 100 TCID50, MOI = 10, Tsingke Biotechnology, China) for 24 h; control cells received no treatment. All experiments were performed in five independent biological replicates.

Cell transfection

For SOX2-OT manipulation, pcDNA3.1-SOX2-OT overexpression plasmids and siRNA targeting SOX2-OT (si-SOX2-OT) were used. Negative controls included pcDNA3.1-NC and si-NC (GenePharma Co., China). Additionally, miR-27b-3p mimic (5’-TTCACAGTGGCTAAGTTCTGC-3’), mimic NC (5’-GGTTCCATCGTACACTGTTCA-3’), inhibitor (5’-GCAGAACTTAGCCACTGTGAA-3’), and inhibitor NC (5’-CCATCAGTCCCCATCGCCA-3’) were synthesized (GenePharma Co., China). H9c2 cells were seeded into 6-well plates and transfected with the above oligonucleotides or plasmids using Lipofectamine 2000 (Invitrogen, USA). Transfection efficiency was evaluated after 24 h by real-time reverse transcription PCR (RT-qPCR).

RNA extraction and quantitative analysis

Serum samples were centrifuged at 3000 g for 5 min prior to RNA extraction to remove impurities. Subsequently, total RNA was extracted using 200 µl of serum with the Qiagen miRNeasy Serum/Plasma Kit (Qiagen, USA). Total RNA was extracted from H9c2 cells using TRIzol reagent (Invitrogen, USA). RNA samples with an A260/A280 ratio between 1.8 and 2.2 were selected for subsequent reverse transcription. Then, the extracted RNA was reverse transcribed into complementary DNA (cDNA) using TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, USA) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Quantitative PCR (qPCR) was performed using the ABI7500 system (Applied Biosystems, USA) with SYBR^® Premix Ex Taq™ (Tli RNaseH Plus, (Takara, Japan)). Melt curve analysis using SYBR Green detection confirmed the specificity of qPCR amplification. Furthermore, repeat runs of all samples ensured the reproducibility of measurements. For normalization, β-actin and U6 were used as internal controls for SOX2-OT and miR-27b-3p, respectively. Relative expression levels were calculated using the 2^−ΔΔCt method. The primer sequences used in the present study were as follows: SOX2-OT: forward, 5’-CGAAATGGATTCACGGTGCC-3’, reverse, 5’-TGCCAGATCAGGGTGTTGTC-3’; miR-27b-3p: forward, 5’-CGGCGGTTCACAGTGGCTAA-3’, reverse, 5’-GTGCAGGGTCCGAGGT-3’; β-actin; forward, 5’-TGACGTGGACATCCGCAAAG-3’, reverse, 5’-CTGGAAGGTGGACAGCGAGG-3’; U6: forward, 5’-GGAACGATACAGAGAAGATTAGC-3’, reverse, 5’-TGGAACGCTTCACGAATTTGCG-3’.

Dual-Luciferase Reporter assay (DLR)

Online databases DIANA and LncRNASNP2 predicted the miRNA targeting SOX2-OT and their binding sites. Further validation was performed using DLR. The wild-type (WT) and mutant (MUT) sequences of SOX2-OT containing the miR-27b-3p binding sites were cloned into the pmirGLO luciferase reporter vector (sequence presented in Fig. S1). These constructs (SOX2-OT-WT and SOX2-OT-MUT) were co-transfected with miR-27b-3p mimics, mimic NC, inhibitors, or inhibitor NC into cells. After 48 h, luciferase activity was measured using the dual-luciferase reporter system (Promega, USA).

RNA Immunoprecipitation (RIP)

The Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, USA) was used to assess the interaction between SOX2-OT and miR-27b-3p. Cells were lysed with RIP lysis buffer, then incubated with magnetic beads conjugated with anti-Ago2 antibody. After immunoprecipitation, the bound RNA was purified using the RNeasy MinElute Cleanup Kit and quantified by RT-qPCR.

Automated biochemical analysis

Levels of CK-MB, cTnI, and lactate dehydrogenase (LDH) in the supernatant of H9c2 cell lysates and patient serum were measured using relevant commercial kits (Beckman, USA). All measurements and calculations were performed with an automated biochemical analyzer AU5800 (Beckman, USA).

