A retrospective cohort study published in BMC Immunology reports that serum levels of microRNA-331-3p (miR-331-3p) are significantly lower in children with severe pneumonia compared to healthy controls and children with non-severe pneumonia. The research, conducted at Hebei Children’s Hospital, suggests that this specific microRNA may serve as a diagnostic biomarker for identifying severe cases of the disease. The study enrolled a total of 299 pediatric participants, including 97 healthy children, 101 children diagnosed with non-severe pneumonia, and 101 children with severe pneumonia. By measuring serum miR-331-3p expression using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the researchers observed a progressive decline in expression levels as the severity of the pneumonia increased.

The primary objective of the study was to evaluate the diagnostic value of serum miR-331-3p in pediatric severe pneumonia and to investigate its biological function and regulatory mechanisms. Severe pneumonia is a life-threatening condition that can lead to respiratory failure, cardiac dysfunction, and shock. Current clinical diagnosis relies heavily on physical examination and imaging, which can be limited by delayed presentation and low specificity. Conventional laboratory indicators such as C-reactive protein (CRP) and procalcitonin (PCT) are widely used to assess infection severity but lack tissue specificity and often yield false-positive results in various systemic inflammatory conditions. The need for novel, reliable biomarkers for early diagnosis and risk stratification in children remains a critical clinical challenge.

MicroRNAs are small non-coding RNA molecules that regulate gene expression and are involved in various pathological processes, including inflammation and immune responses. Previous studies have linked dysregulated miRNA expression to the pathogenesis of pneumonia. For instance, aberrant expression of miR-483-3p has been associated with severe pneumonia complicated by respiratory failure in pediatric patients. The current study focused on miR-331-3p, which has been shown to suppress viral replication and alleviate inflammatory responses in other contexts, such as porcine reproductive and respiratory syndrome virus infection and intracerebral hemorrhage. The researchers hypothesized that miR-331-3p might play a protective role in severe pneumonia by targeting specific downstream genes involved in cell death and inflammation.

To investigate the mechanism, the study identified B cell lymphoma 2 antagonist/killer 1 (BAK1) as a potential direct target of miR-331-3p. BAK1 is a pro-apoptotic protein in the BCL-2 family that regulates mitochondrial apoptosis. Bioinformatics analysis using the TargetScan database predicted that miR-331-3p binds to the 3′ untranslated region of BAK1. This predicted interaction was experimentally verified using a dual-luciferase reporter assay in human fetal lung fibroblast cells (MRC-5). The results confirmed that miR-331-3p directly binds to BAK1 and negatively regulates its expression. In clinical samples, BAK1 expression was significantly higher in children with severe pneumonia compared to healthy controls, and a strong negative correlation was observed between miR-331-3p and BAK1 expression levels.

The diagnostic performance of miR-331-3p was evaluated using receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) for distinguishing severe pneumonia from healthy controls was 0.899, with a sensitivity of 76.24% and a specificity of 91.75%. When differentiating severe pneumonia from non-severe pneumonia, the AUC was 0.830, with a sensitivity of 79.2% and a specificity of 74.3%. These metrics indicate that miR-331-3p has favorable differential diagnostic potential for identifying severe pediatric pneumonia. Furthermore, lower levels of miR-331-3p were correlated with higher levels of inflammatory indicators, including CRP, PCT, white blood cell count, and neutrophil count, in pneumonia patients.

In vitro experiments were conducted to explore the functional role of miR-331-3p in severe pneumonia. An LPS-induced MRC-5 cell model was established to simulate severe pneumonia. LPS treatment reduced cell viability and downregulated miR-331-3p expression in a dose-dependent manner. Overexpression of miR-331-3p in these cells promoted cell proliferation, inhibited apoptosis, and suppressed the production of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Conversely, inhibition of miR-331-3p had the opposite effects, leading to reduced proliferation, increased apoptosis, and elevated cytokine levels. These findings suggest that miR-331-3p exerts protective effects on lung fibroblasts by modulating apoptosis and inflammation.

Rescue experiments were performed to confirm that BAK1 mediates the biological functions of miR-331-3p. Co-transfection of miR-331-3p mimic with BAK1 overexpression plasmid reversed the protective effects of miR-331-3p. Specifically, BAK1 overexpression abolished the anti-apoptotic phenotype and increased the levels of TNF-α and IL-6. Western blot analysis showed that miR-331-3p downregulated pro-apoptotic proteins Bax and cleaved caspase-3 while upregulating anti-apoptotic Bcl-2. These changes were reversed by BAK1 overexpression, indicating that miR-331-3p protects lung fibroblasts by repressing BAK1 and thereby breaking the positive feedback loop between mitochondrial apoptosis and inflammatory injury.

The study highlights the importance of the miR-331-3p/BAK1 axis in the pathophysiology of severe pneumonia. BAK1-mediated mitochondrial outer membrane permeabilization is a key step in intrinsic apoptosis and can trigger inflammatory signaling through NF-κB pathways. By targeting BAK1, miR-331-3p may reduce mitochondrial damage and subsequent inflammation. This mechanism provides a theoretical foundation for developing point-of-care assays for miR-331-3p to enable early risk stratification and timely intervention in pediatric severe pneumonia. The findings are consistent with previous studies showing that miRNAs can modulate inflammatory and infectious lung disorders by regulating key apoptotic and inflammatory pathways.

