Abstract
Background
Severe acute pancreatitis (AP) is accompanied by systemic inflammatory process and organ dysfunction. Emerging evidence suggests that miRNA contributes to pancreatic injury. This study investigated miR-215-5p in AP and its mechanistic insight.
Methods
100 healthy controls and 130 patients with AP were enrolled. Based on disease severity, AP is classified into 72 mild acute pancreatitis (MAP) and 58 non-MAP (NMAP). Serum miR-215-5p abundance was quantified by qRT-PCR. The clinical significance was evaluated by logistic regression and ROC curve. A caerulein (CAE)-induced injury model was established in AR42J pancreatic acinar cells. Functional experiments, including cell viability, LDH release, and oxidative stress indicators, were performed. The RNA interaction was validated by dual-luciferase assay.
Results
miR-215-5p was elevated in AP and NMAP subjects. miR-215-5p positively correlated with BISAP score. miR-215-5p exhibits strong performance in distinguishing NMAP from MAP. Multivariate analysis identified miR-215-5p as an independent risk factor for NMAP. In vitro, CAE treatment upregulated miR-215-5p and induced ferroptosis, as substantiated by the accumulation of Fe²⁺, ROS, and MDA and decreased GSH and GPX4 expression. Inhibition of miR-215-5p alleviated cellular injury, including suppressed ferroptosis, and reduced inflammatory cytokine production. miR-215-5p directly binds to SLC7A11. SLC7A11 silencing reversed the protective role of miR-215-5p inhibition on AR42J cells.
Conclusions
miR-215-5p is elevated and associated with disease severity. miR-215-5p may promotes pancreatic acinar cell injury by targeting SLC7A11 and inducing ferroptosis.
Clinical trial number
Not applicable.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12876-026-05334-y.
Keywords: Severe acute pancreatitis, miR-215-5p, SLC7A11, Ferroptosis
Background
Acute pancreatitis (AP) is a digestive disorder marked by the inappropriate activation of pancreatic enzymes, thereby inducing autodigestion of pancreatic tissue and resulting in localized inflammatory damage to the pancreas, as well as varying degrees of involvement of surrounding tissues or organs [1]. The primary risk factors for AP include gallstones, alcohol consumption, and hypertriglyceridemia [2]. It manifests as acute upper abdominal pain and raised serum amylase or lipase [2]. The management of AP relies on timely medical interventions and nursing care, such as early fluid resuscitation monitoring, pain assessment and control, nutritional support, prevention of infection, and observation for complications [3]. The incidence of AP is on the rise. Over 20% of AP may progress to moderately severe AP or severe AP, accompanied by local or systemic complications such as pancreatic or peripancreatic tissue necrosis, and/or multiple organ dysfunction [4]. The pathogenesis of AP is complex and multifaceted, involving the modulation of diverse signaling pathways, such as intracellular calcium dysregulation in pancreatic cells, the generation of reactive oxygen species (ROS) [5, 6]. The precise molecular mechanisms underlying AP progression remain incompletely understood [7]. Recent evidence has demonstrated that ferroptosis, an iron-dependent form of regulated cell death characterized by excessive lipid peroxidation and oxidative stress, contributes to pancreatic acinar cell injury and inflammatory amplification during acute pancreatitis [8]. Further investigation into the pathophysiological mechanisms of AP is essential for improving therapeutic strategies and preventing disease progression.
miRNAs are a class of endogenously expressed non-coding RNAs that dynamically regulate target gene expression by specifically binding to the 3’ UTR of their messenger RNA [9]. miRNAs have been implicated in pathological processes, including cancer, inflammatory responses, and autoimmune diseases [10]. miR-9 attenuates caerulein-induced cell inflammation by targeting FGF10 to regulate the NF-κB pathway [11]. miR-216a promotes the progression of AP in mice by targeting PTEN and Smad7 to activate the AKT pathway [12]. miR-215-5p contributes to the progression of various chronic and inflammatory diseases. miR-215-5p is upregulated in severe AP by transcriptomic analysis [13]. In recurrent AP, miR-215-5p was recognized as an upregulated molecule and participates in ceRNA network regulation [14]. Preliminary data analysis indicates elevated miR-215-5p in the severe AP serum, suggesting its potential involvement in the pathogenesis. Bioinformatics prediction results indicate that SLC7A11 may be a potential target of miR-215-5p. SLC7A11 expression is downregulated in a mouse model of severe AP [15]. Suppression of SLC7A11 promotes ferroptosis and exacerbates pancreatic injury in severe acute pancreatitis [15]. Ferroptosis-related pathways, particularly the SLC7A11/GPX4 antioxidant axis, are critical for maintaining intracellular redox homeostasis and protecting against oxidative damage [16]. Since bioinformatics prediction identified SLC7A11 as a potential downstream target of miR-215-5p, we hypothesized that miR-215-5p may participate in severe AP progression through regulation of ferroptosis-related signaling.
This study systematically investigated the clinical significance and molecular mechanisms of miR-215-5p in AP. We evaluated the diagnostic value of miR-215-5p expression and its association with AP. A caerulein-induced cellular injury model was established to examine the function of miR-215-5p in pancreatic acinar cell lines and identified SLC7A11 as its downstream effector molecule. The study elucidates the miR-215-5p/SLC7A11 regulatory axis in AP and provides novel insights into potential therapeutic targets for this life-threatening condition.
