Clinical significance of LINC01093 in children with severe pneumonia and its regulation of miR‑326/MAPK1 axis

Abstract

Objective

Severe pneumonia (SP) poses a significant threat to the life safety of children. This study aims to investigate the diagnostic and prognostic significance as well as the molecular mechanism of LINC01093 in children with SP.

Methods

Ninety-eight children with SP and ninety-two healthy children were included in the study. The diagnostic potential of LINC01093 was evaluated using the receiver operating characteristic (ROC) curve. The prognosis and the factors influencing the prognosis were analyzed through the Kaplan-Meier survival curve and the multivariate Cox regression model. An in vitro SP model was constructed by inducing MRC-5 cells with lipopolysaccharide (LPS). The relative expression of genes was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability, apoptosis and levels of inflammatory factors were detected by cell counting kit-8 (CCK-8), flow cytometry and Enzyme-linked immunosorbent assay (ELISA).

Results

Compared with healthy children, LINC01093 and MAPK1 were elevated in the serum of children with SP, while miR-326 was reduced. LINC01093 had a good diagnostic potential between children with SP and healthy children. High LINC01093 expression correlated with poor prognosis. Silencing LINC01093 upregulated the viability of MRC-5 cells induced by LPS, inhibited apoptosis and the levels of inflammatory factors (interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α)). Inhibiting miR-326 or overexpressing MAPK1 partially reversed the protective effect of LINC01093 silencing on LPS-induced cell damage.

Conclusion

Silencing LINC01093 exerts a protective effect against LPS-induced damage by regulating the miR-326/MAPK1 axis. This study provides new experimental evidence for understanding the pathogenesis of SP.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12887-026-07148-z.

Introduction

Pneumonia is an inflammatory reaction that occurs in the terminal airways, alveoli and pulmonary interstitium. It is mostly caused by microbial infections such as bacteria, viruses and fungi [1, 2]. The main clinical manifestations include fever, cough, and expectoration [3]. The severity of pneumonia depends on the degree of inflammation throughout the body and locally, as well as the extent of the inflammation’s spread in the lungs [4]. Severe pneumonia (SP) is the critical stage of pneumonia, characterized by acute respiratory failure, circulatory dysfunction and severe hypoxemia, among other symptoms [5]. It is characterized by rapid onset, rapid changes, severe condition, and tends to cause serious complications. It is a common severe infectious disease in children, with a relatively high mortality rate [6, 7]. Currently, the diagnosis and assessment of children with SP mainly rely on clinical symptoms, imaging features, and traditional infection indicators [8]. However, the clinical manifestations in children often lack specificity, and traditional inflammatory markers have certain limitations in terms of accuracy [9]. Therefore, timely diagnosis of SP, accurate assessment of the condition and appropriate treatment play a pivotal role in improving the prognosis of the children.

Long non-coding RNAs (lncRNAs) are non-coding RNAs that are longer than 200 nucleotides and can be transcribed but not translated [10]. A large number of fundamental studies on lncRNAs and pneumonia have been published [11]. These studies have proven that lncRNAs exert a critical effect in the occurrence and development of pneumonia, and some of these lncRNAs have been identified as potential diagnostic markers for pneumonia [12]. The level of CASC2 in the serum of children with SP was decreased. Children with low CASC2 levels had a lower survival rate, and this low level of CASC2 was an independent factor influencing the poor prognosis of children with SP [13]. Previous studies screened a series of abnormally expressed lncRNAs in patients with severe community-acquired pneumonia (SCAP) compared with healthy controls [14]. LINC01093 was upregulated in SCAP. It has been reported that in other diseases, the dysregulation of LINC01093 affects the development of the disease and predicts the prognosis of patients. For instance, LINC01093 was downregulated in liver cell carcinoma (HCC) tissues and was negatively correlated with cancer embolus and tumor node metastasis (TNM) stage. It is a prognostic predictor for HCC patients [15]. LINC01093 was expressed at a low level in mice with alcoholic hepatitis. Overexpression of LINC01093 increased the cell viability and reduced apoptosis [16]. Furthermore, LINC01093 showed a significantly higher expression in the high-altitude pulmonary edema group compared with the control group [17]. Therefore, the dysregulation of LINC01093 in the SCAP is also speculated to be a biomarker for the early detection and prognosis prediction of SP.

