The Clinical Significance and the Potential Regulatory Mechanism of the LncRNA OIP5-AS1 in Paediatric Severe Community-Acquired Pneumonia Blood Through the MiR-150-5p/PDCD4 Axis

ABSTRACT Background This study aimed to elucidate the clinical significance and regulatory mechanism of the long non-coding RNA OIP5-AS1 in severe community-acquired pneumonia (SCAP) among paediatric patients. Methods qRT-PCR was used to assess the mRNA levels of OIP5-AS1. ROC curve analysis was used to assess the diagnostic significance of OIP5-AS1. Short-term prognostic significance was evaluated through Kaplan-Meier survival. An in vitro cell model was developed using LPS-induced MRC-5 cells. CCK-8, flow cytometry, and ELISA were conducted to measure cell viability, apoptosis, and inflammatory factor levels. The association between miR-150-5p and PDCD4 was confirmed through DLR assays. Results Elevated OIP5-AS1 were observed in paediatric patients with SCAP, which enabled effective differentiation from healthy individuals. High expression of OIP5-AS1 correlated with reduced survival rates. OIP5-AS1 knockdown attenuated cell viability suppression and the promotion of apoptosis and inflammatory factors induced by LPS. However, this attenuation was reversed by reduced levels of miR-150-5p. miR-150-5p was identified as a target of PDCD4 and OIP5-AS1. Conclusion Increased OIP5-AS1 levels show potential as a valuable diagnostic and prognostic biomarker for paediatric patients with SCAP. This study illustrates its role in regulating cell viability, apoptosis, and the inflammatory response via the miR-150-5p/PDCD4 axis, acting as a ceRNA.


Introduction
Pneumonia, an inflammatory immune disorder caused by invasive pathogens (Fan et al., 2023), affects over 150 million children annually (Mekuria et al., 2023), with 11-200 million experiencing severe disease necessitating hospitalization.Unfortunately, approximately 5% hospitalisatio10% of hospitalized individuals succumb to pneumonia within 30 days of hospitalization (Feng et al., 2021).Severe community-acquired pneumonia (SCAP) represents the most prevalent and severe manifestation, carrying a mortality rate of 30-50% in intensive care units (Q.Li et al., 2021).Secondary complications such as sepsis, septic shock, and acute respiratory distress syndrome (ARDS) frequently accompany it.Despite advancements in prevention and treatment strategies, paediatric pneumonia recurs and leads to complications due to delayed or incomplete treatment.
This study aimed to investigate the levels of OIP5-AS1 in children with severe pneumonia.Furthermore, to further elucidate the diagnostic and prognostic significance of OIP5-AS1.Finally, further investigation of the molecular mechanisms of OPI5-AS1 in pneumonia cell models induced by LPS.

Experimental design
The current research is mainly divided into three stages.The first stage involves examining the expression of OIP5-AS1 in the subjects using RT-qPCR.Subsequently, the diagnostic and prognostic value of OIP5-AS1 in SCAP patients is verified through the chi-square test, receiver operating characteristic (ROC), and Kaplan-Meier method.The second step involves exploring the mechanism by which OIP5-AS1 affects SCAP through an in vitro cell model.In this, CCK-8, ELISA kit, and flow cytometry were used to respectively assess the cell viability, inflammation, and apoptosis levels after inhibiting OPI5-AS1.In the third step, analysis predicted the target miRNA of OIP5-AS1 as miR-150-5p using the online in silico tool the Encyclopedia of RNA interactomes (ENCORI).Subsequently, further predictions of the target gene of miR-150-5p as programmed cell death 4 (PDCD4) were made through miRDB.After dual-luciferase reporter and RIP kit validation, further confirmation was conducted in clinical subjects and in vitro cell models.

