Abstract
Objective
The prognosis of sepsis, a pathological condition associated with high mortality and rapid progression, can be improved with precise diagnosis, effective treatment, and nursing care. This study investigated the expression and clinical significance of the long non-coding RNA (lncRNA) EPB41L4A-AS1 in sepsis.
Methods
The serum EPB41L4A-AS1 levels in patients with sepsis were quantified using quantitative real-time polymerase chain reaction (RT-qPCR). The diagnostic value of EPB41L4A-AS1 was determined using the receiver operating characteristic curves. The correlation of EPB41L4A-AS1 with clinical parameters was evaluated using Pearson correlation. Kaplan–Meier assessment of prognostic value. Lipopolysaccharide (LPS)-treated THP-1 cells served as an in vitro cell model for sepsis. The mRNA and protein levels of inflammatory factors were examined using RT-qPCR analysis and enzyme-linked immunosorbent assay, respectively. Finally, the binding of EPB41L4A-AS1 to miR-146a-5p was determined using the dual-luciferase reporter and RNA immunoprecipitation assays.
Results
EPB41L4A-AS1 was significantly downregulated in patients with sepsis. The downregulation was inversely correlated with inflammatory cytokines and severity scores (APACHE II and SOFA). EPB41L4A-AS1 expression had a high diagnostic value in sepsis. The overexpression of EPB41L4A-AS1 downregulated inflammatory factor expression and release. However, miR-146a-5p mitigated the inhibitory effects of EPB41L4A-AS1 overexpression on inflammatory factors expression and release. EPB41L4A-AS1 was a target of miR-146a-5p.
Conclusion
lncRNA EPB41L4A-AS1 alleviates sepsis-related inflammation by targeting miR-146a-5p and may serve as a diagnostic biomarker for sepsis.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40001-025-02991-9.
Keywords: EPB41L4A-AS1, Sepsis, miR-146a-5p, Molecular mechanism
Introduction
Infections can lead to sepsis, a potentially fatal syndrome of systemic inflammation [1, 2]. Sepsis often presents with multiple organ dysfunction [3] and is one of the major causes of mortality among critically ill patients worldwide [4]. The pathophysiology of sepsis is complex, involving inflammatory dysregulation, immune suppression, and metabolic disturbances [5]. Although progress has been achieved in the management of sepsis, early diagnosis and accurate prognosis prediction of sepsis are major clinical challenges [6]. Therefore, the identification of new biomarkers and therapeutic targets is critical for improving the prognosis of patients with sepsis. Delayed treatment of sepsis often causes irreversible organ damage and death. Thus, early diagnosis and timely intervention (including accurate nursing surveillance and personalized care delivery) can decrease the mortality rate among patients with sepsis [7].
Long non-coding RNAs (lncRNAs), which are non-coding RNAs with a length of > 200 nucleotides [8], are involved in gene expression regulation [9]. The functions of lncRNAs in diseases, especially cancer, cardiovascular, and inflammatory diseases, have piqued the interest of the scientific community [10–12]. EPB41L4A-AS1, an lncRNA, is located on human chromosome 5q22.1. Previous studies have examined the expression and function of EPB41L4A-AS1 in different diseases [13]. EPB41L4A-AS1 is reported to be involved in inflammation regulation [14]. For example, EPB41L4A-AS1 downregulation accelerates diabetes progression by promoting inflammation [15]. Additionally, the level of EPB41L4A-AS1 is downregulated in the gingival crevicular fluid of patients with chronic periodontitis. The overexpression of EPB41L4A-AS1 alleviated lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells [16]. Liu et al. analyzed the differentially expressed lncRNAs related to neonatal sepsis and demonstrated that EPB41L4A-AS1 was downregulated in patients with sepsis [17]. The potential role of EPB41L4A-AS1 in sepsis has not been elucidated. The elucidation of the mechanism of EPB41L4A-AS1 in sepsis can promote its application as a biomarker and a novel therapeutic target for sepsis.
This study hypothesized that lncRNA EPB41L4A-AS1 is a biomarker of sepsis and can modulate the inflammatory response. The clinical diagnostic relevance of EPB41L4A-AS1 was evaluated in patients with sepsis. Additionally, the molecular mechanism underlying the function of EPB41L4A-AS1 was elucidated. This study aimed to present innovative concepts and potential therapeutic targets for the diagnosis and management of sepsis.
