Diagnostic and prognostic value of deregulated miR-493-5p in patients with severe pneumonia

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Aims This study endeavors to explore the expression levels and clinical significance of miR-493-5p in pediatric patients diagnosed with severe pneumonia. Methods A total of 125 children diagnosed with severe pneumonia and 100 children with common pneumonia were enrolled in this study, alongside 100 healthy children who served as controls. Real-time quantitative PCR was used to was used to detect gene expression levels. The receiver operating characteristic curve was used to evaluate the diagnostic value of miR-493-5p in children with severe pneumonia. Kaplan-Meier and COX regression analyses were used to evaluate the prognostic value of miR-493-5p in children with severe pneumonia. Pearson correlation analysis was used for correlation analysis. Furthermore, pneumonia model cells were constructed in vitro to explore the underlying mechanism of miR-493-5p in severe pneumonia. Results Serum miR-493-5p was highly expressed in children with severe pneumonia. miR-493-5p has a certain diagnostic value for severe pneumonia and is related to the prognosis of the disease. miR-493-5p is positively correlated with the levels of inflammatory factors in children with severe pneumonia. miR-493-5p affects the function and inflammation of pneumonia model cells. IRF1 could partially reverse the effect of miR-493-5p on MRC-5 cell. Conclusions The level of miR-493-5p has a certain value in the diagnosis and prognosis of children with severe pneumonia, and it may play a critical role in the disease process and associated inflammatory responses by targeting the IRF1. severe pneumonia miR-493-5p diagnosis prognosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pneumonia stands as the most prevalent respiratory infectious disease in pediatric populations ( 1 ). According to the severity of the disease, pneumonia is divided into mild pneumonia and severe pneumonia ( 2 ). Mild pneumonia typically presents with subtle symptoms, a lack of complications, and a favorable prognosis ( 3 ). In contrast, severe pneumonia is characterized by a sudden onset, rapid deterioration, significant severity, and a plethora of potential complications, often posing a grave threat to the lives of affected children. It is the main cause of death in children under 5 years old ( 4 , 5 ). Among pediatric patients diagnosed with pneumonia, more than 30% are classified as having severe pneumonia ( 6 ). Severe pneumonia in children arises from the invasion of pathogenic microorganisms into the lung parenchyma ( 7 ). This condition displays distinctive features, including a swift onset, numerous complications, and systemic involvement. Notably, typical manifestations may be obscured or overlooked in clinical settings. In instances where timely intervention is not prioritized, there exists a substantial risk of misdiagnosis and inappropriate treatment, potentially precipitating disease progression and, in dire cases, fatality ( 4 ). Consequently, the accurate diagnosis and appropriate management of severe pneumonia in children constitute critical challenges within the field of pediatrics. MicroRNA (miRNA) constitutes a class of gene regulatory molecules intimately connected to critical biological processes such as cell proliferation, apoptosis, invasion, and differentiation ( 8 ). A wealth of research has elucidated the close association between miRNAs and various pulmonary conditions, including lung development, pneumonia, lung cancer, and pulmonary fibrosis ( 9 , 10 ). Studies have demonstrated that miR-146a and miR-16-5p serve as prognostic indicators for community-acquired pneumonia ( 11 ). MiR-181b was found to be a potential diagnostic and prognostic indicator for severe community-acquired pneumonia ( 12 ). The high expression of miR-493-5p was shown to be associated with clinical prognosis of non-small cell lung cancer ( 13 ). In instances of adenovirus infection pneumonia in children, the miRNA expression profile in whole blood has revealed aberrant expression patterns of miR-493-5p ( 14 ). Previous investigations have suggested that the miR-493-5p may play a significant role in pneumonia infections. Nevertheless, its expression and clinical significance in severe pneumonia need to be further explored. The study seeks to investigate the expression and clinical relevance of miR-493-5p in pediatric patients suffering from severe pneumonia. Materials and methods General Information A total of 125 children diagnosed with severe pneumonia were admitted to The Second Affiliated Hospital of Mudanjiang Medical University between January 2022 and December 2023, constituting the severe group. Additionally, 100 children presenting with common pneumonia were randomly selected to form the mild group, while a healthy group was comprised of 100 healthy children. Inclusion criteria were strictly defined: all patients were confirmed through comprehensive laboratory and imaging examinations, and children categorized as having severe pneumonia met the pertinent diagnostic standards. Antibiotics and antiviral drugs were selected according to the clinical conditions of the children. Exclusion criteria included: a history of antibiotic allergy; instances of asphyxia; concurrent aspiration pneumonia or other infectious diseases; and the presence of familial disease. Informed consent was signed by the family members of the selected study subjects, and this study was approved by the ethics committee of the The Second Affiliated Hospital of Mudanjiang Medical University Sample collection and follow-up Clinical data were meticulously gathered from each group. Fasting venous blood samples were collected in the morning and centrifuged to obtain serum. Following this process, the supernatant was carefully pipetted into an EP tube and stored at -80°C to ensure optimal preservation. Assessment of prognosis in children with severe pneumonia the medical records of children with severe pneumonia were tracked. The prognosis was evaluated according to the 28-day survival, and the outcomes included survival and death. Cell culture and treatment MRC-5 cells were procured from Wuhan Pricella Biotechnology Co., Ltd. The growth medium utilized was DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). The cells were maintained under optimal culture conditions of 95% air and 5% CO 2 at 37°C. To establish a pneumonia model, MRC-5 cells were treated with lipopolysaccharide (LPS) for 12 hours. Untreated cells served as the control group, and the concentrations of LPS were set at 0, 5, 10, and 20 µg/mL, respectively ( 15 ). Cell transfection and grouping MRC-5 cells treated with LPS were initially seeded in 6-well plates and incubated in DMEM for a duration of 12 hours. Subsequently, transfection was carried out utilizing Lipofectamine 2000 (Invitrogen, USA). The transfection experiments were categorized into distinct groups: the control group (un-transfected), the miR-493-5p mimic group, the miR-493-5p inhibitor group, and their corresponding negative controls (mimic NC and inhibitor NC) (Ribobio, Guangzhou). The cells were co-transfected into miR-493-5p mimic + OE-IRF1 group (miR-493-5p mimic and IRF1 overexpression plasmid were co-transfected) and its respective negative control (miR-493-5p mimic + OE-NC group). Real Time Quantitative PCR Total RNA was extracted from serum and cells using Trizol (Invitrogen, USA), and the RNA concentration was quantified through ultraviolet spectrophotometry. Following this, the total RNA underwent reverse transcription into complementary DNA (cDNA) utilizing a reverse transcription kit from Thermo Fisher (USA). Real-time PCR analysis was conducted on an ABI 7500 Fast system, in accordance with the protocols outlined in the ChamQ Universal SYBR qPCR Master Mix reagent kit (Vazyme Biotech, Nanjing). Employing U6 as an internal reference, the relative expression level of the miR-493-5p gene was calculated using the 2 −△△Ct method. Measurement of inflammation factors Serum samples were collected from three distinct groups of subjects, along with cell supernatants from each group. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were subsequently measured using a commercially available enzyme-linked immunosorbent assay kit (MLBIO, Shanghai). CCK-8 assay Cell viability was assessed using the CCK-8 assay. Cells from each experimental group were harvested and subsequently seeded into 96-well plates at a density of 2 × 10 ^3 cells per well. Following inoculation, the cells were cultured until they reached the logarithmic growth phase. After this period, 10µL of CCK-8 solution was added to each well, and the cells were incubated for an additional 4 hours. The viability of the cells was quantified by measuring the optical density at 450 nm with a microplate reader. Cell apoptosis assay Cells from each group were routinely subjected to digestion using a 0.25% trypsin solution, followed by centrifugation at 800×g for 5 minutes. The resulting cell pellet was washed three times with phosphate-buffered saline (PBS) before being resuspended in 500µL of 1× Binding Buffer. Subsequently, 5µL of Annexin V-FITC was added and mixed thoroughly, followed by the addition of 10µL of propidium iodide (PI), and the mixture was incubated for 40 minutes. Flow cytometry was then employed to detect fluorescence intensity, enabling the calculation of the apoptosis rate. Luciferase reporter assay The potential target genes of miR-493-5p were identified utilizing the TargetScan online database ( http://www.targe-tscan.org/vert_72/ ). Following the identification of the binding site for miR-493-5p, we constructed wild-type (IRF1-wt) and corresponding mutant (IRF1-mut) vectors that encompassed the predicted binding region. These constructed plasmid vectors were subsequently co-transfected into MRC-5 cells alongside either a miR-493-5p mimic or a miR-493-5p inhibitor. To validate the interaction between miR-493-5p and IRF1, we employed a dual luciferase reporter gene assay, thereby elucidating the targeting relationship between the microRNA and its putative gene target. Statistical methods Statistical analysis and data visualization were conducted using SPSS version 26.0 and GraphPad Prism version 9.0. To assess normal distribution measurement data, independent samples t-tests were employed, while the Wilcoxon Mann-Whitney test was utilized for data exhibiting skewness. Categorical data were analyzed using the chi-square test. The ROC curve was used to evaluate the diagnostic value of miR-493-5p. Pearson correlation analysis was used for correlation analysis. Kaplan-Meier analysis was used to investigate the relationship between miR-493-5p expression and overall survival rate of children with severe pneumonia. COX regression analysis was used for multivariate analysis. Statistical significance was set at P < 0.05. Results Clinical data in three groups The results of the comparative analysis of clinical data across the three groups of subjects are presented in Table 1 . Demographic variables, including age and gender, did not differ markedly between the groups ( P > 0.05). However, a significant difference was observed in white blood cell (WBC) counts, neutrophil levels, and various other clinical indicators ( P < 0.05). Further pairwise comparisons revealed that the clinical indicators for the mild pneumonia group were elevated compared to those of the healthy group, and that the severe pneumonia group exhibited even higher values than the mild pneumonia group ( P < 0.05). Table 1 The basic information of the subjects was compared Variable Healthy (n = 100) Mild pneumonia (n = 100) Severe pneumonia (n = 125) Age (years) 6.66 ± 3.16 6.84 ± 3.37 7.15 ± 3.29 Sex (male/female) 53 / 47 54 / 46 74 / 51 WBC (10 9 /L) 6.94 ± 1.09 9.97 ± 1.53*** 10.93 ± 1.79*** ### Neutrophils (10 9 /L) 2.59 ± 0.54 4.31 ± 0.81*** 5.38 ± 1.20*** ### CRP (mg/L) 2.74 ± 0.53 8.14 ± 1.75*** 12.59 ± 1.53*** ### PCT (ng/mL) 0.08 ± 0.12 0.19 ± 0.17*** 1.91 ± 0.64*** ### Lymphocyte (10 9 /L) 1.95 ± 0.31 2.33 ± 0.38*** 2.43 ± 0.33*** # LDH (U/L) 164.06 ± 21.15 212.51 ± 25.47*** 315.65 ± 52.51*** ### BMI, Body Mass Index; WBC, white blood cell; CRP, C-reactive protein; PCT, Procalcitonin; LDH, Lactate dehydrogenase. *** P <0.001 Compared with the healthy group; # P <0.05, ### P <0.001 Compared with the Mild pneumonia group Diagnostic and prognostic value of serum miR-493-5p in severe pneumonia RT-qPCR analysis revealed that the level of serum miR-493-5p in the severe pneumonia cohort was markedly increased compared to both the mild pneumonia group and the healthy group ( P < 0.001) (Fig. 1 A). Furthermore, ROC curve analysis demonstrated that miR-493-5p possesses significant potential to differentiate children with severe pneumonia from those with mild cases. The AUC was calculated at 0.894 (95% CI: 0.855–0.934), with a sensitivity of 80.8% and a specificity of 82% (Fig. 1 B). Based on the median expression levels of miR-493-5p, children with severe pneumonia were stratified into a high expression group (n = 66) and a low expression group (n = 59). A Kaplan-Meier curve depicting the survival status of these children at 28 days post-birth was constructed, and a Log-rank test was conducted. It was found that the 28-day cumulative survival rate for the high miR-493-5p expression group was substantially lower than that of the low expression group, with the difference attaining statistical significance ( P < 0.05) (Fig. 1 C). Additionally, multivariate COX regression analysis identified the expression level of miR-493-5p and Procalcitonin (PCT) as significant prognostic factors within the severe pneumonia group ( P 0.05) (Table 2 ). These findings suggest that elevated levels of miR-493-5p may be intricately linked to both the development and adverse prognosis of severe pneumonia in pediatric patients. Table 2 Cox regression analysis of the association between clinical parameters and survival in patients with severe pneumonia Variable HR 95% CI for HR P value Lower Upper miR-493-5p expression 3.363 1.252 9.037 0.016* Age 0.965 0.437 2.131 0.929 Sex 1.268 0.564 2.851 0.565 WBC 1.856 0.825 4.174 0.135 Neutrophils 2.019 0.897 4.543 0.089 CRP 1.864 0.798 4.353 0.150 PCT 2.439 1.022 5.819 0.045* Lymphocyte 1.647 0.676 4.011 0.272 LDH 1.222 0.553 2.699 0.620 BMI, Body Mass Index; WBC, white blood cell; CRP, C-reactive protein; PCT, Procalcitonin; LDH, Lactate dehydrogenase. * p <0.05 Serum miR-493-5p correlates with levels of inflammatory factors in severe pneumonia Pearson correlation analysis revealed a significant positive correlation between serum miR-493-5p levels and the pro-inflammatory cytokines IL-6 (r = 0.715), TNF-α (r = 0.787), and IL-1β (r = 0.759) in children afflicted with severe pneumonia (Fig. 1 D-F). These findings imply that miR-493-5p may play a pivotal role in the inflammatory response associated with severe pneumonia. Effect of miR-493-5p on LPS-induced MRC-5 cells The CCK-8 assay demonstrated that LPS treatment markedly inhibited the viability of MRC-5 cells, with a noticeable decline in cell viability corresponding to the increasing concentrations of LPS (Fig. 2 A ) . Further RT-qPCR showed that the expression of miR-493-5p in MRC-5 cells was increased after LPS treatment, and the level showed an upward trend with the increase of LPS concentration (Fig. 2 B ) . We subsequently established cell models characterized by either overexpression or knockdown of miR-493-5p (Fig. 2 C ) . Cell viability and apoptosis assays indicated that the overexpression of miR-493-5p led to a reduction in cell viability and an enhancement of apoptosis in the pneumonia model cells, whereas the knockdown of miR-493-5p yielded opposing effects (Fig. 2 D-E ) . Moreover, the overexpression of miR-493-5p was associated with an increase in the production of inflammatory factors within the pneumonia model cells, while the knockdown of miR-493-5p exhibited the reverse effect (Fig. 2 F ) . These showed that miR-493-5p affected the function and inflammation of pneumonia model cells. Target correlation between miR-493-5p and IRF1 The binding site of miR-493-5p to IRF1 is depicted in Fig. 3 