This experiment aims to detect the expression of LINC01093 in children with SP and its correlation with clinical characteristics, and to evaluate the diagnostic and prognostic significance of LINC01093. Finally, the potential molecular mechanism of the LINC01093/miR-326/MAPK1 axis in the pathogenesis of SP is further explored, aiming to provide a new theoretical basis for the early identification and targeted intervention of this disease. Based on the above research objectives, we have proposed the following core scientific hypotheses: LINC01093 is upregulated in children with SP. By adsorbing miR-326, it removes the inhibitory effect of miR-326 on its target gene MAPK1, thereby promoting inflammatory damage induced by lipopolysaccharide (LPS); conversely, silencing LINC01093 can alleviate this damage process.

Materials and methods

Study subjects

98 children with SP who were admitted to Ezhou Central Hospital were selected as the research subjects. At the same time, 92 healthy children with gender and age matched to the children with SP were also selected. The diagnosis of SP was based on the PIDS/IDSA criteria [18]. Inclusion criteria: (1) Respiratory failure requiring mechanical ventilation; (2) Hypoxemia; (3) Septic shock; (4) Significantly increased respiratory rate; (5) Refusal to eat or signs of dehydration; (6) Pleural effusion; (7) Extrapulmonary complications; (8) Complete clinical data. Exclusion criteria: (1) Congenital or hereditary diseases; (2) Individuals with abnormal immune function; (3) Patients with impaired or incomplete functions of vital organs such as liver and kidney; (4) Patients with infections in other parts. This study was approved by the Ethics Committee of Ezhou Central Hospital (Approval No. 2023-HS-015). All methods were performed in accordance with relevant guidelines, regulations and the Declaration of Helsinki. Written informed consent was signed by guardians.

Collection of blood samples and clinical data

All the subjects had their fasting venous blood drawn on the morning of the day after enrollment. The blood samples were centrifuged at 3000 rpm for 15 min. Then, the collected serum samples were transferred to a -80℃ refrigerator for subsequent studies. Baseline data of the subjects were retrospectively collected, including age, gender, mechanical ventilation duration, antibiotic use, etiology, and complications. The tests for white blood cells (WBC), neutrophils (NEU) and lymphocytes (LYM) were conducted using an automatic blood analyzer (Mindray, China). The tests for C-reactive protein (CRP), lactate dehydrogenase (LDH) and procalcitonin (PCT) were carried out using an automatic biochemical analyzer (Beckman Coulter, USA). The test for D-dimer (D-D) was performed using an automatic coagulation analyzer (Sysmex, Japan).

Follow-up

A 28-day follow-up was conducted for 98 children with SP who received treatment. The follow-up was mainly carried out through phone calls or outpatient visits. Death was regarded as the endpoint event, and the number of deaths was recorded. Based on the mean expression level of LINC01093, the children with SP were divided into the high LINC01093 group (n = 53) and the low LINC01093 group (n = 45). Based on the follow-up data, the Kaplan-Meier survival curve was plotted and the Log Rank test was conducted. At the same time, the Cox regression model was applied to analyze the independent risk factors affecting the prognosis of the children.

Cell cultivation and model construction

The MRC-5 and A549 cells were obtained from the American Type Culture Collection (USA) and cultured in DMEM medium (Gibco, USA) containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. Based on previous studies, an in vitro pneumonia model was established by inducing cell damage with LPS [19, 20]. Different concentrations of LPS (Sigma-Aldrich, USA) (0, 5, 10, 20 µg/mL) were respectively added to the MRC-5 cell culture medium, and further experiments were conducted after 24 h of incubation.