Ethics statement and subjects
Ethical statement: The research protocol of this retrospective study complies with the Declaration of Helsinki and has been approved by the Ethics Committee of Xingtai People's Hospital (Approval number: 2020[03]).Written informed consent was obtained from the legal guardians of all participants.This study is retrospective.The statistical power of this study was 80%, with a one-sided significance level of 5%.Assuming a 15% dropout or loss to follow-up, a minimum of 82 patients' samples would be required (Yan et al., 2019).
A total of 108 children diagnosed with SCAP and admitted to the intensive care unit at Xingtai People's Hospital from January 2020 and June 2021 were included in the study.They met the severe pneumonia diagnostic criteria outlined in the "Guidelines for Management of Community-Acquired Pneumonia in Children (Revised)" (Subspecialty Group of Respiratory Diseases TSoPCMA, Editorial Board CJoP, 2013): which are as follows: a) symptoms including fever, cough, increased respiratory rate, dyspnoea, and cyanosis; b) presence of patchy shadows, increased lung texture, and dry rales on chest radiographs (Yu et al., 2019); c) age ranging from 1 month to 14 years; d) complete clinical data availability.Additionally, exclusion criteria for all subjects were: a) received immunotherapy; b) experienced complications such as chronic obstructive pulmonary disease (COPD), asthma, chronic liver disease, severe acute respiratory syndrome coronavirus 2 infection, hypertension, malignancies, or congenital diseases; c) had bronchial foreign body tuberculosis infection; or d) had hematological diseases.Simultaneously, 90 healthy children matched for sex and age, visiting our hospital for routine physiological examinations were chosen as the control group.The baseline clinical and demographic characteristics are outlined in Table 1.Fasting inferior venous blood samples (10 mL) were collected, centrifuged to obtain serum, and then stored.

Follow-up program
All children diagnosed with severe pneumonia underwent a 28-day follow-up (Ni et al., 2022).They were categorized into two groups based on their serum OPI5-AS1 messenger RNA (mRNA) expression levels: the OIP5-AS1 high expression group and the OIP5-AS1 low expression group.Kaplan-Meier analysis was performed to assess the relationship between short-term prognosis and OIP5-AS1 levels.

Cell culture and pneumonia cell model construction
MRC-5 were cultured in DMEM containing 10% FBS, maintained in an incubator at 37°C, and 5% carbon dioxide.The pneumonia cell model was established through LPS induction, following established protocols.Specifically, MRC-5 cells were subjected to LPS treatment at concentrations of 0, 5, 10, and 20 μg/mL for 12 h.

Cell transfection
After LPS induction, MRC-5 cells were seeded into six-well plates and cultured overnight.
Transfection was performed with the transfection reagent lipofectamine 2000 mixed with si-OIP5-AS1, si-NC, miR-150-5p inhibitor, and inhibitor NC alone in a six-well plate.The culture medium was changed after 6 h.

Quantitative reverse transcription -polymerase chain reaction (qRT-PCR)
A qRT-PCR assay was performed to verify OIP5-AS1 and microRNA (miRNA) levels.In summary, total RNA extraction from patients' serum and cell lines was accomplished using the miRNeasy Serum/Plasma Kit and miRNeasy Kit, respectively.Following RNA extraction, the purity and concentration of the extracted RNA were assessed.Subsequently, the superscript III First Strand cDNA Synthesis Kit and miScript II RT kit were employed to convert the RNA into complementary DNA (cDNA) through reverse transcription.For qRT-PCR amplification, SYBR Green qPCR SuperMix-UDG or miScript SYBR Green PCR Kit, in combination with primers, cDNA, and double-distilled water was used.The relative lncRNA and miRNA levels were determined using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 as internal controls, normalized, and quantified using the 2 −ΔΔCt method.

Cell viability assay
A 100 μL resuspension comprising MRC-5 cells (2 × 10 3 ) was prepared and added to a 96well plate.Cell viability was monitored daily up to the third day.At the same time intervals, the cell culture medium was substituted with a combination of the CCK-8 reagent and DMEM in a 1:9 ratio.Following a 1-h incubation at 37°C, the absorbance value at OD450 nm was measured.

Apoptosis analysis
LPS-treated MRC-5 cells were gathered, and the apoptosis rate was evaluated using the Annexin V -fluorescein isothiocyanate (FITC) apoptosis detection kit.Specifically, a mixture of 5 μL Annexin V-FITC was prepared, and cells were suspended in this solution mixture.Following this, the plate was incubated in darkness for 5 min.Subsequently, the mixture was resuspended in 500 μL of binding buffer for the analysis of apoptosis levels using flow cytometry.

Enzyme-linked immunosorbent assay
The levels of the inflammatory factors IL-6, IL-10, and TNF-α in the serum and MRC-5 cell supernatants were quantified using an ELISA kit.In summary, the samples and protein standards were appropriately diluted and added to the plate.Following this, they underwent incubation with horseradish peroxidase-coupled detection antibodies.Subsequently, a termination solution was added, and the absorbance was measured at 450 nm.