However, the study has several limitations that must be considered. The participants were enrolled from a single center, which may introduce selection bias. The study did not include an independent validation cohort to externally verify the diagnostic performance of serum miR-331-3p. The favorable ROC metrics derived solely from the discovery cohort may overestimate its true clinical predictive value. Additionally, the mechanistic experiments used an LPS-stimulated MRC-5 fibroblast model, which is a simplified system that cannot fully recapitulate the complex pathophysiology of pediatric severe pneumonia. Only TNF-α and IL-6 were measured to assess inflammation, and other important inflammatory mediators such as IL-1β, IL-8, and NF-κB were not detected.

The study was cross-sectional, evaluating diagnostic value at admission without long-term follow-up to explore prognostic utility. Future research should adopt multicenter prospective cohorts to validate the diagnostic threshold of serum miR-331-3p and reduce single-center bias. Expanded in vitro biomarker detection and in vivo pneumonia models are needed to systematically characterize the miR-331-3p/BAK1/NF-κB regulatory network. Despite these limitations, the study provides preliminary evidence that serum miR-331-3p declines with pediatric pneumonia severity and has diagnostic value. Further multicenter verification and expanded pathway detection are necessary to validate its clinical application and fully understand its regulatory network in severe pneumonia.

The study was conducted as a retrospective cohort at Hebei Children’s Hospital, enrolling pediatric patients admitted between January 2024 and May 2025. The researchers divided the participants into three distinct groups: a healthy control group consisting of 97 children, a non-severe pneumonia group with 101 children, and a severe pneumonia group also comprising 101 children. Among the children diagnosed with severe pneumonia, 79 patients had been transferred from other hospitals. The definition of severe pneumonia followed international pediatric respiratory guidelines, requiring pneumonia accompanied by specific criteria such as room-air oxygen saturation below 95%, severe respiratory distress, the need for non-invasive or invasive ventilation, or extra-pulmonary organ complications like circulatory dysfunction or pleural effusion. In contrast, non-severe pneumonia was characterized by symptoms such as cough and tachypnea, with patients maintaining room-air oxygen saturation at or above 95% and without respiratory failure or multi-organ dysfunction. Baseline characteristics including age, sex, and body mass index showed no significant differences across the three cohorts, ensuring a comparable starting point for the analysis.

Pathogen distribution analysis revealed notable differences between the pneumonia groups. Single-pathogen infection dominated the non-severe pneumonia group, whereas mixed infection accounted for a significantly higher proportion in severe pneumonia patients, at 42.57% compared to 13.86%. No significant differences were observed in the composition of single bacterial or viral pathogens between the two pneumonia groups. Serum levels of conventional inflammatory markers, including C-reactive protein, procalcitonin, white blood cell count, and neutrophil count, gradually increased from healthy controls to non-severe pneumonia patients and reached the highest levels in the severe pneumonia group. These findings underscore the progressive nature of inflammatory response associated with disease severity, providing a clinical context for the observed changes in microRNA expression.

The diagnostic performance of miR-331-3p was further contextualized by its correlation with clinical inflammatory markers. In patients with severe pneumonia, those with lower miR-331-3p levels exhibited significantly higher serum levels of C-reactive protein, procalcitonin, white blood cell count, and neutrophil counts. Conversely, no significant correlations were detected between miR-331-3p levels and demographic factors such as age, gender, or body mass index. This specific association with inflammatory indicators suggests that miR-331-3p may reflect the intensity of the inflammatory response rather than general patient characteristics. The study’s cross-sectional design evaluated diagnostic value at admission, meaning it did not track changes over time or assess long-term prognostic utility. The lack of an independent validation cohort remains a significant limitation, as the favorable diagnostic metrics were derived solely from the discovery cohort, which may overestimate the true clinical predictive value of the biomarker.

Mechanistically, the study utilized an LPS-induced MRC-5 cell model to simulate severe pneumonia in vitro. LPS treatment reduced cell viability and downregulated miR-331-3p expression in a dose-dependent manner, with statistical significance observed starting at 5 µg/mL. The researchers used this model to test the hypothesis that miR-331-3p targets BAK1 to regulate apoptosis and inflammation. The dual-luciferase reporter assay confirmed that miR-331-3p directly binds to the 3′ untranslated region of BAK1, suppressing its expression. In clinical samples, BAK1 expression was significantly upregulated in children with severe pneumonia compared to healthy controls. A strong negative correlation was observed between miR-331-3p and BAK1 expression levels, with a correlation coefficient of -0.681. These findings support the notion that miR-331-3p acts as an endogenous suppressor of inflammatory signaling by targeting BAK1, a key pro-apoptotic protein in the BCL-2 family.

Subject of Research: Diagnostic value of serum miR-331-3p in pediatric severe pneumonia

Article Title: MiR-331-3p serves as a diagnostic biomarker in children with severe pneumonia

Article References: Si, X., Guo, W., Wei, X., Liu, N., Zhao, J., & Li, Q. (2026). MiR-331-3p serves as a diagnostic biomarker in children with severe pneumonia. BMC Immunology, 27(1), Article 87. https://doi.org/10.1186/s12865-026-00883-z

Image Credits: AI Generated

DOI: 10.1186/s12865-026-00883-z

Keywords: miR-331-3p, severe pneumonia, pediatric, biomarker, BAK1, diagnostic, microRNA, inflammation, apoptosis, BMC Immunology, serves, children