Cell transfection

The MRC-5 and A549 cells in the logarithmic growth phase were transfected using Lipofectamine 3000 (Invitrogen, USA) as the transfection reagent. Small interfering RNA (si-LINC01093), miR-326 mimic, miR-326 inhibitor, pcDNA-MAPK1 and their negative controls (si-NC, miR-NC, pcDNA) were synthesized by Ribobio (Guangzhou, China).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA of serum and cells was extracted using TRIzol reagent (Takara, Dalian, China). The RNA concentration and purity were detected using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA with an OD260/280 ratio ranging from 1.8 to 2.0 was selected. The integrity of the RNA was verified by agarose gel electrophoresis. The total RNA was reverse-transcribed into complementary DNA using PrimeScript™ RT reagent (Takara). qRT-PCR was performed using TB Green^® Fast qPCR Mix (Takara). GAPDH and U6 were used as internal references for lncRNA, mRNA and miRNA, and the relative expression was calculated according to the 2^−ΔΔCt formula. The primer sequences are presented in Table S1.

Cell viability

Cell viability was measured using the cell counting kit-8 (CCK-8) kit (Dojindo, Tokyo, Japan). MRC-5 and A549 cells (5 × 10³ cells/well) were seeded in a 96-well plate and incubated for 24 h. 10 µL of CCK-8 reagent was added to each well, and the plate was incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a microplate reader.

Cell apoptosis

Cell apoptosis was assessed using the Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Detection Kit (BD Biosciences). The collected cells were washed with phosphate buffered saline (PBS). The cells were resuspended in the binding buffer containing Annexin V-FITC and PI. The cell suspension was incubated at room temperature in the dark for 15 min, and then analyzed using a flow cytometer (BD Biosciences).

Enzyme-linked immunosorbent assay (ELISA)

The supernatant from the processed MRC-5 and A549 cells were collected, and the concentrations of interleukin-1 beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α) were assessed using an ELISA kit (R&D Systems, USA).

Bioinformatics analysis

The differentially expressed lncRNAs were analyzed using the GSE196399 dataset. This dataset included 21 healthy subjects and 56 SCAP patients. According to the disease and control groups, with |log (2 fold change)| > 1 and adjusted P < 0.05, the gene differential expression analysis was conducted using the Limma software package. The potential target miRNAs of LINC01093 were predicted using the lncRNASNP2 database. The potential target mRNAs of miR-326 were analyzed using the TargetScan, miRWalk, ENCORI, EVmiRNA and miRDB databases. Based on these results, the genes in the intersection of the databases were screened using the Venn diagram. The complementary sequences of LINC01093 and miR-326, as well as miR-326 and MAPK1, were predicted using the lncRNASNP2 and TargetScan databases.

Luciferase reporter gene assay

The wild-type (WT) sequences of LINC01093 and MAPK1 3’UTR containing the predicted miR-326 binding site, along with their corresponding mutant (MUT) sequences, were synthesized and inserted into the pmirGLO reporter vector. For the luciferase assay, MRC-5 cells were co-transfected with the reporter plasmid (WT-LINC01093, MUT-LINC01093, WT-MAPK1 or MUT-MAPK1) and either the miR-326 mimic/inhibitor or a negative control (miR-NC) using Lipofectamine 3000 reagent. After 48 h of incubation at 37℃, the luciferase activity was detected using the Dual-Luciferase assay system (Promega, USA).

Statistical analysis

Data analysis was performed using SPSS 23.0 and GraphPad Prism 7.0 software. Comparisons of continuous and categorical variables between the two groups were conducted using Student’s t-test and chi-square test, while comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA). The correlation between LINC01093 and clinical indicators was assessed using the chi-square test. The diagnostic potential of LINC01093 for SP was evaluated using the receiver operating characteristic (ROC) curve. The correlation of the data was analyzed using the Pearson correlation coefficient. A difference was considered statistically significant if P < 0.05.