Nuclear/Cytoplasmic fractionation assay
The nuclear/cytoplasmic fractionation assay was performed using the commercial PARIS kit .Cells were collected using pre-cooled phosphate-buffered saline (PBS) and then treated with cell grading buffer (450 μL) on ice.The upper cytoplasmic RNA was obtained after centrifugation.The precipitated fraction was resuspended with PBS, nuclear isolation buffer, and RNase-free water followed by a minute of incubation to collect the nuclear RNA.The distribution of OIP5-AS1 was assessed through qRT-PCR assay using U6 and GAPDH as controls for the nuclear and cytoplasmic RNA, respectively.
Radioimmunoprecipitation assay lysate was added to the cells for 5 min of lysis on ice.The resulting supernatant was then collected and incubated overnight at 4°C with 5 μL of the specified antibodies (protein argonaute-2 [Ago2] and NC immunoglobulin G [IgG]) at 4°C.Proteinase K buffer was employed to digest the samples and extract RNA for subsequent qRT-PCR assay.

Statistical analysis
The experiments were performed in triplicate, and the results are presented as the mean ± standard deviation.Statistical comparisons between two or more groups were conducted using Student's t-test and analysis of variance, followed by Tukey post hoc analysis.SPSS 23.0 or GraphPad Prism 7.0 was used for calculating statistically significant differences.ROC analysis and relative area under the curve (AUC) statistics were used, and the ratio closest to the point with the maximum sensitivity and specificity was selected as the optimal cut-off value.Multivariate Cox hazard regression models were used, and the hazard ratio (HR) with the corresponding 95% confidence interval (CI) was utilized for description.Kaplan-Meier and log-rank tests were employed to evaluate patient survival, with statistical significance set at p < .05.

Demographic and clinical data of the participants
As presented in the supplementary table , 108 patients with SCPA were included in the study, with an average age, of 7.38 ± 2.67 years (male/female, 65/43).Furthermore, 90 control participants were enrolled in the study, with an average age of 7.18 ± 1.11 years (male/female, 63/27).Parameters such as age, gender, and lactate dehydrogenase exhibited no statistically significant differences between the groups (p > .05).However, markers including white blood cell (WBC) count, absolute neutrophil count, C-reactive protein (CRP), and procalcitonin (PCT) levels were notably high in patients with SCAP (p < .05).

Poorer prognosis associated with SCAP in paediatric patients with elevated OIP5-AS1 levels
Subsequently, the short-term prognostic value of OIP5-AS1 was explored in these patients.

LPS suppressed cell viability and enhanced apoptosis and inflammation in MRC-5 cells
As the concentration of LPS increased, there was a gradual decrease in MRC-5 cell viability (p < .05, Figure 2a).Concurrently, OIP5-AS1 levels exhibited a dosedependent increase in response to LPS (p < .05, Figure 2b).Considering these findings alongside previous research, a concentration of 10 μg/mL LPS was chosen for subsequent investigation.LPS significantly augmented the apoptosis rate of MRC-5 cells (p < .05, Figure 2c).Additionally, the pro-inflammatory factors TNF-α and IL-6, in terms of concentration and mRNA levels in MRC-5 cells were elevated by LPS, while the anti-inflammatory factor IL-10 was suppressed (p < .05, Figure 2d-e).