Enzyme-Linked Immunosorbent Assay (ELISA)

The culture medium supernatant of H9c2 cells was collected to analyze inflammatory cytokines. According to the manufacturer’s instructions, ELISA kits (R&D Systems USA) were used to detect the concentrations of TNF-α, IL-6, and IL-1β.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). All experiments were repeated at least three times to ensure reliability, with parallel testing conducted. Clinical data of patients were collected and organized using SPSS 22.0 software, while other data were analyzed and plotted using GraphPad Prism 7. Comparisons between two groups were performed using an independent samples t-test, and multiple groups were compared using one-way ANOVA. The diagnostic value of SOX2-OT and miR-27b-3p was assessed using receiver operating characteristic (ROC) curve analysis. Logistic regression models were used to analyze the risk factors for children with viral myocarditis. P < 0.05 was considered statistically significant.

Results

Clinical baseline data analysis

The study collected and compared clinical data from healthy children and children with VMC. As shown in Table 1, there were no significant differences in gender, age, or BMI between the groups (P > 0.05). The serum levels of cTnI and CK-MB in the VMC group were 0.42 ± 0.09 µg/L and 30.35 ± 5.56 U/L, respectively, both significantly higher than those in the control group (0.07 ± 0.01 µg/L and 8.93 ± 1.89 U/L, P < 0.001). Additionally, the left ventricular fractional shortening (FS) and left ventricular ejection fraction (EF) in the VMC group were 24.17 ± 4.14% and 37.90 ± 6.96%, both lower than in the control group (31.93 ± 3.32% and 58.78 ± 6.53%, P < 0.001).

Table 1.

Clinical baseline characteristics of VMC

Index	control (n = 100)	VMC (n = 110)	P value
Age (year)	6.83 ± 2.61	6.57 ± 3.13	0.511
BMI (kg/m2)	14.26 ± 2.28	14.44 ± 2.06	0.546
Gender			0.675
Female	52	54
Male	48	56
cTnI (µg/L)	0.07 ± 0.01	0.42 ± 0.09	< 0.001
CK-MB (U/L)	8.93 ± 1.89	30.35 ± 5.56	< 0.001
FS (%)	31.93 ± 3.32	24.17 ± 4.14	< 0.001
EF (%)	58.78 ± 6.53	37.90 ± 6.96	< 0.001

Annotation: VMC Viral myocarditis, BMI Body mass index, cTnI Cardiac troponin I, CK-MB Creatine kinase myocardial band, FS Left ventricular fractional shortening, EF Left ventricular ejection fraction

Diagnostic value of SOX2-OT and miR-27b-3p in VMC

In the VMC group, the relative expression level of SOX2-OT was significantly increased (P < 0.001, Fig. 1A), while the expression of miR-27b-3p was markedly decreased (P < 0.001, Fig. 1B), indicating that SOX2-OT and miR-27b-3p may play roles in the occurrence and progression of VMC. ROC curve analysis (Fig. 1C) showed that serum SOX2-OT could predict VMC with an area under the curve (AUC) of 0.878, a sensitivity of 73.64%, and a specificity of 96.00%. Meanwhile, miR-27b-3p had an AUC of 0.888 for predicting VMC, with a sensitivity of 78.18% and a specificity of 84.00%. When combined, the ROC AUC increased to 0.945, with a sensitivity of 85.45% and a specificity of 90.00%, indicating that the combined diagnosis performed better than individual markers. Logistic regression analysis revealed that alterations in SOX2-OT and miR-27b-3p expression levels constitute risk factors for VMC occurrence (P < 0.001, Table 2).

Fig. 1.

Diagnostic value analysis of SOX2-OT and miR-27b-3p in VMC. RT-qPCR detected levels of SOX2-OT (A) and miR-27b-3p (B) in children with VMC (n = 110) and healthy controls (n = 100). C ROC curve analysis showing the diagnostic performance of SOX2-OT, miR-27b-3p and their combination for VMC. Data are presented as mean ± SD; **** P < 0.0001; unpaired two-tailed Student’s t-test

Table 2.