Results

Basic information of study subjects

Clinical data (Table 1) showed that there was no statistical significance in age, gender and LYM between children with SP and control children (P > 0.05). Children with SP had higher levels of WBC, NEU, CRP, LDH, PCT and D-D than the control group (P < 0.001). The duration of mechanical ventilation for children with SP was (12.05 ± 3.13) days. Among them, 78 cases required antibiotic treatment. Among 98 children with SP, 76 cases had positive results from pathogen detection, including 44 cases of bacterial infection, 20 cases of viral infection, 5 cases of mycoplasma infection, and 7 cases of mixed infection. In terms of complications, 42 children with SP developed respiratory complications, and 27 developed cardiovascular complications.

Table 1.

Clinical characteristics of the study population

Characteristics	Control (n = 92)	Severe pneumonia (n = 98)	P value
Age (years)	5.22 ± 1.04	5.35 ± 1.11	0.408
Gender			0.607
Male	52	59
Female	40	39
WBC (109/L)	7.78 ± 1.34	9.85 ± 1.92	< 0.001
NEU (109/L)	3.55 ± 0.87	12.89 ± 1.78	< 0.001
CRP (mg/L)	3.65 ± 0.92	11.92 ± 2.55	< 0.001
LDH (U/L)	245.06 ± 49.42	305.00 ± 67.52	< 0.001
LYM (109/L)	2.47 ± 0.43	2.58 ± 0.45	0.076
PCT (ng/mL)	0.16 ± 0.06	2.05 ± 0.81	< 0.001
D-D (mg/L)	0.23 ± 0.07	1.83 ± 0.33	< 0.001
Mechanical ventilation duration (days)	-	12.05 ± 3.13
Antibiotic use	-	98
Etiology
Bacteria	-	44
Viruses	-	20
Mycoplasma	-	5
Mixed	-	7
Negative	-	22
Complications
Respiratory complications	-	42
Cardiovascular complications	-	27
Others	-	29

Significant difference at P < 0.05 by Student’s t-test and chi-square test

WBC white blood cells, NEU neutrophils, CRP C-reactive protein, LDH lactate dehydrogenase, LYM lymphocyte, PCT procalcitonin, D-D D-dimer

Expression level of LINC01093

The GSE196399 dataset showed that LINC01093 was significantly upregulated in SCAP patients (Fig. 1A). The expression of LINC01093 in the serum of children with SP was determined using qRT-PCR. Compared with healthy children, serum LINC01093 expression was significantly higher in children with SP (P < 0.0001, Fig. 1B). This suggests that the abnormal expression of LINC01093 may be associated with the occurrence of SP in children. The levels of LINC01093 in bacterial pneumonia, viral pneumonia, mycoplasma pneumonia, and mixed infection pneumonia showed no significant differences (P > 0.05), but when compared with the control group, the differences were extremely significant (P < 0.001, Figure S1). The diagnostic value of LINC01093 for SP was analyzed through the ROC curve (Fig. 1C). The area under the curve (AUC) was 0.903, with a sensitivity of 82.7% and a specificity of 91.3%. This indicates that LINC01093 has a good discriminatory ability between children with SP and healthy children.

Fig. 1.

Expression and diagnostic value of LINC01093. A Volcano plot of differential gene expression. B The level of LINC01093 in healthy children and children with SP. C ROC curve for evaluating the diagnostic value of LINC01093. ^****P < 0.0001

Relationship between LINC01093 and clinicopathologic features

Further analysis was conducted to investigate the association between LINC01093 and multiple clinical characteristics of SP. The results showed that the expression of LINC01093 was not related to age, gender and LYM (P > 0.05), but was correlated with WBC, NEU, CRP, LDH, PCT, D-D, mechanical ventilation duration, etiology and complications (P < 0.05, Table 2).

Table 2.