Materials and methods
Participants
This study enrolled 128 patients with sepsis admitted to the Affiliated Hospital of Hebei University of Engineering between January 2021 and June 2023. Sepsis was diagnosed based on the criteria of the 2021 Global Guidelines for the Governance and Control of Sepsis and Septic Shock. The exclusion criteria were as follows: (1) patients aged <18 or >80 years; (2) patients with cancerous neoplasm, autoimmune disorder, or severe hepatic and renal insufficiency; (3) pregnant or lactating patients; (4) patients who previously received immunosuppressive therapy. In this study, 102 age-matched and gender-matched healthy volunteers were recruited into the control group. Each participant provided their signature on an informed consent form. The research plan was then given the green light by the Ethics Committee affiliated with the Affiliated Hospital of Hebei University of Engineering.
Data collection
Detailed personal information and medical history were recorded during the study. The levels of serum creatinine (Scr), albumin, white blood cell count (WBC), and C-reactive protein (CRP) were determined using biochemical analysis. Additionally, the levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and IL-8 were measured. The Acute Physiology and Chronic Health Evaluation II (APACHE II) and Sequential Organ Failure Assessment (SOFA) scores were evaluated for each patient after admission. The SOFA score evaluates the functional status of the following six organ systems: respiratory, coagulation, liver, circulatory, central nervous, and renal systems. Each organ system was scored from 0 to 4 points based on the degree of functional impairment. The total score, which ranged from 0 to 24 points, was directly proportional to the severity of the condition. The APACHE II score evaluates acute physiological indicators, age, and chronic health conditions. The total score, which ranges from 0 to 71 points, is directly proportional to the severity of the condition. Severity classification was conducted using two different scoring systems. Based on the SOFA score, which is widely used to reflect the degree of organ dysfunction in sepsis patients according to clinical practice and previous studies [18], severity was classified into mild (SOFA 0–3), moderate (4–6), and severe (≥ 7). Additionally, based on the APACHE II score, a commonly employed tool in clinical research for assessing the overall severity of critically ill patients, severity was classified into mild (APACHE II ≤ 15), moderate (16–25), and severe (> 25) [19]. The absence of severe sepsis cases in the APAHCE II severe group is because we excluded patients with tumors, autoimmune diseases, or severe hepatic/renal insufficiency.
The peripheral venous blood (2 mL) samples were obtained from the upper limbs of all participants. The blood samples were centrifuged at 12,000 rpm for 15 min. The upper serum layer was used for analyzing the expression of the nucleic acids (lncRNA, miRNA, and inflammatory cytokines-encoding genes). The serum was pooled and stored at − 80 °C until analysis. All patients were followed up for 28 days to record the survival outcomes.
Cell culture
THP-1 cells (ATCC, USA) were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin at 37 °C and 5% CO2. The growth status of the cells was regularly observed. The culture medium was replaced every 2–3 days. To establish the sepsis model in vitro, THP-1 cells were treated with different concentrations (0, 0.5, 1, or 2 μg/mL) of LPS for 0, 12, 24, or 48 h as described previously [20]. Based on the experimental results and previous studies, the induction conditions for the in vitro model were as follows: LPS concentration: 1 μg/mL, treatment duration: 48 h.
Cell transfection
THP-1 cells were cultured in culture plates and following conventional culture conditions. Transfection was initiated when the cells reached 70%-80% confluency. EPB41L4A-AS1 overexpressing plasmid pcDNA3.1-EPB41L4A-AS1 (oe-EPB41L4A-AS1) and blank plasmid pcDNA3.1 (empty vector) were constructed. TPH-1 cells were transfected with 50 nM oe-EPB41L4A-AS1 or empty vector plasmid using Lipofectamine™ 2000. To perform rescue experiments, oe-EPB41L4A-AS1-transfected or empty vector-transfected THP-1 cells were transfected with 100 nM miR-146a-5p mimic or negative control (scramble mimic). At 6 h post-transfection, the medium was replaced with fresh medium. The cells were cultured for 48 h, harvested.
Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from THP-1 cells or serum samples by using the mirVana™ miRNA Isolation Kit and TRIzol™ Reagent. The isolated RNA was reverse-transcribed into complementary DNA (cDNA) using the miScript II RT Kit and SuperScript™ IV Reverse Transcriptase Kit. The cDNA was mixed with specific primers and RNase-free H2O and subjected to RT-qPCR assays using the miScript SYBR Green PCR kit or the SYBR Green Quantitative RT-qPCR kit. The expression levels of target genes were determined based on the fluorescence signal. The relative expression levels of genes were determined using the 2−ʌʌCT method. The internal reference gene for lncRNA and inflammation-related genes was GAPDH, while that for miRNAs was U6.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α, IL-6, and IL-8 in the culture supernatant were determined using an Invitrogen ELISA kit, following the manufacturer’s instructions. Experiments were performed with three replicated samples.
Subcellular fractionation
The THP-1 cells were washed with a pre-cooled phosphate-buffered solution and lysed with a lysis buffer. The cytoplasmic and nuclear fractions were obtained using the PARIS™ Kit (Thermo Fisher Scientific). The total RNA was isolated and subjected to RT-qPCR analysis to quantify the expression of EPB41L4A-AS1. GAPDH and U6 served as the endogenous reference genes for the cytoplasmic and nuclear genes, respectively.
RNA immunoprecipitation (RIP) assay
THP-1 cells were maintained in RPMI-1640 medium until the target cell density was attained. The cells were lysed with RIP assay lysis buffer. The lysates were incubated with magnetic beads pre-coupled with anti-Ago2 antibodies or anti-IgG antibodies (negative control). The immunoprecipitated samples were subjected to RT-qPCR analysis to determine the levels of EPB41L4A-AS1 and miR-146a-5p.
Dual-luciferase reporter (DLR) assay
GenePharma synthesized the wild-type EPB41L4A-AS1 dual-luciferase reporter recombinant plasmid containing the wild-type (EPB41L4A-AS1-Wt) or mutant (EPB41L4A-AS1-Mt) miR-146a-5p-binding site. After the cells achieved 70%-80% confluency, the recombinant plasmids were co-transfected into the cells with miR-146a-5p mimic or scramble mimic using Lipofectamine™ 3000. At 48 h post-transfection, the cells were collected and lysed. The luciferase activity of the samples was detected using the dual-luciferase reporter gene assay kit (Beyotime, China).
Statistical analysis
All statistical analyses were performed using SPSS 23.0 software and GraphPad Prism 9.0. Means between two groups were compared using a t-test, while those between more than two groups were compared using one-way analysis of variance. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of EPB41L4A-AS1 in sepsis. The correlation between the two variables was examined using Pearson correlation analysis. Meanwhile, the correlation of EPB41L4A-AS1 expression with the prognosis of patients was determined using Kaplan–Meier curves. The potential risk factors affecting the prognosis of patients with sepsis were identified using Cox proportional hazards regression analysis. Differences were considered significant at P < 0.05.
Results
Baseline clinical characteristics of study participants
As shown in Table 1, the demographic and medical history characteristics were not significantly different between the control and sepsis groups (P > 0.05). The levels of Scr, WBC, CRP, TNF-α, IL-6 and IL-8 were upregulated, whereas those of Albumin were downregulated in the sepsis group (P < 0.001).
Table 1.