Discussion
The significance of OIP5-AS1 in lung disease conditions has received significant attention.Moreover, OIP5-AS1 has been identified as a suppressor of non-small cell lung cancer cell proliferation, acting through the inhibition of the G1 cell cycle (Kotake et al., 2021).In ARDS, a prevalent complication of SCAP (R. Li et al., 2023), OIP5-AS1 exhibited significant upregulation and correlation with cellular damage (Ji et al., 2022).Similarly, in sepsis, another frequent complication of SCAP, OIP5-AS1 consistently correlated with ALI.Additionally, OIP5-AS1 has demonstrated the ability to stimulate inflammatory responses across various cell types.For instance, its suppression alleviates inflammation induced by oxidized-low-density lipoprotein in human umbilical vein endothelial cells atherosclerosis (Ren et al., 2020).Furthermore, OIP5-AS1 enhances the inflammatory response triggered by house dust mites in bronchial epithelial cells (Cai et al., 2020).Collectively, these findings highlight the potentially crucial role of OIP5-AS1 in pneumonia.To substantiate our hypothesis, the serum OIP5-AS1 levels were examined in paediatric patients with SCAP, which were significantly elevated, aligning with the levels observed in common pulmonary and pneumonia-related complications.
Previous studies have underscored the potential of lncRNA as a prognostic and diagnostic biomarker in patients with pneumonia.For instance, increased maternally expressed 3 expression serves as a prognostic biomarker in severe paediatric pneumonia (Guo et al., 2021).Moreover, the diagnostic significance of OIP5-AS1 in COPD (Hao et al., 2021) and multiple sclerosis (Gharesouran et al., 2019) has been validated.Thus, the potential clinical relevance of OIP5-AS1 in paediatric SCAP was analyzed.As anticipated, serum OIP5-AS1 effectively distinguished paediatric patients with SCAP from their healthy counterparts, demonstrating promising diagnostic utility in our study.Currently, diagnosing SCAP relies on clinical assessments and laboratory tests including chest radiography.In suspected bacterial infections, acute blood cultures and complete cell counts are recommended.However, establishing the etiology of most bacterial community-acquired pneumonia (CAP) cases remains challenging, given that routine lung tissue culture is uncommon, and pneumonia is typically attributed to a blend that often results from a mix of viral and bacterial infections.Furthermore, severe pneumonia is characterized by an acute onset and rapid progression, often leading to pediatric fatalities due to delayed or ineffective treatment.Our investigation suggests the possibility of alternative molecules serving as biomarkers for pediatric SCAP.
Data from a prospective multicentre trial revealed an overall mortality rate of 17.3% among patients with CAP during an 18-month follow-up (Hermann et al., 2020).Another study highlighted a high 28-day mortality rate of 13.6% in patients with CAP (Acar et al., 2021).It is worth noting that these statistics are specific to adult patients, while children, due to their immature immune systems, are more susceptible to pathogenic infections and exhibit poorer prognoses.In our investigation into the short-term (28-day) prognosis of paediatric patients with SCAP, increased OIP5-AS1 expression was associated with a poorer prognosis.Both OIP5-AS1, PLR, and PSI scores were identified as independent risk factors for poor prognosis.Briefly, the prognostic relevance of OIP5-AS1 in paediatric patients with SCAP was validated.The use of LncRNA as a clinical biomarker offers a certain degree of stability, accessibility, cost-effectiveness, and non-invasiveness.This suggests that utilizing OIP5-AS1 as a biomarker for SCAP diagnosis or prognosis holds certain advantages.
LPS, a potent endotoxin derived from Gram-negative bacteria, induces severe cellular damage (S.Wang et al., 2023).Currently, LPS-induced lung fibroblasts serve as a widely adopted cellular model for pneumonia, employed in studies focusing on pathogenesis and pharmacological efficacy.An in vitro cell model was established through LPS induction in MRC-5 to further understand the functional mechanism of OIP5-AS1 in pneumonia.This confirmed a general increase in OIP5-AS1 levels.Moreover, it was observed that OIP5-AS1 knockdown notably mitigated the inhibitory and apoptosis-promoting effects of LPS on MRC-5 cell activity.As an inflammatory condition, LPS promoted an increase in the protein and mRNA levels of pro-inflammatory factors IL-6 and TNF-α, while inhibiting the expression of the pro-inflammatory factor IL-10. OIP5-AS1 knockdown significantly restrained their expression.
LncRNAs possess the ability to modulate miRNAs, effectively acting as miRNA sponges to regulate mRNA levels.miR-150-5p has been associated with the transformation of lung fibroblasts into myofibroblasts and the onset of iron death (Y.Yang et al., 2020).Small extracellular vesicles, inclusive of miR-150-5p, have shown potential in distinguishing between benign and malignant indeterminate lung nodules (Zheng et al., 2022).Abnormal expression of miR-150-5p serves as an indicator of sepsis severity (Ye et al., 2023).Dysregulated miR-150-5p has been identified in malignant pleural effusions, which are common clinical complications of various pulmonary or systemic diseases such as lung cancer and tuberculosis (Shojaee et al., 2022).Notably, plasma miR-150-5p levels in patients with coronavirus disease 2019 declined significantly, exacerbating severe acute respiratory syndrome coronavirus 2 infection (Akula et al., 2022).Moreover, Huang et al. identified abnormal miR-150-5p expression in children with adenovirus-infected pneumonia (Huang et al., 2018).In our investigation, OIP5-AS1 was reported as a sponge for miR-150-5p, suppressing its expression in LPS-induced MRC-5 cells.The connection between miR-150-5p and OIP5-AS1 was confirmed through RIP and DLR assays.Importantly, miR-150-5p levels exhibited a negative correlation with OIP5-AS1 levels in paediatric patients with SCAP.Additionally, it was observed that the mitigating effects of OIP5-AS1 knockdown on LPS-induced cytotoxicity were partially counteracted by suppressed miR-150-5p.
Additionally, we conducted an in-depth exploration into the actions of miR-150-5p was conducted.PDCD4 was identified as a target gene of miR-150-5p.PDCD4 levels were significantly elevated in paediatric patients with SCAP, exhibiting a positive correlation with OIP5-AS1 and a negative correlation with miR-150-5p levels in these patients.LPS induction triggered an increase in PDCD4 levels, typically suppressed by OIP5-AS1 knockdown.However, decreased miR-150-5p expression partially restored PDCD4 levels.The targeted binding of miR-150-5p to PDCD4 has been demonstrated in Alzheimer's disease (Chia et al., 2022) and hypoxia-induced endometriosis (Chen et al., 2023).Furthermore, PDCD4's role in inflammation regulation (Liu et al., 2021) was confirmed.Notably, consistent with our findings, Zhou et al. also reported a significant elevation of PDCD4 in paediatric patients with pneumonia and LPS-treated MRC-5 cells (Zhou et al., 2021).The present study has the following undeniable limitations in analyzing the role of the OIP5-AS1/miR-150-5p/PDCD4 axis in the regulation of SCAP.First, this was a single-center retrospective study with a limited sample size.There may be bias in some of the results.In addition, the lack of in vivo animal data is another limitation of this study at this stage.Furthermore, cell invasion and metastasis, which are important hallmarks of malignant disease progression, were not focused on in this study.In future research, we will expand the sample size and validate the clinical value of OIP5-AS1 in SCAP across multiple research centers.Furthermore, we will delve into the molecular mechanisms and signaling pathways through animal model studies to further explore the impact of OIP5-AS1 on SCAP.Additionally, the effect of OIP5-AS1 on cell migration and invasion will also be carried out in subsequent studies.
In summary, we first discovered that elevated serum OIP5-AS1 is a potential diagnostic and prognostic biomarker for paediatric SCAP.Functioning as a competing endogenous RNA, OIP5-AS1 was also first found to regulate cell viability, apoptosis, and inflammatory response through the miR-150-5p/PDCD4 axis, participating in the progression of SCAP.Consequently, the elimination of OIP5-AS1 might serve as a protective intervention for paediatric SCAP.This preliminary study broadens the scope of potential targets for treating paediatric SCAP.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Expression and clinical significance of long non-coding ribonucleic acid opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in the serum of paediatric patients with severe community-acquired pneumonia (SCAP).(a) Quantitative reverse transcription -polymerase chain reaction was performed to assess serum OIP5-AS1 expression levels in the participants.(b) Receiver operating characteristic curve analysis to evaluate the diagnostic significance of OIP5-AS1 in paediatric patients with SCAP.(c) Kaplan-Meier analysis illustrating the survival differences between the high OIP5-AS1 expression and low OIP5-AS1 expression groups during the short-term (28 days) follow-up.****p < .0001,compared with the controls (unpaired student's test or Kaplan-Meier analysis).