A. This targeting relationship between miR-493-5p and IRF1 was further corroborated through a dual luciferase reporter gene assay (Fig. 3 B ) . In MRC-5 cells treated with LPS, the expression of IRF1 exhibited a decline, demonstrating a downward trend in correlation with increasing concentrations of LPS (Fig. 3 C ) . Within the pneumonia model cells, overexpression of miR-493-5p led to a suppression in IRF1 levels, whereas the knockdown of miR-493-5p produced an opposing effect (Fig. 3 D ) . The expression level of IRF1 in the severe pneumonia group was elevated compared to both the mild pneumonia group and the healthy control group (Fig. 3 E ) . Pearson correlation analysis revealed a significant negative correlation between miR-493-5p and IRF1 expression in children suffering from severe pneumonia (r= -0.722, P<0.001) (Fig. 3 F ) . Effect of miR-493-5p targeting IRF1 on LPS-induced MRC-5 cells To delve deeper into the impact of miR-493-5p in modulating IRF1 within pneumonia model cells, we conducted co-transfection experiments to restore IRF1 expression (Fig. 4 A ) . The CCK-8 cell viability assay revealed that IRF1 effectively mitigated the inhibitory effects of miR-493-5p on the viability of LPS-induced MRC-5 cells (Fig. 4 B ) . Apoptosis assay showed that IRF1 could partially counteract the pro-apoptotic influence of miR-493-5p under LPS stimulation in MRC-5 cells (Fig. 4 C ) . Furthermore, IRF1 was shown to effectively suppress the enhancing effect of miR-493-5p on the LPS-induced production of inflammatory factors in MRC-5 cells (Fig. 4 D ) . Collectively, these findings suggest that miR-493-5p may influence the progression and inflammatory response associated with severe pneumonia by modulating the expression of IRF1. Discussion Severe pneumonia represents a prevalent and critical illness in pediatrics, characterized by challenging clinical management, rapid progression, poor prognostic outcomes, and elevated mortality rates ( 16 ). An early and thorough evaluation of a patient's condition and prognosis is essential for devising an effective treatment strategy that can enhance patient outcomes. Clinical research has demonstrated that elevated expression levels of miR-10a-3p are observed in patients suffering from severe pneumonia, suggesting its potential role as a diagnostic and prognostic biomarker for this condition ( 17 ). Our investigation reveals that serum levels of miR-493-5p are markedly elevated in children with severe pneumonia, exhibiting a positive correlation with inflammatory response levels in these patients. This finding shows that miR-493-5p may serve as a valuable tool for assessing the severity and prognosis of severe pneumonia in pediatric patients. The overexpression of miR-493-5p appears to be associated with severe pneumonia, underscoring its potential as an early diagnostic and prognostic marker in clinical settings. Inflammation represents a fundamental pathological mechanism in patients experiencing severe pneumonia ( 18 ). In this context, inflammatory cytokines play a pivotal role in the progression and severity of the disease. IL-6 serves as a key mediator of the body's inflammatory response, acting as a critical transmitter within this process ( 19 ). IL-1β is produced by cell types such as fibroblasts during infection events and plays an important role in inflammatory responses by promoting the activation, proliferation, and differentiation of B cells ( 20 ). TNF-α, synthesized by activated macrophages and monocytes, exhibits a range of complex biological activities and is integral to the regulation of infection, inflammation, and immune responses ( 21 ). Elevated levels of IL-6, IL-1β, and TNF-α have been consistently observed in cases of pneumonia ( 22 ). MiRNAs have been closely associated with the regulation of the inflammatory response. Recent studies have demonstrated that miR-155 exerts significant biological functions across various inflammatory cell types ( 23 ). MiR-146b has been implicated in the inflammatory injury associated with pneumonia in pediatric patients ( 24 ). Additionally, findings indicate that miR-195-5p is involved in cell proliferation, apoptosis, and the inflammatory responses triggered by LPS ( 25 ). In our investigation, we found that overexpressing miR-493-5p inhibited LPS-induced MRC-5 cell proliferation and promoted cell apoptosis and the production of inflammatory factors. This suggests that miR-493-5p may have a regulatory role in the context of severe pneumonia. IRF1 is a pivotal molecule that governs downstream inflammatory responses and apoptosis ( 26 ). Accumulating evidence highlights the critical role of IRF1 in antiviral immunity ( 27 ). Notably, prior researches have established that miR-130b-3p influences the polarization of M1 macrophages by targeting IRF1 ( 28 ). Furthermore, research has demonstrated that miR-370-3p modulates LPS-induced acute pneumonia by targeting TLR4 ( 29 ). MiR-429 exacerbates inflammatory injury in neonatal pneumonia through the targeting of KLF4 ( 30 ). Our findings indicate that the overexpression of miR-493-5p substantially impacts LPS-induced functional alterations and inflammatory responses in MRC-5 cells via the down-regulation of IRF1. This implicates the targeting of IRF1 by miR-493-5p as a contributing factor in the progression of severe pneumonia. The limitations of our study include the singularity of patient inclusion from a single center, which may influence the overall robustness of our findings. The exploration of miR-493-5p in conjunction with additional biomarkers for the diagnosis of severe pneumonia warrants further investigation to enhance clinical diagnostic reference. Additionally, the application of serum miR-493-5p expression levels to inform individualized treatment strategies for children suffering from severe pneumonia remains ambiguous. Furthermore, the specific mechanisms by which miR-493-5p participates in the pathophysiology of severe pneumonia require more in-depth research. These aspects will constitute the focal points of our future investigations. In conclusion, the level of serum miR-493-5p was increased in children with severe pneumonia and was associated with the prognosis of pneumonia. It may be used as a molecular marker for early diagnosis of severe pneumonia and a potential therapeutic target. This study provides a new reference for the clinical study of severe pneumonia in children and the improvement of prognosis. Declarations Statement of author contributions All authors contributed to the study conception and design. Study concept and design: L.L. L., and L.Y. W.; analysis and interpretation of data: L.L. L., and L. H.; drafting of the manuscript: L.L. L.; critical revision of the manuscript for important intellectual content: L.L. L., L. H., and L.Y. W.; statistical analysis: L.L. L. and H. Z.. Acknowledgements Not applicable. Competing Interests The authors have no relevant financial or non-financial interests to disclose. 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Zhang L., Yan H., Wang H., Wang L., Bai B., Ma Y., Tie Y., and Xi Z., Current neurovascular research 17, 102-109, 2020. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5769928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":398998863,"identity":"655bef41-0d58-4432-a704-cbdc3cc9e661","order_by":0,"name":"Lili Liu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Mudanjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Liu","suffix":""},{"id":398998864,"identity":"9aca3365-7d17-489b-9638-68e7af5ee714","order_by":1,"name":"Lei Han","email":"","orcid":"","institution":"The Second Affiliated Hospital of Mudanjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Han","suffix":""},{"id":398998865,"identity":"84022e56-865b-4026-9371-a8e9165e97e5","order_by":2,"name":"Huan Zhao","email":"","orcid":"","institution":"The Second Affiliated Hospital of Mudanjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Zhao","suffix":""},{"id":398998866,"identity":"b60526e7-f705-47b6-afe1-630f31705dc6","order_by":3,"name":"Liying Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYBACgwNA4oEBAw8DA/+HAx8MbOyI05IA1sJg+HBGQVoykVogbGNjng+HGBsIajl+9vCLhILDMub8C9KkbQwOMDOwHz66AZ8WszN5aRYJBod5LGc8OCadY3CHj4EnLe0GXi0HcswMQFoMbhxsA2p5xswgwWOGX8v5NzAth9mkLQwOMzYQ0mJ/I8f4AVjL+TZmYwZitFjeeGMGDOR0oC08jA97DNKS2Qj5xeB8jvGHD3+s7Q3On2E48OOPjR0/++FjeLUAAZsEA0MzA4NEApRLQDkIMH9gYKgDppcDRKgdBaNgFIyCEQkAuFBSOgI81rYAAAAASUVORK5CYII=","orcid":"","institution":"The Second Affiliated Hospital of Mudanjiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Liying","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-01-06 02:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5769928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5769928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73674847,"identity":"e1bb3732-3515-4f3b-b5f0-858048f5ff87","added_by":"auto","created_at":"2025-01-13 12:59:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":449744,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic and prognostic value of serum miR-493-5p in severe pneumonia. The serum miR-493-5p in the severe pneumonia group was markedly higher than that in the mild pneumonia group and the healthy group (\u003cstrong\u003eA\u003c/strong\u003e). ROC curve was used to evaluate the diagnostic value of miR-493-5p for severe pneumonia (\u003cstrong\u003eB\u003c/strong\u003e). A K-M curve was used to analyze the effect of miR-493-5p on the prognosis of children with severe pneumonia (\u003cstrong\u003eC\u003c/strong\u003e). Serum miR-493-5p was positively correlated with inflammatory factors in children with severe pneumonia (\u003cstrong\u003eD-F\u003c/strong\u003e). ** \u003cem\u003eP\u003c/em\u003e＜0.01, *** \u003cem\u003eP\u003c/em\u003e＜0.001, Compared with the healthy group; \u003csup\u003e###\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e＜0.001, Compared with mild pneumonia group\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5769928/v1/f18c63b50a72b7f39f6723c3.png"},{"id":73672964,"identity":"28b68421-ea82-40c6-8c12-31cc6df76baa","added_by":"auto","created_at":"2025-01-13 12:51:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":600999,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of miR-493-5p on LPS-induced MRC-5 cells. LPS inhibited the viability of MRC-5 cells (\u003cstrong\u003eA\u003c/strong\u003e). The expression of miR-493-5p in MRC-5 cells was markedly increased after LPS treatment, and increased with the increase of LPS concentration (\u003cstrong\u003eB\u003c/strong\u003e). miR-493-5p inhibited cell viability and promoted cell apoptosis and the production of inflammatory factors in pneumonia model cells (\u003cstrong\u003eC-F\u003c/strong\u003e). * \u003cem\u003eP\u003c/em\u003e＜0.05, ** \u003cem\u003eP\u003c/em\u003e＜0.01, *** \u003cem\u003eP\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5769928/v1/9313ab6215b69b8ac4a4b1aa.png"},{"id":73674848,"identity":"bc974fa9-7742-4691-a0e2-d1da0b120f19","added_by":"auto","created_at":"2025-01-13 12:59:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":507332,"visible":true,"origin":"","legend":"\u003cp\u003eVerification of the targeting relationship between miR-493-5p and IRF1. Binding site of miR-493-5p and IRF1 (\u003cstrong\u003eA\u003c/strong\u003e). Dual luciferase reporter gene assay confirmed the targeting relationship between miR-493-5p and IRF1 (\u003cstrong\u003eB\u003c/strong\u003e). After LPS treatment, the expression of IRF1 in MRC-5 cells decreased and showed a gradually decreasing trend with the increase of LPS concentration (\u003cstrong\u003eC\u003c/strong\u003e). Within the pneumonia model cells, overexpression of miR-493-5p led to a suppression in IRF1 levels, whereas the knockdown of miR-493-5p produced an opposing effect (\u003cstrong\u003eD\u003c/strong\u003e). The expression of IRF1 in severe pneumonia group was markedly lower than that in mild pneumonia group and healthy group (\u003cstrong\u003eE\u003c/strong\u003e). There was a significant negative correlation between miR-493-5p and IRF1 in children with severe pneumonia (r= -0.722, P \u0026lt; 0.001) (\u003cstrong\u003eF\u003c/strong\u003e). * \u003cem\u003eP\u003c/em\u003e＜0.05, ** \u003cem\u003eP\u003c/em\u003e＜0.01, *** \u003cem\u003eP\u003c/em\u003e＜0.001 Compared with the healthy group; \u003csup\u003e###\u003c/sup\u003e\u003cem\u003e P\u003c/em\u003e＜0.001, Compared with mild pneumonia group\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5769928/v1/e275dc9a01268c339a1ef3ff.png"},{"id":73672961,"identity":"97657f4f-a182-4d44-9241-762f05acd409","added_by":"auto","created_at":"2025-01-13 12:51:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":638919,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of miR-493-5p targeting IRF1 on pneumonia model cells. IRF1 expression was restored by co-transfection experiments (\u003cstrong\u003eA\u003c/strong\u003e). IRF1 could partially reverse the effects of mir-493-5p on cell viability, apoptosis and inflammation in pneumonia model cells (\u003cstrong\u003eB-D\u003c/strong\u003e). ** \u003cem\u003eP\u003c/em\u003e＜0.01, *** \u003cem\u003eP\u003c/em\u003e＜0.001\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5769928/v1/817e804e82efb17f5ca535f0.png"},{"id":73780842,"identity":"0f84c413-2985-4256-9c96-100843b8398a","added_by":"auto","created_at":"2025-01-14 15:17:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3190617,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5769928/v1/2e9eebba-6207-4396-a457-6574eff4e004.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDiagnostic and prognostic value of deregulated miR-493-5p in patients with severe pneumonia \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePneumonia stands as the most prevalent respiratory infectious disease in pediatric populations (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). According to the severity of the disease, pneumonia is divided into mild pneumonia and severe pneumonia (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Mild pneumonia typically presents with subtle symptoms, a lack of complications, and a favorable prognosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In contrast, severe pneumonia is characterized by a sudden onset, rapid deterioration, significant severity, and a plethora of potential complications, often posing a grave threat to the lives of affected children. It is the main cause of death in children under 5 years old (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Among pediatric patients diagnosed with pneumonia, more than 30% are classified as having severe pneumonia (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Severe pneumonia in children arises from the invasion of pathogenic microorganisms into the lung parenchyma (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). This condition displays distinctive features, including a swift onset, numerous complications, and systemic involvement. Notably, typical manifestations may be obscured or overlooked in clinical settings. In instances where timely intervention is not prioritized, there exists a substantial risk of misdiagnosis and inappropriate treatment, potentially precipitating disease progression and, in dire cases, fatality (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Consequently, the accurate diagnosis and appropriate management of severe pneumonia in children constitute critical challenges within the field of pediatrics.\u003c/p\u003e \u003cp\u003eMicroRNA (miRNA) constitutes a class of gene regulatory molecules intimately connected to critical biological processes such as cell proliferation, apoptosis, invasion, and differentiation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). A wealth of research has elucidated the close association between miRNAs and various pulmonary conditions, including lung development, pneumonia, lung cancer, and pulmonary fibrosis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Studies have demonstrated that miR-146a and miR-16-5p serve as prognostic indicators for community-acquired pneumonia (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). MiR-181b was found to be a potential diagnostic and prognostic indicator for severe community-acquired pneumonia (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The high expression of miR-493-5p was shown to be associated with clinical prognosis of non-small cell lung cancer (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In instances of adenovirus infection pneumonia in children, the miRNA expression profile in whole blood has revealed aberrant expression patterns of miR-493-5p (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Previous investigations have suggested that the miR-493-5p may play a significant role in pneumonia infections. Nevertheless, its expression and clinical significance in severe pneumonia need to be further explored.