Clinical baseline characteristics of subjects in both groups
| Items | Controls (n = 102) | Sepsis patients (n = 128) | P value |
|---|---|---|---|
| Demographics | |||
| Age (years), mean ± SD | 56.81 ± 8.81 | 54.96 ± 8.85 | 0.115 |
| Gender, no. (%) | 0.289 | ||
| Female | 42 (41.18) | 62 (48.44) | |
| Male | 60 (58.82) | 66 (51.56) | |
| BMI (kg/m2), mean ± SD | 23.48 ± 3.57 | 24.02 ± 2.54 | 0.182 |
| Smoke, no. (%) | 34 (33.33) | 38 (29.69) | 0.570 |
| Drink, no. (%) | 35 (34.31) | 40 (31.25) | 0.672 |
| Medical history | |||
| Hypertension, no. (%) | 38 (37.25) | 48 (37.50) | 0.970 |
| Hyperlipidemia, no. (%) | 35 (34.31) | 39 (30.47) | 0.571 |
| Diabetes, no. (%) | 33 (32.35) | 40 (31.25) | 0.887 |
| Biochemical indicators | |||
| Scr (mg/dL), median (IQR) | 0.75 (0.64, 0.82) | 1.64 (1.37, 1.91) | < 0.001 |
| Albumin (g/L), median (IQR) | 42.07 (39.67, 44.61) | 31.94 (26.80, 36.21) | < 0.001 |
| WBC (× 109/L), median (IQR) | 6.51 (5.84, 7.27) | 19.65 (14.99, 23.21) | < 0.001 |
| CRP (mg/L), median (IQR) | 5.23 (3.82, 6.50) | 98.35 (70.58, 126.60) | < 0.001 |
| Inflammatory cytokine | |||
| TNF-α (pg/mL), median (IQR) | 31.68 (27.72, 34.65) | 178.20 (137.12, 232.40) | < 0.001 |
| IL-6 (pg/mL), median (IQR) | 17.82 (14.60, 22.03) | 61.38 (46.53, 74.75) | < 0.001 |
| IL-8 (pg/mL), median (IQR) | 68.81 (52.22, 82.17) | 122.76 (99.00, 144.29) | < 0.001 |
| Disease severity | |||
| APACHE II score, median (IQR) | – | 16.00 (13.00, 21.00) | – |
| SOFA score, median (IQR) | – | 7.00 (5.00, 10.00) | – |
BMI, body mass index; Scr, serum creatinine; WBC, white blood cell; CRP, C-reactive protein; SOFA, sequential organ failure assessment; APACHE II, acute physiology and chronic health evaluation II; IL, interleukin; TNF, tumor necrosis factor; SD, standard deviation; IQR, interquartile range
EPB41L4A-AS1 is a potential diagnostic biomarker for patients with sepsis
The serum levels of EPB41L4A-AS1 in the sepsis group were lower than those in the control group (P < 0.001, Fig. 1A). The levels of EPB41L4A-AS1 expression were negatively correlated with those of IL-6, TNF-α, and IL-8 (P < 0.05, Fig. 1B–D). Additionally, the EPB41L4A-AS1 level was negatively correlated with APACHE II and SOFA scores (P < 0.001, Fig. 1E, F). Notably, regardless of whether sepsis severity was assessed using the SOFA score and APAHCHE II score, the expression of EPB41L4A-AS1 exhibited a progressive decline as the severity of sepsis increased (P < 0.0001, Fig. 1G and supplementary Figure). Finally, ROC curve analysis revealed that the area under the curve, sensitivity, and specificity of EPB41L4A-AS1 for identifying patients with sepsis were 0.860 (95% confidence interval: 0.810–0.910), 92.19%, and 71.57%, respectively (Fig. 1H).
Fig. 1.
EPB41L4A-AS1 is a potential diagnostic biomarker for sepsis. A The serum EPB41L4A-AS1 levels were significantly downregulated in patients with sepsis. The serum EPB41L4A-AS1 levels in patients with sepsis were negatively correlated with the levels of IL-6 (B), TNF-α (C), and IL-8 (D). The serum EPB41L4A-AS1 levels in patients with sepsis were negatively correlated with Acute Physiology and Chronic Health Evaluation II (APACHE II) score (E) and Sequential Organ Failure Assessment (SOFA) scores (F). G Patients with sepsis were grouped by severity based on the SOFA score, and the levels of EPB41L4A-AS1 were compared across different groups. H The diagnostic value of EPB41L4A-AS1 in sepsis was determined using the receiver operating characteristic (ROC) curve. ****P < 0.0001
EPB41L4A-AS1 downregulation may predict poor prognosis in patients with sepsis
After 28 days of follow-up, the mortality rate in the study population was 27.34% (n = 35). As shown in Fig. 2A, the expression level of EPB41L4A-AS1 in deceased patients was significantly lower than that in surviving patients (P < 0.0001). Patients were divided into the high-EPB41L4A-AS1 expression (n = 63) and low-EPB41L4A-AS1 expression groups (n = 65) based on the mean value (0.51 ± 0.25) of EPB41L4A-AS1 expression. Kaplan–Meier survival analysis revealed that the survival rates in the low EPB41L4A-AS1 expression group were higher than those in the high-EPB41L4A-AS1 expression group (P < 0.01, Fig. 2B). Cox regression analysis revealed that SOFA score, APACHE II score, and EPB41L4A-AS1 were risk factors for poor prognosis in patients with sepsis (P < 0.05, Table 2).