Logistic regression analysis of risk factors for VMC

Parameters	OR	95% CI	P value
Age (year)	1.474	0.627–3.469	0.374
BMI (kg/m2)	1.077	0.457–2.537	0.866
Gender	0.474	0.190–1.186	0.111
SOX2-OT	0.031	0.011–0.086	< 0.001
miR-27b-3p	15.066	5.794–39.172	< 0.001

Correlation between SOX2-OT and miR-27b-3p levels and clinical pathological indices of VMC

Spearman correlation analysis revealed that serum SOX2-OT levels were positively correlated with cTnI and CK-MB concentrations (r = 0.624, P < 0.001; r = 0.514, P < 0.001, Fig. 2A and B), and negatively correlated with FS and EF (r = -0.517, P < 0.001; r = -0.572, P < 0.001, Fig. 2C and D). Conversely, serum miR-27b-3p levels were negatively correlated with cTnI and CK-MB (r = -0.540, P < 0.001; r = -0.592, P < 0.001, Fig. 2E and F), and positively correlated with FS and EF (r = 0.537, P < 0.001; r = 0.527, P < 0.001, Fig. 2G and H).

Fig. 2.

Correlation of SOX2-OT and miR-27b-3p levels with clinical pathological indices of VMC. SOX2-OT levels are correlated with cTnI (A), CK-MB (B), FS (C), and EF (D) in children with VMC.miR-27b-3p levels are correlated with cTnI (E), CK-MB (F), FS (G), and EF (H) in children with VMC. Statistical significance was determined by Spearman correlation analysis

Correlation of SOX2-OT and miR-27b-3p levels with VMC prognosis

Based on follow-up results, 110 VMC children were divided into a good prognosis group (n = 63) and a poor prognosis group (n = 47). RT-qPCR results showed that SOX2-OT was significantly higher in the poor prognosis group (P < 0.001, Fig. 3A), while miR-27b-3p was markedly lower (P < 0.001, Fig. 3B). Serum SOX2-OT demonstrated predictive value for prognosis, with an AUC of 0.822, a sensitivity of 78.72%, and a specificity of 73.02%. Conversely, miR-27b-3p predicted prognosis with an AUC of 0.774, a sensitivity of 65.96%, and a specificity of 79.37%. When combined, the AUC increased to 0.869, with a sensitivity of 85.11% and a specificity of 79.37% (Fig. 3C), showing that joint detection had better diagnostic performance than individual markers.

Fig. 3.

SOX2-OT and miR-27b-3p levels and their association with the prognosis of VMC. RT-qPCR detected levels of SOX2-OT (A) and miR-27b-3p (B) in good prognosis group (n = 63) and poor prognosis group (n = 47). C ROC curve analysis evaluating the predictive value of SOX2-OT, miR-27b-3p and their combination for poor prognosis in VMC. Data are presented as mean ± SD; **** P < 0.0001; unpaired two-tailed Student’s t-testgood prognosis groupgood prognosis group

Interaction between SOX2-OT and miR-27b-3p

Online databases DIANA and LncRNASNP2 predicted the target miRNAs of SOX2-OT (Fig. 4A), and the binding site between SOX2-OT and miR-27b-3p was shown in Fig. 4B. The dual-luciferase reporter assay results demonstrated that, in the SOX2-OT-WT group, the luciferase activity in the miR-27b-3p mimic group was significantly lower than that in the mimic NC group, while there was no significant difference in luciferase signals between the SOX2-OT-WUT group and its corresponding negative control (P < 0.001, Fig. 4C). Furthermore, RIP analysis showed that endogenous SOX2-OT and miR-27b-3p in H9c2 cells were significantly enriched with the anti-Ago2 antibody compared to IgG control (P < 0.001, Fig. 4D). Additionally, overexpression of SOX2-OT suppressed miR-27b-3p levels, while silencing SOX2-OT resulted in a significant increase in miR-27b-3p (P < 0.001, Fig. 4E). In summary, these results confirm the binding relationship between SOX2-OT and miR-27b-3p.

Fig. 4.