\u003c/p\u003e \u003cp\u003eThe study seeks to investigate the expression and clinical relevance of miR-493-5p in pediatric patients suffering from severe pneumonia.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Information\u003c/h2\u003e \u003cp\u003eA total of 125 children diagnosed with severe pneumonia were admitted to The Second Affiliated Hospital of Mudanjiang Medical University between January 2022 and December 2023, constituting the severe group. Additionally, 100 children presenting with common pneumonia were randomly selected to form the mild group, while a healthy group was comprised of 100 healthy children. Inclusion criteria were strictly defined: all patients were confirmed through comprehensive laboratory and imaging examinations, and children categorized as having severe pneumonia met the pertinent diagnostic standards. Antibiotics and antiviral drugs were selected according to the clinical conditions of the children. Exclusion criteria included: a history of antibiotic allergy; instances of asphyxia; concurrent aspiration pneumonia or other infectious diseases; and the presence of familial disease.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eInformed consent\u003c/strong\u003e \u003cp\u003e was signed by the family members of the selected study subjects, and this study was approved by the ethics committee of the The Second Affiliated Hospital of Mudanjiang Medical University\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection and follow-up\u003c/h3\u003e\n\u003cp\u003eClinical data were meticulously gathered from each group. Fasting venous blood samples were collected in the morning and centrifuged to obtain serum. Following this process, the supernatant was carefully pipetted into an EP tube and stored at -80\u0026deg;C to ensure optimal preservation.\u003c/p\u003e \u003cp\u003eAssessment of prognosis in children with severe pneumonia the medical records of children with severe pneumonia were tracked. The prognosis was evaluated according to the 28-day survival, and the outcomes included survival and death.\u003c/p\u003e\n\u003ch3\u003eCell culture and treatment\u003c/h3\u003e\n\u003cp\u003eMRC-5 cells were procured from Wuhan Pricella Biotechnology Co., Ltd. The growth medium utilized was DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). The cells were maintained under optimal culture conditions of 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. To establish a pneumonia model, MRC-5 cells were treated with lipopolysaccharide (LPS) for 12 hours. Untreated cells served as the control group, and the concentrations of LPS were set at 0, 5, 10, and 20 \u0026micro;g/mL, respectively (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eCell transfection and grouping\u003c/h3\u003e\n\u003cp\u003eMRC-5 cells treated with LPS were initially seeded in 6-well plates and incubated in DMEM for a duration of 12 hours. Subsequently, transfection was carried out utilizing Lipofectamine 2000 (Invitrogen, USA). The transfection experiments were categorized into distinct groups: the control group (un-transfected), the miR-493-5p mimic group, the miR-493-5p inhibitor group, and their corresponding negative controls (mimic NC and inhibitor NC) (Ribobio, Guangzhou). The cells were co-transfected into miR-493-5p mimic\u0026thinsp;+\u0026thinsp;OE-IRF1 group (miR-493-5p mimic and IRF1 overexpression plasmid were co-transfected) and its respective negative control (miR-493-5p mimic\u0026thinsp;+\u0026thinsp;OE-NC group).\u003c/p\u003e\n\u003ch3\u003eReal Time Quantitative PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from serum and cells using Trizol (Invitrogen, USA), and the RNA concentration was quantified through ultraviolet spectrophotometry. Following this, the total RNA underwent reverse transcription into complementary DNA (cDNA) utilizing a reverse transcription kit from Thermo Fisher (USA). Real-time PCR analysis was conducted on an ABI 7500 Fast system, in accordance with the protocols outlined in the ChamQ Universal SYBR qPCR Master Mix reagent kit (Vazyme Biotech, Nanjing). Employing U6 as an internal reference, the relative expression level of the miR-493-5p gene was calculated using the 2\u003csup\u003e\u0026minus;△△Ct\u003c/sup\u003e method.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of inflammation factors\u003c/h2\u003e \u003cp\u003eSerum samples were collected from three distinct groups of subjects, along with cell supernatants from each group. The concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were subsequently measured using a commercially available enzyme-linked immunosorbent assay kit (MLBIO, Shanghai).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCK-8 assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the CCK-8 assay. Cells from each experimental group were harvested and subsequently seeded into 96-well plates at a density of 2 \u0026times; 10\u003csup\u003e^3\u003c/sup\u003e cells per well. Following inoculation, the cells were cultured until they reached the logarithmic growth phase. After this period, 10\u0026micro;L of CCK-8 solution was added to each well, and the cells were incubated for an additional 4 hours. The viability of the cells was quantified by measuring the optical density at 450 nm with a microplate reader.\u003c/p\u003e\n\u003ch3\u003eCell apoptosis assay\u003c/h3\u003e\n\u003cp\u003e Cells from each group were routinely subjected to digestion using a 0.25% trypsin solution, followed by centrifugation at 800\u0026times;g for 5 minutes. The resulting cell pellet was washed three times with phosphate-buffered saline (PBS) before being resuspended in 500\u0026micro;L of 1\u0026times; Binding Buffer. Subsequently, 5\u0026micro;L of Annexin V-FITC was added and mixed thoroughly, followed by the addition of 10\u0026micro;L of propidium iodide (PI), and the mixture was incubated for 40 minutes. Flow cytometry was then employed to detect fluorescence intensity, enabling the calculation of the apoptosis rate.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eThe potential target genes of miR-493-5p were identified utilizing the TargetScan online database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.targe-tscan.org/vert_72/\u003c/span\u003e\u003cspan address=\"http://www.targe-tscan.org/vert_72/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Following the identification of the binding site for miR-493-5p, we constructed wild-type (IRF1-wt) and corresponding mutant (IRF1-mut) vectors that encompassed the predicted binding region. These constructed plasmid vectors were subsequently co-transfected into MRC-5 cells alongside either a miR-493-5p mimic or a miR-493-5p inhibitor. To validate the interaction between miR-493-5p and IRF1, we employed a dual luciferase reporter gene assay, thereby elucidating the targeting relationship between the microRNA and its putative gene target.