Fig. 2.
Effect of serum EPB41L4A-AS1 levels on the prognosis of patients with sepsis. A Serum EPB41L4A-AS1 expression levels in deceased and surviving patients. B Kaplan–Meier curve analysis was performed to examine the correlation of EPB41L4A-AS1 levels with the prognosis of patients. ****P < 0.0001
Table 2.
Cox regression analysis identified independent risk factors for 28-day mortality in sepsis patients
| Parameters | HR | 95% CI | P value |
|---|---|---|---|
| Age | 1.215 | 0.564–2.616 | 0.619 |
| Male | 1.628 | 0.684–3.876 | 0.271 |
| BMI | 1.389 | 0.649–2.971 | 0.397 |
| Smoke | 1.100 | 0.420–2.876 | 0.846 |
| Drink | 1.180 | 0.450–3.100 | 0.736 |
| Hypertension | 1.765 | 0.793–3.928 | 0.164 |
| Hyperlipidemia | 1.081 | 0.465–2.513 | 0.856 |
| Diabetes | 1.103 | 0.440–2.764 | 0.835 |
| Scr | 1.183 | 0.444–3.152 | 0.737 |
| Albumin | 0.865 | 0.409–1.830 | 0.704 |
| WBC | 1.339 | 0.582–3.079 | 0.492 |
| CRP | 2.115 | 0.717–6.236 | 0.175 |
| TNF-α | 1.072 | 0.373–3.082 | 0.897 |
| IL-6 | 1.440 | 0.537–3.858 | 0.468 |
| IL-8 | 1.603 | 0.598–4.301 | 0.348 |
| SOFA score | 2.978 | 1.040–8.524 | 0.042 |
| APAHCE II score | 4.273 | 1.247–14.642 | 0.021 |
| EPB41L4A-AS1 | 0.057 | 0.012–0.280 | 0.000 |
Statistically significant differences between groups are shown in bold print
BMI, body mass index; APACHE, acute physiology and chronic health evaluation, SOFA, sequential organ failure assessment; Scr, serum creatinine; WBC, white blood cells; CRP, C-reactive protein; PCT, procalcitonin; TNF-α, tumor necrosis factor-α; IL, interleukin
EPB41L4A-AS1 regulates LPS-induced inflammatory response
The effect of LPS on the expression level of EPB41L4A-AS1 in THP-1 cells and the suppressing effect of EPB41L4A-AS1 on inflammation are shown in Fig. 3. At 48 h post-induction, LPS concentration-dependently downregulated the expression of EPB41L4A-AS1 (P < 0.001, Fig. 3A). Additionally, treatment with 1 μg/ml LPS, time-dependently downregulated the expression of EPB41L4A-AS1 (P < 0.001, Fig. 3B). Transfection with oe-EPB41L4A-AS1 significantly upregulated the expression of EPB41L4A-AS1 than the empty vector (P < 0.001, Fig. 3C, D). Additionally, transfection with oe-EPB41L4A-AS1 significantly mitigated the LPS-induced expression and secretion of IL-6, TNF-α, and IL-8 (P < 0.001, Fig. 3E, F).
Fig. 3.