SOX2-OT targets miR-27b-3p. A. Prediction of SOX2-OT target miRNAs using the online databases LncRNASNP2 and DIANA. B The binding sites of SOX2-OT and miR-27b-3p. C Dual-luciferase reporter assay. D RIP assay. E Regulation of SOX2-OT levels can inversely affect miR-27b-3p expression. n = 5; Data are presented as mean ± SD; *** P < 0.001, **** P < 0.0001; one-way ANOVA with Tukey’s post-hoc test or unpaired two-tailed Student’s t-test

Inhibition of miR-27b-3p reverses the protective effect of SOX2-OT silencing against myocardial injury and inflammation

To investigate the regulatory role of the SOX2-OT/miR-27b-3p axis in VMC, an in vitro VMC model was established by infecting H9c2 cells with CVB3. The results showed that, following CVB3 treatment, SOX2-OT levels increased while miR-27b-3p decreased. Transfection with si-SOX2-OT led to decreased SOX2-OT and increased miR-27b-3p, while in the CVB3 + si-SOX2-OT + miR-27b-3p inhibitor group, miR-27b-3p levels decreased (P < 0.05, Fig. 5A and B). CVB3 induced a decrease in H9c2 cell viability, which rebounded after inhibiting SOX2-OT. However, further transfection with miR-27b-3p inhibitor caused cell viability to decline again (P < 0.01, Fig. 5C). Additionally, the levels of myocardial damage markers CK-MB, cTnI, and LDH were significantly elevated in the CVB3 group; silencing SOX2-OT reduced these markers, but further silencing miR-27b-3p caused their levels to rise again (P < 0.05, Fig. 5D-F). Meanwhile, CVB3-stimulated release of IL-6, IL-1β, and TNF-α was suppressed by transfection with si-SOX2-OT, and this anti-inflammatory effect was partially abrogated by miR-27b-3p inhibition (P < 0.05, Fig. 5G).

Fig. 5.

Inhibition of miR-27b-3p reverses the protective effect of SOX2-OT silencing against myocardial injury and inflammation. The effects of transfecting si-SOX2-OT and miR-27b-3p inhibitors on SOX2-OT (A), miR-27b-3p levels (B), cell viability (C), CK-MB (D), cTnI (E), LDH (F), and pro-inflammatory cytokines (G) in CVB3-infected H9c2 cells. n = 5; Data are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA with Tukey’s post-hoc test

miR-27b-3p overexpression reverses SOX2-OT-induced myocardial injury and inflammation

To further investigate the causal effects of the SOX2-OT/miR-27b-3p axis, we conducted gain-of-function experiments in CVB3-infected cardiomyocytes. As shown in Fig. 6A-B, transfection with pcDNA3.1-SOX2-OT further elevated SOX2-OT while reducing miR-27b-3p, whereas transfection with miR-27b-3p mimic restored miR-27b-3p levels (P < 0.05). Correspondingly, SOX2-OT overexpression exacerbated CVB3-induced decreases in cardiomyocyte viability, increases in myocardial injury markers, and inflammation (P < 0.01, Fig. 6C-G). Co-transfection with miR-27b-3p mimic effectively counteracted the adverse effects of SOX2-OT overexpression, including reduced cell viability, cardiomyocyte injury, and inflammation (P < 0.05, Fig. 6C-G).

Fig. 6.

miR-27b-3p overexpression reverses SOX2-OT-induced myocardial injury and inflammation. Effects of transfection with pcDNA3.1-SOX2-OT and miR-27b-3p mimic on SOX2-OT (A), miR-27b-3p levels (B), cell viability (C), CK-MB (D), cTnI (E), LDH (F), and pro-inflammatory cytokines (G) in CVB3-infected H9c2 cells. n = 5; Data are presented as mean ± SD; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA with Tukey’s post-hoc test

Prediction and functional enrichment analysis of potential miR-27b-3p target genes

To elucidate the downstream molecular mechanisms underlying the protective role of miR-27b-3p in viral myocarditis, we conducted a systematic analysis of its potential target genes using bioinformatics methods. Using TargetScan and miRDB for cross-platform prediction, a total of 84 candidate genes were jointly identified as miR-27b-3p targets (Fig. S2A). Gene Ontology (GO) enrichment analysis of these 84 target genes revealed high enrichment in processes related to viral protein processing, immunological synapse, and phosphatidic acid binding (Fig. S2B). KEGG pathway analysis indicated significant enrichment of these target genes in multiple signaling pathways, including the MAPK signaling pathway, Rap1 signaling pathway, and P13K-Akt signaling pathway (Fig. S2C). These pathways play central roles in regulating cell proliferation and inflammatory responses, suggesting that miR-27b-3p may influence cardiomyocyte fate by coordinating these critical networks.