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical methods\u003c/h2\u003e \u003cp\u003eStatistical analysis and data visualization were conducted using SPSS version 26.0 and GraphPad Prism version 9.0. To assess normal distribution measurement data, independent samples t-tests were employed, while the Wilcoxon Mann-Whitney test was utilized for data exhibiting skewness. Categorical data were analyzed using the chi-square test. The ROC curve was used to evaluate the diagnostic value of miR-493-5p. Pearson correlation analysis was used for correlation analysis. Kaplan-Meier analysis was used to investigate the relationship between miR-493-5p expression and overall survival rate of children with severe pneumonia. COX regression analysis was used for multivariate analysis. Statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical data in three groups\u003c/h2\u003e \u003cp\u003eThe results of the comparative analysis of clinical data across the three groups of subjects are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Demographic variables, including age and gender, did not differ markedly between the groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, a significant difference was observed in white blood cell (WBC) counts, neutrophil levels, and various other clinical indicators (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Further pairwise comparisons revealed that the clinical indicators for the mild pneumonia group were elevated compared to those of the healthy group, and that the severe pneumonia group exhibited even higher values than the mild pneumonia group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe basic information of the subjects was compared\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMild pneumonia (n\u0026thinsp;=\u0026thinsp;100)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSevere pneumonia (n\u0026thinsp;=\u0026thinsp;125)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.66\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (male/female)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53 / 47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54 / 46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74 / 51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC (10\u003csup\u003e9\u003c/sup\u003e /L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79***\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils (10\u003csup\u003e9\u003c/sup\u003e /L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20***\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP (mg/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53***\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCT (ng/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64***\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte (10\u003csup\u003e9\u003c/sup\u003e /L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33***\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDH (U/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e164.06\u0026thinsp;\u0026plusmn;\u0026thinsp;21.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e212.51\u0026thinsp;\u0026plusmn;\u0026thinsp;25.47***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e315.65\u0026thinsp;\u0026plusmn;\u0026thinsp;52.51***\u003csup\u003e###\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eBMI, Body Mass Index; WBC, white blood cell; CRP, C-reactive protein; PCT, Procalcitonin; LDH, Lactate dehydrogenase. ***\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 Compared with the healthy group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.001 Compared with the Mild pneumonia group\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDiagnostic and prognostic value of serum miR-493-5p in severe pneumonia\u003c/h2\u003e \u003cp\u003eRT-qPCR analysis revealed that the level of serum miR-493-5p in the severe pneumonia cohort was markedly increased compared to both the mild pneumonia group and the healthy group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, ROC curve analysis demonstrated that miR-493-5p possesses significant potential to differentiate children with severe pneumonia from those with mild cases. The AUC was calculated at 0.894 (95% CI: 0.855\u0026ndash;0.934), with a sensitivity of 80.8% and a specificity of 82% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on the median expression levels of miR-493-5p, children with severe pneumonia were stratified into a high expression group (n\u0026thinsp;=\u0026thinsp;66) and a low expression group (n\u0026thinsp;=\u0026thinsp;59). A Kaplan-Meier curve depicting the survival status of these children at 28 days post-birth was constructed, and a Log-rank test was conducted. It was found that the 28-day cumulative survival rate for the high miR-493-5p expression group was substantially lower than that of the low expression group, with the difference attaining statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Additionally, multivariate COX regression analysis identified the expression level of miR-493-5p and Procalcitonin (PCT) as significant prognostic factors within the severe pneumonia group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). While neutrophils were noted to potentially influence the prognosis of children suffering from severe pneumonia, this did not reach statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings suggest that elevated levels of miR-493-5p may be intricately linked to both the development and adverse prognosis of severe pneumonia in pediatric patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCox regression analysis of the association between clinical parameters and survival in patients with severe pneumonia\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e95% CI for HR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLower\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUpper\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emiR-493-5p expression\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.016*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.929\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.565\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.825\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.135\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.353\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.045*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.272\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.553\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.699\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.620\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eBMI, Body Mass Index; WBC, white blood cell; CRP, C-reactive protein; PCT, Procalcitonin; LDH, Lactate dehydrogenase. * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSerum miR-493-5p correlates with levels of inflammatory factors in severe pneumonia\u003c/h2\u003e \u003cp\u003ePearson correlation analysis revealed a significant positive correlation between serum miR-493-5p levels and the pro-inflammatory cytokines IL-6 (r\u0026thinsp;=\u0026thinsp;0.715), TNF-α (r\u0026thinsp;=\u0026thinsp;0.787), and IL-1β (r\u0026thinsp;=\u0026thinsp;0.759) in children afflicted with severe pneumonia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). These findings imply that miR-493-5p may play a pivotal role in the inflammatory response associated with severe pneumonia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of miR-493-5p on LPS-induced MRC-5 cells\u003c/h2\u003e \u003cp\u003eThe CCK-8 assay demonstrated that LPS treatment markedly inhibited the viability of MRC-5 cells, with a noticeable decline in cell viability corresponding to the increasing concentrations of LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Further RT-qPCR showed that the expression of miR-493-5p in MRC-5 cells was increased after LPS treatment, and the level showed an upward trend with the increase of LPS concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. We subsequently established cell models characterized by either overexpression or knockdown of miR-493-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Cell viability and apoptosis assays indicated that the overexpression of miR-493-5p led to a reduction in cell viability and an enhancement of apoptosis in the pneumonia model cells, whereas the knockdown of miR-493-5p yielded opposing effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E\u003cb\u003e)\u003c/b\u003e. Moreover, the overexpression of miR-493-5p was associated with an increase in the production of inflammatory factors within the pneumonia