Difference of clinical characteristics between LINC01093 expression

Characteristics	Severe pneumonia (n = 98)	LINC01093 expression		P value
		Low (n = 45)	High (n = 53)
Age (years)				0.598
< 5.35	56	27	29
≥ 5.35	42	18	24
Gender				0.707
Male	59	28	31
Female	39	17	22
WBC (109/L)				0.028
< 9.71	47	27	20
≥ 9.71	51	18	33
NEU (109/L)				0.030
< 12.89	45	26	19
≥ 12.89	53	19	34
CRP (mg/L)				0.005
< 11.92	48	29	19
≥ 11.92	50	16	34
LDH (U/L)				0.030
< 305.00	45	26	19
≥ 305.00	53	19	34
LYM (109/L)				0.230
< 2.58	48	25	23
≥ 2.58	50	20	30
PCT (ng/mL)				0.016
< 2.05	48	28	20
≥ 2.05	50	17	33
D-D (mg/L)				0.048
< 1.83	46	26	20
≥ 1.83	52	19	33
Mechanical ventilation duration				0.037
< 12.05	52	29	23
≥ 12.05	46	16	30
Etiology				0.034
Bacteria	44	15	29
Viruses	20	13	7
Mycoplasma	5	1	4
Mixed	7	2	5
Negative	22	14	8
Complications				0.027
Respiratory complications	42	13	29
Cardiovascular complications	27	14	13
Others	29	18	11

Significant difference at P < 0.05 by chi-square test

WBC white blood cells, NEU neutrophils, CRP C-reactive protein, LDH lactate dehydrogenase, LYM lymphocyte, PCT procalcitonin, D-D D-dimer

Prognostic value of LINC01093

During the follow-up period, a total of 19 deaths occurred. Among them, 15 deaths were in the high LINC01093 group and 4 deaths were in the low LINC01093 group. The Kaplan-Meier curve showed that in children with SP, children with low expression of LINC01093 had a higher survival rate (Log Rank P = 0.027, Fig. 2). The results of multivariate Cox regression analysis showed that LINC01093 (hazard ratio (HR) = 9.479, 95% confidence interval (CI) = 2.211–50.17, P = 0.004), CRP (HR = 5.844, 95% CI = 1.566–30.35, P = 0.016) and mechanical ventilation duration (HR = 6.933, 95% CI = 1.250–52.94, P = 0.038) were risk factors affecting the prognosis of children with SP. Taking respiratory complications as the reference group, cardiovascular complications (HR = 6.278, 95% CI: 1.262–34.61, P = 0.027) and other complications (HR = 6.732, 95% CI: 1.415–35.48, P = 0.018) remained independent risk factors that affected the prognosis of children with SP (Table 3).

Fig. 2.

Kaplan-Meier survival curve of LINC01093 level

Table 3.

Multivariate Cox analysis of clinicopathological variables

Characteristics	Multivariate Cox analysis		P value
	HR	95% CI
LINC01093	9.479	2.211–50.17	0.004
Age	1.249	0.271–5.944	0.774
Gender	0.715	0.206–2.408	0.588
WBC	1.229	0.309–4.897	0.765
NEU	1.121	0.243–5.225	0.882
CRP	5.844	1.566–30.35	0.016
LDH	1.237	0.298–5.471	0.769
LYM	1.453	0.490–4.596	0.505
PCT	2.117	0.436–12.66	0.369
D-D	1.031	0.320–3.437	0.959
Mechanical ventilation duration	6.933	1.250–52.94	0.038
Etiology
Bacteria	1 (reference)
Viruses	0.868	0.178–3.915	0.855
Mycoplasma	3.769	0.337–42.03	0.257
Mixed	0.936	0.032–8.881	0.961
Negative	0.407	0.064–1.991	0.294
Complications
Respiratory complications	1 (reference)
Cardiovascular complications	6.278	1.262–34.61	0.027
Others	6.732	1.415–35.48	0.018

HR hazard ratio, 95% CI 95% confidence interval, WBC white blood cells, NEU neutrophils, CRP C-reactive protein, LDH lactate dehydrogenase, LYM lymphocyte, PCT procalcitonin, D-D D-dimer