Discussion

VMC is an inflammation of the myocardium caused by viral infection, predominantly occurring in children and adolescents, with a certain mortality rate [23]. Clinical evidence indicates that approximately 20% of childhood deaths are due to VMC or lethal ventricular arrhythmias leading to sudden death [24]. Loss of myocardial cells and other physiological alterations result in symptoms such as fever, lethargy, congestive heart failure, cardiogenic shock, or new-onset arrhythmias [25]. These manifestations may initially appear mild, but without timely intervention, symptoms can progressively worsen. The clinical presentation of VMC is highly variable and complex, often leading to misdiagnosis and delayed treatment, which can result in severe heart failure or death [26]. Traditional diagnostic methods are invasive, have limited sensitivity, or lack specificity, restricting early detection and clinical monitoring [27]. Recently, with advancements in viral detection technology and elucidation of cardiac biomarkers, early diagnosis and prognosis assessment of myocarditis have become key focuses of clinical research.

Most current studies are centered on the functions of individual non-coding RNAs, but the interaction mechanisms between lncRNAs and miRNAs remain poorly understood, especially in the context of viral myocarditis. Exploring this regulatory network could uncover critical control points in the disease process, offering new strategies for precise diagnosis and treatment. For example, some studies have demonstrated that NEAT1 combined with miR-425-3p exhibits high diagnostic value for VMC in children [11]; TUG1, by binding to miR-140-3p, forms a “competitive endogenous RNA” network that regulates inflammatory response and apoptosis of cardiomyocytes [28]. Such mechanisms enrich our understanding of the pathogenesis of myocarditis and provide potential molecular targets for targeted RNA-based therapies. In this study, we focused on SOX2-OT, a known lncRNA associated with neurodevelopment, which has also been found to be dysregulated in cardiovascular diseases such as ischemic heart failure and arrhythmias [14, 29]. Notably, SOX2-OT can act as a “sponge” molecule by binding to specific miRNAs, such as miR-146a-5p and miR-27a-3p, thereby regulating downstream gene expression and controlling pathological processes like inflammation, apoptosis, and oxidative stress in myocarditis [30, 31]. Our clinical data analysis revealed that serum SOX2-OT levels were elevated, while miR-27b-3p levels were decreased in children with VMC. The combined detection of these two molecules showed high diagnostic value, with an AUC of 0.945, significantly outperforming single markers, suggesting substantial clinical application potential. Additionally, serum SOX2-OT levels positively correlated with myocardial injury markers (cTnI, CK-MB) and negatively with cardiac function indices (FS, EF), whereas miR-27b-3p showed opposite correlations, indicating that their dysregulation reflects the extent of myocardial damage. As the prognosis worsened, SOX2-OT levels increased while miR-27b-3p levels decreased, highlighting their potential as indicators for disease severity and prognosis. These expression changes may also reflect the pathological progression of VMC, offering novel biomarkers for clinical assessment.