model cells, while the knockdown of miR-493-5p exhibited the reverse effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e. These showed that miR-493-5p affected the function and inflammation of pneumonia model cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTarget correlation between miR-493-5p and IRF1\u003c/h2\u003e \u003cp\u003eThe binding site of miR-493-5p to IRF1 is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. This targeting relationship between miR-493-5p and IRF1 was further corroborated through a dual luciferase reporter gene assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. In MRC-5 cells treated with LPS, the expression of IRF1 exhibited a decline, demonstrating a downward trend in correlation with increasing concentrations of LPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Within the pneumonia model cells, overexpression of miR-493-5p led to a suppression in IRF1 levels, whereas the knockdown of miR-493-5p produced an opposing effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The expression level of IRF1 in the severe pneumonia group was elevated compared to both the mild pneumonia group and the healthy control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cb\u003e)\u003c/b\u003e. Pearson correlation analysis revealed a significant negative correlation between miR-493-5p and IRF1 expression in children suffering from severe pneumonia (r= -0.722, P\u0026lt;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of miR-493-5p targeting IRF1 on LPS-induced MRC-5 cells\u003c/h2\u003e \u003cp\u003eTo delve deeper into the impact of miR-493-5p in modulating IRF1 within pneumonia model cells, we conducted co-transfection experiments to restore IRF1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. The CCK-8 cell viability assay revealed that IRF1 effectively mitigated the inhibitory effects of miR-493-5p on the viability of LPS-induced MRC-5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Apoptosis assay showed that IRF1 could partially counteract the pro-apoptotic influence of miR-493-5p under LPS stimulation in MRC-5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Furthermore, IRF1 was shown to effectively suppress the enhancing effect of miR-493-5p on the LPS-induced production of inflammatory factors in MRC-5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Collectively, these findings suggest that miR-493-5p may influence the progression and inflammatory response associated with severe pneumonia by modulating the expression of IRF1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSevere pneumonia represents a prevalent and critical illness in pediatrics, characterized by challenging clinical management, rapid progression, poor prognostic outcomes, and elevated mortality rates (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). An early and thorough evaluation of a patient's condition and prognosis is essential for devising an effective treatment strategy that can enhance patient outcomes. Clinical research has demonstrated that elevated expression levels of miR-10a-3p are observed in patients suffering from severe pneumonia, suggesting its potential role as a diagnostic and prognostic biomarker for this condition (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Our investigation reveals that serum levels of miR-493-5p are markedly elevated in children with severe pneumonia, exhibiting a positive correlation with inflammatory response levels in these patients. This finding shows that miR-493-5p may serve as a valuable tool for assessing the severity and prognosis of severe pneumonia in pediatric patients. The overexpression of miR-493-5p appears to be associated with severe pneumonia, underscoring its potential as an early diagnostic and prognostic marker in clinical settings.\u003c/p\u003e \u003cp\u003eInflammation represents a fundamental pathological mechanism in patients experiencing severe pneumonia (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). In this context, inflammatory cytokines play a pivotal role in the progression and severity of the disease. IL-6 serves as a key mediator of the body's inflammatory response, acting as a critical transmitter within this process (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). IL-1β is produced by cell types such as fibroblasts during infection events and plays an important role in inflammatory responses by promoting the activation, proliferation, and differentiation of B cells (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). TNF-α, synthesized by activated macrophages and monocytes, exhibits a range of complex biological activities and is integral to the regulation of infection, inflammation, and immune responses (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Elevated levels of IL-6, IL-1β, and TNF-α have been consistently observed in cases of pneumonia (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). MiRNAs have been closely associated with the regulation of the inflammatory response. Recent studies have demonstrated that miR-155 exerts significant biological functions across various inflammatory cell types (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). MiR-146b has been implicated in the inflammatory injury associated with pneumonia in pediatric patients (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Additionally, findings indicate that miR-195-5p is involved in cell proliferation, apoptosis, and the inflammatory responses triggered by LPS (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In our investigation, we found that overexpressing miR-493-5p inhibited LPS-induced MRC-5 cell proliferation and promoted cell apoptosis and the production of inflammatory factors. This suggests that miR-493-5p may have a regulatory role in the context of severe pneumonia.\u003c/p\u003e \u003cp\u003eIRF1 is a pivotal molecule that governs downstream inflammatory responses and apoptosis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Accumulating evidence highlights the critical role of IRF1 in antiviral immunity (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Notably, prior researches have established that miR-130b-3p influences the polarization of M1 macrophages by targeting IRF1 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Furthermore, research has demonstrated that miR-370-3p modulates LPS-induced acute pneumonia by targeting TLR4 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). MiR-429 exacerbates inflammatory injury in neonatal pneumonia through the targeting of KLF4 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Our findings indicate that the overexpression of miR-493-5p substantially impacts LPS-induced functional alterations and inflammatory responses in MRC-5 cells via the down-regulation of IRF1. This implicates the targeting of IRF1 by miR-493-5p as a contributing factor in the progression of severe pneumonia.\u003c/p\u003e \u003cp\u003eThe limitations of our study include the singularity of patient inclusion from a single center, which may influence the overall robustness of our findings. The exploration of miR-493-5p in conjunction with additional biomarkers for the diagnosis of severe pneumonia warrants further investigation to enhance clinical diagnostic reference. Additionally, the application of serum miR-493-5p expression levels to inform individualized treatment strategies for children suffering from severe pneumonia remains ambiguous. Furthermore, the specific mechanisms by which miR-493-5p participates in the pathophysiology of severe pneumonia require more in-depth research. These aspects will constitute the focal points of our future investigations.\u003c/p\u003e \u003cp\u003eIn conclusion, the level of serum miR-493-5p was increased in children with severe pneumonia and was associated with the prognosis of pneumonia. It may be used as a molecular marker for early diagnosis of severe pneumonia and a potential therapeutic target. This study provides a new reference for the clinical study of severe pneumonia in children and the improvement of prognosis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eStatement of author contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Study concept and design: L.L. L., and L.Y. W.; analysis and interpretation of data: L.L. L., and L. H.; drafting of the manuscript: L.L. L.; critical revision of the manuscript for important intellectual content: L.L. L., L. H., and L.Y. W.; statistical analysis: L.L. L. and H. Z..