EPB41L4A-AS1 regulates lipopolysaccharide (LPS)-induced inflammatory response in THP-1 cells. A Expression level of EPB41L4A-AS1 in THP-1 cells induced by different concentrations of LPS for 48 h. B Effect of different durations of 1 μg/mL LPS treatment on the expression levels of EPB41L4A-AS1 in THP-1 cells. C Effect of EPB41L4A-AS1 overexpression plasmid transfection on EPB41L4A-AS1 expression in THP-1 cells. D Effect of EPB41L4A-AS1 overexpression plasmid transfection on EPB41L4A-AS1 levels in LPS-induced THP-1 cells. Effects of EPB41L4A-AS1 overexpression plasmid transfection on mRNA (E) and protein (F) levels of inflammatory factors in LPS-induced THP-1 cells. *P < 0.05; **P < 0.01; ***P < 0.001
EPB41L4A-AS1 targets miR-146a-5p
The cellular distribution of EPB41L4A-AS1 was predicted using the LncATLAS database. As shown in Fig. 4A, the cytoplasm and nucleus relative concentration index values in multiple cell lines were > 0. This indicates that EPB41L4A-AS1 is primarily localized to the cytoplasm. Subcellular fractionation assays confirmed that the content of EPB41L4A-AS1 in the cytoplasm of THP-1 cells was higher than that in the nucleus (Fig. 4B). LncBook2 database revealed that EPB41L4A-AS1 comprised sequences complementary to miR-146a-5p (Fig. 4C). The dual-luciferase reporter gene assay results indicated that the luciferase activity of EPB41L4A-AS1-Wt was negatively regulated by miR-146a-5p mimic and positively regulated by miR-146a-5p inhibitor (P < 0.0001, Fig. 4D). RIP analysis demonstrated that, the EPB41L4A-AS1 and miR-146a-5p levels in the anti-Ago2 group were higher than those in the anti-IgG group (P < 0.0001Fig. 4E). The serum miR-146a-5p levels in the sepsis group were higher than those in the control group. Additionally, the miR-146a-5p levels were negatively correlated with the EPB41L4A-AS1 level (P < 0.0001, Fig. 4F, G). LPS markedly upregulated the miR-146a-5p levels in THP-1 cells (P < 0.0001, Fig. 4H). Transfection with oe-EPB41L4A-AS1 significantly downregulated miR-146a-5p (P < 0.01, Fig. 4I).
Fig. 4.
EPB41L4A-AS1 is a target of miR-146a-5p. A Cellular localization of EPB41L4A-AS1 was predicted using the lncATLAS database. B The subcellular localization of EPB41L4A-AS1 in THP-1 cells was determined after subcellular fractionation. C The binding sequences between EPB41L4A-AS1 and miR-146a-5p. Dual luciferase reporter assay (D) and RNA immunoprecipitation assay (E) validated the targeted binding between EPB41L4A-AS1 and miR-146a-5p. F The serum miR-146a-5p levels in patients with sepsis. G The correlation between miR-146a-5p and EPB41L4A-AS1 levels in patients with sepsis. H Effect of 1 1 μg/mL LPS treatment on the levels of miR-146A-5p in THP-1 cells. I Effect of EPB41L4A-AS1 overexpression plasmid transfection on miR-146a-5p expression levels in LPS-induced THP-1 cells. *P < 0.05; **P < 0.01; ***P < 0.001
EPB41L4A-AS1 regulates LPS-induced inflammation by modulating miR-146a-5p
The miR-146a-5p level in the miR-146a-5p mimic-transfected cells was higher than that in the scramble mimic-transfected cells (P < 0.001, Fig. 5A). However, EPB41L4A-AS1 overexpression significantly suppressed miR-146a-5p expression (P < 0.001, Fig. 5B). The synthesis and secretion of IL-6, TNF-α, and IL-8 were downregulated upon EPB41L4A-AS1 overexpression. Transfection with miR-146a-5p mimic partially mitigated the inhibitory effect of EPB41L4A-AS1 overexpression on inflammatory factors (P < 0.001, Fig. 5C, D). Thus, miR-146a-5p mitigates the effects of EPB41L4A-AS1 overexpression on miR-146a-5p and inflammatory cytokines.
Fig. 5.
EPB41L4A-AS1 mediates inflammatory response in sepsis through miR-146a-5p. A Transfection of miR-146a-5p mimic upregulated miR-146a-5p expression in THP-1 cells. B The levels of miR-146a-5p under different treatment conditions. C Co-regulatory effects of EPB41L4A-AS1 and miR-146a-5p on the levels of inflammatory cytokines. D Co-regulatory effects of EPB41L4A-AS1 and miR-146a-5p on the secretion of inflammatory factors. *P < 0.05; **P < 0.01; ***P < 0.001
Discussion
EPB41L4A-AS1 is reported to target miR-146a-5p [13]. During the osteogenic differentiation of bone marrow-resident mesenchymal stem cells, EPB41L4A-AS1 regulates miR-146a-5p expression. Thus, EPB41L4A-AS1 was hypothesized to be involved in sepsis progression by targeting miR-146a-5p. This hypothesis offers new insights into the pathogenesis of sepsis and potential therapeutic targets for sepsis. This study revealed that EPB41L4A-AS1 expression is significantly downregulated in patients with sepsis. This downregulation was negatively correlated with the level of inflammation and could predict poor prognosis. EPB41L4A-AS1 overexpression effectively suppressed the inflammatory response by targeting miR-146a-5p, providing novel guidance for sepsis diagnosis and treatment.