Silencing of LINC01093 attenuates LPS-induced cellular damage

As the concentration of LPS increased, the level of LINC01093 gradually rose, and cell viability decreased in a dose-dependent manner (P < 0.05, Fig. 3A-B). The transfection efficiency was verified by qRT-PCR, and silencing of LINC01093 significantly reduced the level of LINC01093 in the cells (P < 0.0001, Figure S2A). Subsequent related experiments were conducted using an LPS concentration of 10 µg/mL. Si-LINC01093 was transfected into LPS-induced MRC-5 cells to further investigate the role of LINC01093 in the SP. Transfection of si-LINC01093 in cells significantly reduced the LPS-induced level of LINC01093 (P < 0.0001, Fig. 3C). Silencing LINC01093 significantly enhanced LPS-induced cell viability (P < 0.01, Fig. 3D). Moreover, LPS induction promoted cell apoptosis and increased the concentrations of IL-1β, IL-6, and TNF-α, while reducing the level of LINC01093 could inhibit apoptosis and alleviate the inflammatory response (P < 0.01, Fig. 3E-H). Silencing LINC01093 in LPS-induced A549 cells significantly enhanced cell viability, reduced apoptosis, and decreased the levels of IL-1β, IL-6, and TNF-α, which were consistent with the findings in MRC-5 cells (P < 0.01, Figure S3).

Fig. 3.

Silencing LINC01093 can alleviate the cell damage induced by LPS. A LPS induced the upregulation of LINC01093 in cells. B LPS induced the decrease in cell viability. C Silencing LINC01093 in LPS-induced cells reduced the level of LINC01093. D Silencing LINC01093 in LPS-induced cells increased cell viability. E Silencing LINC01093 in LPS-induced cells reduced apoptosis. F-H Silencing LINC01093 in LPS-induced cells reduced the levels of inflammatory factors (IL-1β, IL-6, TNF-α). ns P > 0.05, ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001

miR-326 is the target of LINC01093

Through the lncRNASNP2 database prediction, it was found that there is a complementary binding sequence between LINC01093 and miR-326 (Fig. 4A). The results of the luciferase reporter gene assay showed that in the cells transfected with WT-LINC01093, overexpression of miR-326 reduced the luciferase activity, while inhibition of miR-326 increased the luciferase activity (P < 0.0001). However, in the cells transfected with MUT-LINC01093, there was no significant effect on the luciferase activity (Fig. 4B). Furthermore, the level of miR-326 in the serum of children with SP was lower than that in the healthy controls, and it was negatively correlated with the level of LINC01093 (r = -0.642, P < 0.0001, Fig. 4C-D). The transfection efficiency was verified by qRT-PCR. Overexpression of miR-326 significantly increased the miR-326 level in the cells (P < 0.0001, Figure S2B). In cells induced by LPS, miR-326 was also expressed at a low level in comparison to the control group. After further silencing LINC01093, miR-326 expression was significantly increased (P < 0.0001, Fig. 4E).

Fig. 4.

miR-326 is the target of LINC01093. A The complementary binding sequences of LINC01093 and miR-326. B Luciferase reporter gene assay. C The levels of miR-326 in healthy children and children with SP. D Pearson correlation analysis of LINC01093 and miR-326. E Silencing LINC01093 in LPS-induced cells upregulated the level of miR-326. ^****P < 0.0001

MAPK1 is the target of miR-326

By drawing a Venn diagram, it was found that the 5 databases jointly identified 35 common genes (Fig. 5A). Through the TargetScan database, it was predicted that there was a complementary binding sequence between miR-326 and MAPK1 (Fig. 5B). The results of the luciferase reporter gene assay showed that the miR-326 mimic reduced the luciferase activity of WT-MAPK1, while the miR-326 inhibitor increased the luciferase activity of WT-MAPK1 (P < 0.0001), but had no effect on MUT-MAPK1 (Fig. 5C). In addition, the expression of MAPK1 in the serum of SP children was increased, and it was negatively correlated with the level of miR-326 (r = -0.689, P < 0.0001, Fig. 5D-E). The transfection efficiency was verified by qRT-PCR. Overexpression of MAPK1 significantly increased the MAPK1 level in the cells (P < 0.0001, Figure S2C). In cells induced by LPS, MAPK1 was highly expressed. After further transfection with miR-326 mimic, MAPK1 expression was downregulated (P < 0.0001, Fig. 5F).

Fig. 5.