\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eIshiwada N., Shinjoh M., Kusama Y., Arakawa H., Ohishi T., Saitoh A., Suzuki A., Tsutsumi H., Nishi J., Hoshino T., Mitsuda T., Miyairi I., Iwamoto-Kinoshita N., Kobayashi H., Satoh K., Shimizu A., Takeshita K., Tanaka T., Tamura D., Tokunaga O., Tomita K., Nagasawa K., Funaki T., Furuichi M., Miyata I., Yaginuma M., Yamaguchi Y., Yamamoto S., Uehara S., Kurosaki T., Okada K., and Ouchi K., The Pediatric infectious disease journal 42, e369-e376, 2023.\u003c/li\u003e\n\u003cli\u003eXie W., Ruan J., Jiang Q., Zheng J., Lin W., and Lyu G., Frontiers in pediatrics 12, 1411365, 2024.\u003c/li\u003e\n\u003cli\u003eYang S., Lu S., Guo Y., Luan W., Liu J., and Wang L., BMC infectious diseases 24, 449, 2024.\u003c/li\u003e\n\u003cli\u003eGong W., Gao K., Shan Z., Yang L., Fang P., Li C., Yang J., and Ni J., Frontiers in pediatrics 12, 1417644, 2024.\u003c/li\u003e\n\u003cli\u003eZhang K., Yang X.M., Sun H., Cheng Z.S., Peng J., Dong M., Chen F., Shen H., Zhang P., Li J.F., Zhang Y., Jiang C., Huang J., Chan J.F., Yuan S., Luo Y.S., and Shen X.C., Antiviral research 231, 106007, 2024.\u003c/li\u003e\n\u003cli\u003eGoyal J.P., Kumar P., Mukherjee A., Das R.R., Bhat J.I., Ratageri V., Vyas B., and Lodha R., Indian pediatrics 58, 1036-1039, 2021.\u003c/li\u003e\n\u003cli\u003eGui Y., Sun L., Liu R., and Luo J., Allergologia et immunopathologia 49, 87-93, 2021.\u003c/li\u003e\n\u003cli\u003eSpecjalski K., and Niedoszytko M., Current opinion in pulmonary medicine 26, 285-292, 2020.\u003c/li\u003e\n\u003cli\u003eAlipoor S.D., Adcock I.M., Garssen J., Mortaz E., Varahram M., Mirsaeidi M., and Velayati A., European journal of pharmacology 791, 395-404, 2016.\u003c/li\u003e\n\u003cli\u003eLobera E.S., Varela M.A., Jimenez R.L., and Moreno R.B., Molecular biology reports 50, 9521-9527, 2023.\u003c/li\u003e\n\u003cli\u003eGalv\u0026aacute;n-Rom\u0026aacute;n J.M., Lancho-S\u0026aacute;nchez \u0026Aacute;., Luquero-Bueno S., Vega-Piris L., Curbelo J., Manzaneque-Pradales M., G\u0026oacute;mez M., de la Fuente H., Ortega-G\u0026oacute;mez M., and Aspa J., PloS one 15, e0240926, 2020.\u003c/li\u003e\n\u003cli\u003eLi Q., Wu T., and Li S., Genetics and molecular biology 44, e20200431, 2021.\u003c/li\u003e\n\u003cli\u003eLiang Z., Kong R., He Z., Lin L.Y., Qin S.S., Chen C.Y., Xie Z.Q., Yu F., Sun G.Q., Li C.G., Fu D., Jiang G.X., Chen J., and Ma Y.S., Oncotarget 8, 47389-47399, 2017.\u003c/li\u003e\n\u003cli\u003eHuang F., Zhang J., Yang D., Zhang Y., Huang J., Yuan Y., Li X., and Lu G., Mediators of inflammation 2018, 2320640, 2018.\u003c/li\u003e\n\u003cli\u003eTang X., Wang T., Qiu C., Zheng F., Xu J., and Zhong B., Medical science monitor : international medical journal of experimental and clinical research 26, e920928, 2020.\u003c/li\u003e\n\u003cli\u003eGowardman J., and Trent L., The New Zealand medical journal 113, 161-164, 2000.\u003c/li\u003e\n\u003cli\u003eXie J., Li Y., Wang M., He W., and Zhao X., Journal of inflammation research 15, 6097-6104, 2022.\u003c/li\u003e\n\u003cli\u003eFernandez J.F., and Restrepo M.I., Respirology (Carlton, Vic) 18, 889-890, 2013.\u003c/li\u003e\n\u003cli\u003eUnver N., and McAllister F., Cytokine \u0026amp; growth factor reviews 41, 10-17, 2018.\u003c/li\u003e\n\u003cli\u003eChen X., OuYang L., Qian B., Qiu Y., Liu L., Chen F., Jiang W., Zheng M., Hu Z., Min X., Wen L., Wang Q., Yu D., Jia S., Lu Q., and Zhao M., Clinical immunology (Orlando, Fla) 269, 110396, 2024.\u003c/li\u003e\n\u003cli\u003eAl-Rashed F., Ahmad Z., Thomas R., Melhem M., Snider A.J., Obeid L.M., Al-Mulla F., Hannun Y.A., and Ahmad R., Scientific reports 10, 16802, 2020.\u003c/li\u003e\n\u003cli\u003eMyers M.J., Farrell D.E., Snider T.G., 3rd, and Post L.O., Pathobiology : journal of immunopathology, molecular and cellular biology 71, 35-42, 2004.\u003c/li\u003e\n\u003cli\u003eHu J., Huang S., Liu X., Zhang Y., Wei S., and Hu X., Journal of immunology research 2022, 7437281, 2022.\u003c/li\u003e\n\u003cli\u003eZhang L., Dong L., Tang Y., Li M., and Zhang M., Infectious diseases (London, England) 52, 23-32, 2020.\u003c/li\u003e\n\u003cli\u003eZheng Y., Wang Y., Zhu X., Diao Y., Chen Y., and Chen G., Bioscience, biotechnology, and biochemistry 85, 1639-1649, 2021.\u003c/li\u003e\n\u003cli\u003eSharma B.R., Karki R., Rajesh Y., and Kanneganti T.D., The Journal of biological chemistry 299, 105141, 2023.\u003c/li\u003e\n\u003cli\u003eZhou H., Tang Y.D., and Zheng C., Cytokine \u0026amp; growth factor reviews 64, 1-6, 2022.\u003c/li\u003e\n\u003cli\u003eGuo Q., Zhu X., Wei R., Zhao L., Zhang Z., Yin X., Zhang Y., Chu C., Wang B., and Li X., Journal of cellular physiology 236, 2008-2022, 2021.\u003c/li\u003e\n\u003cli\u003eYu X., Qian X., Sun R., Yang B., Zheng H., Jiang P., and Li X., Panminerva medica 64, 121-122, 2022.\u003c/li\u003e\n\u003cli\u003eZhang L., Yan H., Wang H., Wang L., Bai B., Ma Y., Tie Y., and Xi Z., Current neurovascular research 17, 102-109, 2020.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"severe pneumonia, miR-493-5p, diagnosis, prognosis","lastPublishedDoi":"10.21203/rs.3.rs-5769928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5769928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eA large number of studies have shown that microRNAs (miRNAs) are closely associated with pneumonia.\u003c/p\u003e\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003eThis study endeavors to explore the expression levels and clinical significance of miR-493-5p in pediatric patients diagnosed with severe pneumonia.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA total of 125 children diagnosed with severe pneumonia and 100 children with common pneumonia were enrolled in this study, alongside 100 healthy children who served as controls. Real-time quantitative PCR was used to was used to detect gene expression levels. The receiver operating characteristic curve was used to evaluate the diagnostic value of miR-493-5p in children with severe pneumonia. Kaplan-Meier and COX regression analyses were used to evaluate the prognostic value of miR-493-5p in children with severe pneumonia. Pearson correlation analysis was used for correlation analysis. Furthermore, pneumonia model cells were constructed in vitro to explore the underlying mechanism of miR-493-5p in severe pneumonia.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSerum miR-493-5p was highly expressed in children with severe pneumonia. miR-493-5p has a certain diagnostic value for severe pneumonia and is related to the prognosis of the disease. miR-493-5p is positively correlated with the levels of inflammatory factors in children with severe pneumonia. miR-493-5p affects the function and inflammation of pneumonia model cells. IRF1 could partially reverse the effect of miR-493-5p on MRC-5 cell.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe level of miR-493-5p has a certain value in the diagnosis and prognosis of children with severe pneumonia, and it may play a critical role in the disease process and associated inflammatory responses by targeting the IRF1.\u003c/p\u003e","manuscriptTitle":"Diagnostic and prognostic value of deregulated miR-493-5p in patients with severe pneumonia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 12:51:32","doi":"10.21203/rs.3.rs-5769928/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ec213e8c-3bd1-4ce6-b5e2-a8c4023c7b25","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-14T15:08:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 12:51:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5769928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5769928","identity":"rs-5769928","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}