The SOFA score and APACHE II scores, which are commonly used for clinical assessment of sepsis severity [21], assess disease severity by integrating multiple physiological indicators, past medical history, and the overall physical condition [22]. In this study, EPB41L4A-AS1 expression was negatively correlated with SOFA and APACHE II scores. We conducted severity subgrouping based on SOFA and APACHE II scores. Our findings consistently indicate that EPB41L4A-AS1 expressions are negatively correlated with both SOFA and APACHE II scores. Thus, EPB41L4A-AS1 downregulation was associated with severe sepsis, implying its potential value in evaluating disease severity. Clinicians can plan for aggressive and targeted treatment regimens for patients exhibiting EPB41L4A-AS1 downregulation. ROC curve analysis demonstrated that EPB41L4A-AS1 exhibited high sensitivity and specificity for differentiating patients with sepsis from healthy individuals and could predict poor prognosis. The downregulated serum level of EPB41L4A-AS1 is strongly linked to poor prognosis. Thus, monitoring EPB41L4A-AS1 expression can aid in the development of novel clinical treatment strategies. LPS, an endotoxin, activates inflammation in epithelial cells, endothelial cells, monocytes, and macrophages through various signal transduction pathways [23]. Thus, LPS is widely used for simulating sepsis in vitro. In this study, EPB41L4A-AS1 overexpression markedly mitigated LPS-induced inflammatory cytokine release, exerting anti-inflammatory effects.
LncRNAs have a critical role in the intricate regulatory network of gene expression [24]. Additionally, lncRNAs promote disease pathogenesis and progression by targeting miRNAs [25]. This study revealed that EPB41L4A-AS1 physically interacts with miR-146a-5p, offering novel insights for further elucidation of the underlying biological mechanisms. miR-146a-5p, a critical miRNA involved in inflammation regulation, mediates the pathophysiology of various diseases [26]. In particular, miR-146a-5p regulates inflammatory response and epithelial cell injury through the TNF-α receptor. Previous studies have demonstrated that miR-146a-5p knockdown alleviates inflammation and delays disease progression in osteoarthritis [27]. Additionally, the miR-146a-5p levels are reported to vary in patients with sepsis. In particular, miR-146a-5p is up-regulated in patients with sepsis but declines during recovery [28]. Experiments with septic mice demonstrated that miR-146a-5p knockdown mitigates inflammatory reactions and suppresses T cell activation [29]. Furthermore, miR-146a-5p regulates macrophage inflammation in response to LPS [30, 31]. Huang et al. revealed that extracellular miR-146a-5p can activate TLR7, a novel mechanism that triggers lung inflammation and leads to sepsis-associated acute respiratory distress syndrome [32].