Studies show that various viruses, including coxsackievirus, adenovirus, influenza virus, and cytomegalovirus, can cause VMC, with CVB3 being the primary pathogen [32, 33]. It has been reported that CVB3 infection is the main cause of myocarditis and dilated cardiomyopathy in children and adolescents, accounting for approximately one-quarter of cases [34]. To further investigate the roles of SOX2-OT and miR-27b-3p in VMC, we first validated their target relationship through bioinformatics prediction and dual luciferase reporter assays. Subsequently, we established an in vitro VMC model using CVB3-infected H9c2 cells. Inhibiting SOX2-OT suppressed CVB3-induced myocardial injury. Furthermore, concurrently suppressing miR-27b-3p during SOX2-OT knockdown partially reversed the protective effect induced by SOX2-OT knockdown, manifested as a rebound in myocardial injury markers and inflammatory cytokine levels. Furthermore, gain-of-function experiments demonstrated that overexpression of SOX2-OT significantly exacerbated the decline in cardiomyocyte viability, release of injury markers, and elevated levels of inflammatory cytokines. Co-transfection with miR-27b-3p mimic effectively reversed these adverse effects induced by SOX2-OT overexpression. This clearly reveals that SOX2-OT promotes injury by negatively regulating miR-27b-3p during the VMC process. Notably, while previous studies have demonstrated that SOX2-OT participates in other cardiac pathologies through distinct mechanisms—such as regulating the EZH2/Nrf-2/NLRP3 axis in inflammatory responses or modulating other signaling pathways during myocardial injury [35, 36]. Our identification of the SOX2-OT/miR-27b-3p axis reveals a unique mechanism specifically relevant to the pathogenesis of VMC. This context-dependent nature of SOX2-OT function, wherein different miRNA partners engage depending on specific disease etiologies (viral infection or other cardiac injury), enriches our understanding of the regulatory networks mediated by lncRNAs in cardiovascular disease. To further explore the downstream mechanisms of miR-27b-3p, we predicted its potential target genes through bioinformatics analysis. Functional enrichment analysis indicated that miR-27b-3p may play a central regulatory role in myocardial cell inflammation and survival by modulating signaling pathways such as MAPK, Rap1, and PI3K-Akt. However, this study has the following limitations: Although the H9c2 rat cardiomyocyte model used is a commonly employed system with biologically relevant conserved seed sequences, it cannot fully mimic the pathological characteristics of human cardiomyocytes; The direct downstream targets of miR-27b-3p in VMC remain incompletely characterized. Furthermore, as a potential ceRNA, SOX2-OT may regulate multiple miRNAs, and its complete regulatory network and diagnostic value warrant further exploration. Future studies will validate these findings in human cardiomyocyte models and systematically elucidate the downstream network of the SOX2-OT/miR-27b-3p axis through high-throughput technologies combined with in vivo experiments to assess its therapeutic potential.

Conclusions

In summary, serum levels of SOX2-OT are elevated while miR-27b-3p are decreased in children with VMC. The combined detection of these two molecules significantly improves diagnostic accuracy and provides a molecular basis for early diagnosis and disease monitoring. Furthermore, SOX2-OT functions as a “sponge” for miR-27b-3p, playing a crucial role in the development of VMC and offering a potential molecular target for intervention.

Abbreviations

AUC

Area under the curve

cDNA

Complementary DNA

CVB3

Coxsackievirus group B type 3

CK-MB

Creatine kinase-MB

cTnI

Cardiac troponin I

DLR

Dual-Luciferase Reporter Assay

DMEM

Dulbecco’s Modified Eagle Medium

EF

Ejection fraction

ELISA

Enzyme-Linked Immunosorbent Assay

EMB

Endomyocardial biopsy

FBS

Fetal bovine serum

FS

Fractional shortening

LDH

Lactate dehydrogenase

lncRNAs

Long non-coding RNAs

miRNAs

MicroRNAs

MUT

Mutant

ncRNAs

Non-coding RNAs

qPCR

Quantitative PCR

RIP

RNA Immunoprecipitation

ROC

Receiver operating characteristic

SD

Standard deviation

SOX2-OT

SRY-box 2 overlapping transcript

VMC

Viral myocarditis

WT

Wild-type

Authors’ contributions

T.Y. Y and W. W have given substantial contributions to the conception or the design of the manuscript. T.Y. Y, W. W and H.C. W to acquisition, analysis and interpretation of the data. T.Y. Y and W. W have participated to drafting the manuscript, H.C. W revised it critically. All authors read and approved the final version of the manuscript.

Funding

No funds, grants, or other support was received.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. All procedures were approved by the Pu’er People’s Hospital’s Ethics Committee (No. 2020-0048), and written informed consent was obtained from the guardians of all children.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.