Figure 2 .
Figure 2. Lipopolysaccharide (LPS) increases the mRNA level of long non-coding ribonucleic acid opainteracting protein 5 antisense RNA 1 (OIP5-AS1) in Medical Research Council cell strain 5 (MRC-5)cells and regulates cell viability, apoptosis, and inflammation.(a) Cell counting kit-8 was used to examine the effects of LPS on MRC-5 cells.(b) Quantitative reverse transcription -polymerase chain reaction assay detected the regulation of LPS on OIP5-AS1 mRNA expression levels in MRC-5.Flow cytometry (c), enzyme-linked immunosorbent assay (d), and quantitative reverse transcription -polymerase chain reaction (e) was performed to explore apoptosis, inflammatory factor protein, and inflammatory factor mRNA expression levels, respectively.*p < .05,**p < .01,and ***p < .001 vs.Control (ANOVA followed by Tukey's multiple comparison, or Student's T test).

Table 1 .
Correlation between LncRNA OIP5-AS1 levels and clinical features in SCAP patients.
WBC, white blood cells, CRP, C-reactive protein; LDH, Lactate dehydrogenase; PCT, procalcitonin; PSI, pneumonia severity index; PLR, platelet to lymphocyte ratio.χ2 test was used to analyze the relationship between OIP5-AS1 expression and clinical parameters of patients.

Table 2 .
Correlation between clinical parameters and survival of SCAP by multivariate COX analysis.
WBC, white blood cells, CRP, C-reactive protein; LDH, Lactate dehydrogenase; PCT, procalcitonin; PSI, pneumonia severity index; PLR, platelet to lymphocyte ratio.Cox proportional risk regression model was used to evaluate independent prognostic factors.