Inflammation-related biomarkers have emerged as crucial tools for predicting disease prognosis across a wide range of malignancies. These biomarkers not only reflect the underlying inflammatory state of the tumor microenvironment but also provide valuable insights into the overall disease progression and patient outcomes. Recent studies have further validated the prognostic significance of various immune and inflammation-based indices in different diseases. For instance, in colorectal cancer patients, the combined NP and LHb index is a reliable prognostic indicator [33]. This index integrates multiple inflammatory factors, highlighting the complexity and multi-faceted nature of inflammation in cancer prognosis. Similarly, the modified Glasgow prognostic score has demonstrated its prognostic value in breast cancer by combining serum C-reactive protein and albumin levels [34]. In another study on colorectal cancer, pan-immune-inflammation values were found to be closely associated with patient survival and disease recurrence [35]. These findings suggest that comprehensive inflammatory indices can effectively capture the systemic inflammatory response and predict disease outcomes. This demonstrated that EPB41L4A-AS1 overexpression significantly downregulates miR-146a-5p expression and suppresses inflammatory factor release. Conversely, siRNA-mediated EPB41L4A-AS1 knockdown upregulated miR-146a-5p levels and inflammatory factor release. The overexpression of miR-146a-5p counteracted the inhibitory effect of EPB41L4A-AS1 overexpression on inflammatory cytokines. These findings suggest that EPB41L4A-AS1 negatively regulates miR-146a-5p to attenuate the inflammatory response. Based on these findings, we hypothesize that EPB41L4A-AS1 regulates the inflammatory response of sepsis by modulating miR-146a-5p expression. This mechanism provides a new perspective on the function of EPB41L4A-AS1 in sepsis and highlights the significance of miR-146a-5p as a potential therapeutic target. In patients with sepsis exhibiting EPB41L4A-AS1 upregulation, the implementation of effective interventions and nursing care, including strategies to downregulate EPB41L4A-AS1 expression, can attenuate the inflammatory response and delay disease progression. Previous studies have demonstrated that EPB41L4A-AS1 is involved in the diabetic inflammatory response through the NF-κB pathway [15]. Qingfei Lianhua Tang mitigates pneumonia progression by targeting the miR-146a-5p-mediated NF-κB pathway [36]. In sepsis, inflammatory cell activation and pro-inflammatory cytokine overproduction are associated with NF-κB activation [37]. Additionally, miR-146a-5p secreted by hepatic stellate cells modulates macrophage inflammatory responses through the JNK pathway [31]. Thus, we hypothesize that EPB41L4A-AS1 may contribute to the sepsis inflammatory response by regulating the NF-κB or JNK pathway through miR-146a-5p. In addition, integrated immune transcriptome regulators can provide a deeper mechanism of insight into the biological functions of EPB41L4A-AS1. A recent study using single-cell and transcriptomic methods has found that IL27RA is an important immunomodulator [38]. IL27RA is involved in regulating the activation of immune cells and the production of cytokines, which are key processes in the inflammatory response. EPB41L4A-AS1 may interact with IL27RA or other immune-related molecules to regulate the expression and function of miR-146a-5p. The mechanisms of EPB41L4A-AS1 will be further explored in the future. Evaluating the molecular mechanism of the EPB41L4A-AS1/miR-146a-5p axis in sepsis is a potential future research direction. This study has some limitations. The study did not use an animal model to validate the role of EPB41L4A-AS1 in the inflammatory response to sepsis. Additionally, this study did not examine the targets and signaling pathways through which the EPB41L4A-AS1/miR-146a-5p axis functions in sepsis. These limitations will be addressed in future studies.
In conclusion, this study demonstrated that EPB41L4A-AS1 can potentially serve as a diagnostic biomarker and a prognostic biomarker for sepsis and that it suppresses LPS-induced inflammation. The overexpression of miR-146a-5p suppressed the anti-inflammatory effects of EPB41L4A-AS1. Thus, EPB41L4A-AS1 regulates inflammation by targeting miR-146a-5p. These findings provide new insights for the treatment of sepsis and the elucidation of the pathological mechanisms of sepsis.
Supplementary Information
Supplementary Figure: Patients with sepsis were grouped by severity based on the APACHE II score, and the levels of EPB41L4A-AS1 were compared across different groups. Additionally, the absence of severe sepsis cases in the APAHCE II severe group is because we excluded patients with tumors, autoimmune diseases, or severe hepatic/renal insufficiency. ****P < 0.0001.
Acknowledgements
Not applicable.
Author contributions
WGZ, CL and RZ conceptualized and designed the study. WGZ, CL, and HXY collected, organized, and drafted the information. HXY and CL analyzed the data. WGZ wrote the manuscript. RZ and NNZ performed manuscript revision. All the authors have read and approved the manuscript.
Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.
Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Affiliated Hospital of Hebei University of Engineering. Each participant provided their signature on an informed consent form.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Weigui Zhou and Chan Li contributed equally to this work.
Contributor Information
Ningning Zhang, Email: zhangningning056@163.com.
Rui Zhang, Email: zhangruirj@163.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplementary Figure: Patients with sepsis were grouped by severity based on the APACHE II score, and the levels of EPB41L4A-AS1 were compared across different groups. Additionally, the absence of severe sepsis cases in the APAHCE II severe group is because we excluded patients with tumors, autoimmune diseases, or severe hepatic/renal insufficiency. ****P < 0.0001.
Data Availability Statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.