MAPK1 is the target gene of miR-326. A Venn diagram of the predicted target genes from 5 databases. B The complementary binding sequences of miR-326 and MAPK1. C Luciferase reporter gene assay. D The levels of MAPK1 in healthy children and children with SP. E Pearson correlation analysis of miR-326 and MAPK1. F Overexpression of miR-326 in LPS-induced cells reduced the level of MAPK1. ^***P < 0.001, ^****P < 0.0001

The miR-326/MAPK1 axis is involved in the protective effect of LINC01093 silencing on LPS-induced cell damage

To explore whether LINC01093 silencing exerts the protective role via the miR-326/MAPK1 axis, rescue experiments were conducted in LPS-induced MRC-5 cells. As expected, silencing LINC01093 enhanced cell viability, reduced apoptosis and inflammatory responses. On this basis, further inhibition of miR-326 or overexpression of MAPK1 significantly decreased cell viability and weakened the protective effect of LINC01093 silencing on LPS-induced apoptosis and inflammatory responses (P < 0.05, Fig. 6A-E).

Fig. 6.

The miR-326/MAPK1 axis is involved in the protective effect of LINC01093 silencing on LPS-induced cell damage. A In LPS-induced cells, inhibiting miR-326 or overexpressing MAPK1 could reverse the promoting effect of si-LINC01093 on cell viability. B In LPS-induced cells, inhibiting miR-326 or overexpressing MAPK1 could counteract the inhibitory effect of si-LINC01093 on cell apoptosis. C-E In LPS-induced cells, inhibiting miR-326 or overexpressing MAPK1 could reverse the inhibitory effect of si-LINC01093 on the levels of inflammatory factors. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001

Discussion

SP is a type of severe respiratory infectious diseases, which is common and prevalent in pediatrics and is the leading cause of death for children under 5 years old [21]. Moreover, SP not only affects the respiratory function of the patients but also causes multiple organ dysfunction, and even poses a threat to life [22]. Therefore, early diagnosis and treatment of children with SP are particularly important for preventing severe diseases and poor prognosis. LncRNAs not only participate in the function of genes encoding regulatory proteins but also regulate signaling pathways and target genes [23]. In recent years, lncRNAs have attracted attention due to their different expressions and involvement in lung-related diseases [24].

Previous studies have highlighted the potential of lncRNAs as prognostic and diagnostic biomarkers for pneumonia patients [12]. For instance, MEG3 was highly expressed in children with SP and could distinguish children with SP and healthy children. The high expression of MEG3 indicated a poorer prognosis and was a potential independent biomarker [25]. The MALAT1 expression in SCAP children was elevated, and its diagnostic AUC for SCAP was 0.927. Additionally, the MALAT1 expression in surviving patients was lower than that in non-surviving patients, and it was positively correlated with the risk of death [26]. In this study, the expression of LINC01093 was higher in children with SP. Further ROC curve analysis revealed that LINC01093 had good diagnostic value. The abnormal expression of LINC01093 suggests that it may be involved in the progression of SP. Interestingly, one study showed that during the 28-day follow-up period, the total mortality rate of children with SP was 13.4% [27]. Another study indicated that the 28-day mortality rate of children with SP was as high as 20.0% [13]. These statistics demonstrate that children with SP have a poorer prognosis. During our 28-day follow-up of the children with SP, we found that high expression of LINC01093 had a poorer prognosis and was an independent risk factor affecting the prognosis of SP. Our current study indicates that the increased expression of LINC01093 may exacerbate the inflammatory responses in children with SP and lead to a poor prognosis.

To further understand the molecular mechanism of LINC01093 in SP, an in vitro SP model was established by inducing MRC-5 cells with LPS. As expected, LINC01093 was upregulated in the cells induced by LPS. Furthermore, LPS induction led to a decrease in cell viability, promoted apoptosis and inflammatory responses, while silencing LINC01093 reduced the cell damage induced by LPS. Increasing evidence suggests that dysregulation of lncRNA is associated with the pathological progression of inflammatory diseases. UCA1 was highly expressed in children with SP. Inhibiting UCA1 partially reversed the decrease in cell viability, the increase in apoptosis and inflammatory factors induced by LPS [28]. CRNDE was highly expressed in cells and lung tissue induced by LPS. Silencing CRNDE increased cell viability, inhibited apoptosis and reduced inflammatory factors levels [29].

LncRNAs regulate the progression of diseases or biological activities by binding to target genes or miRNAs [30]. miR-326 was the target miRNA of LINC01093, and its expression was downregulated in MRC-5 cells stimulated by LPS. The downregulation of miR-326 partially counteracted the protective effect of LINC01093 silencing on LPS-induced cell damage. This suggests that LINC01093 may “sponge” miR-326 and thereby relieve its inhibitory effect on downstream target genes. Studies have shown that miR-326 exerts a critical effect in the progression of lung-related diseases [31]. The absence of miR-326 reversed the protective effect of circESPL1 silencing on LPS-induced lung cell damage [32]. Silencing circ-UQCRC2 upregulated miR-326, thereby alleviating LPS-induced cytotoxicity in MRC-5 cells [33]. Additionally, through multi-database intersection analysis, MAPK1 was identified as the target mRNA of miR-326. In children with SP, the expression of MAPK1 was upregulated. Overexpression of miR-326 could downregulate MAPK1 expression. Overexpression of MAPK1 partially reversed the protective effect of LINC01093 silencing on LPS-induced cell damage, confirming that LINC01093 participates in the pathological process of SP by regulating the miR-326/MAPK1 axis. Consistent with our research results, Cai et al. reported that the serum MAPK1 levels in SCAP patients were elevated. Knockout of MANCR could inhibit apoptosis and inflammatory factors through the miR-20a-5p/MAPK1 axis [19].

This study has several limitations. First, the host inflammatory responses and lncRNA expression profiles may vary depending on the pathogen causing the pneumonia. This retrospective study included a total of 98 children with SP. However, because the sample sizes for each pathogen subgroup were small, conducting statistical subgroup analyses would not have been meaningful; therefore, no in-depth stratified analysis was performed based on different pathogen types. Future prospective studies with larger sample sizes and a stratified design based on pathogen type are needed to validate the diagnostic and prognostic value of LINC01093 in pneumonia caused by different pathogens. Second, this study employed a case-control design, comparing only the SP group with a healthy control group; it did not include a control group with non-SP conditions, which limits the generalizability of the findings. Future prospective studies should include a non-SP control group to further validate the specific diagnostic value of LINC01093 for SP. Third, the in vitro experiments used LPS-induced MRC-5 cells to establish a pneumonia model, which primarily simulates the inflammatory response caused by Gram-negative bacterial infections; however, this model cannot fully represent the molecular mechanisms underlying viral or mycoplasma pneumonia, nor can it fully replicate the complexity of SP in children. Future studies will utilize animal models to further validate the regulatory mechanism of the LINC01093/miR-326/MAPK1 axis in SP.

In conclusion, the upregulation of LINC01093 is a potential diagnostic and prognostic biomarker for children with SP. LINC01093 regulates cell viability, apoptosis and inflammatory responses through the miR-326/MAPK1 axis. This provides a new theoretical basis for the early identification, prognosis assessment and molecular targeted therapy of SP.

Authors’ contributions

Conceptualization: P.T. and N.W.; Data curation: P.T.; Formal analysis: P.T.; Funding acquisition: P.T.; Investigation: P.T.; Methodology: N.W.; Project administration: P.T. and N.W.; Resources: P.T.; Software: P.T.; Supervision: P.T.; Validation: P.T.; Visualization: N.W.; Writing - original draft: P.T.; Writing - review & editing: N.W.

Funding

No funding was received to assist with the preparation of this manuscript.

Data availability

The datasets generated and/or analysed during the current study are available in the GSE196399 dataset, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE196399 .

Declarations

Ethics approval and consent to participate

This study was approved by the ethics Committee of Ezhou Central Hospital. All patients or their legal representatives signed informed consent forms, and the research procedures complied with the ethical principles of the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.