Methods
Study subjects
130 AP patients admitted to Rudong People’s Hospital during the full year of 2023–2024 were consecutively enrolled. 100 healthy individuals who underwent routine physical examinations at Health Examination Center were recruited as control. The study was conducted in accordance with the Declaration of Helsinki. This study was approved by the Ethics Committee of Rudong People’s Hospital (Approval No. 2022-031, 9/16/2022). Written informed consent was obtained from all participants prior to enrollment after they were fully informed about the study purpose, procedures, potential risks, and benefits. Participants were assured that all data would be used solely for research purposes and confidentiality would be strictly maintained.
Inclusion criteria: [1] Diagnosis of AP based on the 2012 Revised Atlanta Classification (RAC), necessitating not less than two of the three criteria listed below: (i) characteristic clinical presentation (sudden, severe epigastric pain commonly radiating posteriorly to the back); (ii) serum pancreatic enzyme levels (amylase or lipase) ≥ 3-fold higher than reference upper limit; (iii) imaging features consistent with acute pancreatitis; [2] Age between 18 and 75 years; [3] Admission ≤ 24 h of symptom onset.
Exclusion criteria: [1] Past pancreatic tumors or acute flare-up of chronic pancreatitis; [2] Pre-existing severe dysfunction of major organs prior to AP onset; [3] Presence of infectious diseases, hematological disorders, metabolic diseases, or malignancies; [4] Pregnancy or lactation; [5] Pancreatitis secondary to trauma, surgery, tumors, or endoscopic retrograde cholangiopancreatography; [6] Prior use of antibiotics, corticosteroids, or hematopoietic stimulants before hospital admission.
Patients with AP were classified according to the 2012 Revised Atlanta Classification criteria. Mild acute pancreatitis (MAP) was defined as the absence of organ failure and local or systemic complications. Moderately severe acute pancreatitis (MSAP) was defined as transient organ failure (< 48 h) and/or local or systemic complications without persistent organ failure, whereas severe acute pancreatitis (SAP) was defined as persistent organ failure (> 48 h). For statistical analysis, MSAP and SAP patients were combined into a non-mild acute pancreatitis (NMAP) group because the SAP subgroup size was limited. Accordingly, the 130 AP volunteers were divided into 72 MAP and 58 NMAP (including both 53 MSAP and 5 SAP).
Blood sample collection
Approximately 5 mL of blood was drawn from all participants and allowed to clot at room temperature for 30 min, followed by centrifugation at 3,000 rpm for 10 min at 4 °C. The supernatant serum was acquired, aliquoted, and preserved at − 80 °C until further analysis.
Cell culture and AP model establishment
The rat pancreatic acinar cell line AR42J was obtained from ATCC (USA). Cells were grown in DMEM (Gibco, USA) contained 10% FBS (Gibco) at 37 °C. To establish an AP model, 2⋅105 AR42J cells/well were seeded in 6-well plates and treated with 10− 7 M caerulein (CAE; Sigma-Aldrich, USA) for 24 h. For ferroptosis inhibition experiments, cells were pretreated with ferrostatin-1 (Fer-1; Sigma-Aldrich, USA) at a final concentration of 2 µM for 2 h prior to CAE stimulation, followed by co-incubation with CAE (10⁻⁷ M) for 24 h. In all experiments involving Fer-1, an equivalent volume of vehicle (DMSO) was used as the control.
Cell transfection
2⋅105 AR42J cells/well were incubated in 6-well plates and transfected at 60–70% confluence using Lipofectamine 3000 (Invitrogen, USA). miR-215-5p inhibitor (miR-inhibitor), siRNA targeting SLC7A11 (si-SLC7A11), and matched negative controls (miR-NC/si-NC) were synthesized by GenePharma (Shanghai, China). The transfection doses were 50 nM for miRNA inhibitors and 100 nM for siRNAs. After transfection for 24 h, cells were treated with 10⁻⁷ M CAE for an additional 24 h prior to subsequent functional assays and molecular analyses. The sequences of transfection are provided in Supplementary Table S2. si-SLC7A11 #1 exhibited greater knockdown efficiency and more pronounced biological effects, it was selected for subsequent mechanistic experiments.
qRT-PCR
Total RNA was extracted from serum samples and AR42J cells using TRIzol (Invitrogen). The expression levels of miR-215-5p, SLC7A11, and GPX4 were determined by qRT-PCR analysis. Reverse transcription was performed using a miRNA-specific reverse transcription kit/PrimeScript RT reagent kit (Takara, Japan). qPCR was conducted using SYBR Green Master Mix (Takara). Results were expressed as relative changes using the 2⁻ΔΔCt method. U6 was used as the internal control for miR-215-5p, while GAPDH was used to normalize mRNA expression, including SLC7A11 and GPX4. The primer sequences were synthesized by Sangon (Shanghai, China). The sequences of qRT-PCR primers are provided in Supplementary Table S2.
Cell viability
5000 AR42J cells/well were plated in 96-well format plates. After 48 h of treatment, 10 µL/well of CCK-8 (Dojindo, Japan) was added and maintained for 2 h in incubator. 450 nm absorbance was assessed using a microplate reader (Bio-Rad, USA).
ELISA
The concentrations of pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cell culture supernatants were quantified using ELISA kits (R&D Systems, USA) according to the manufacturer’s instructions.
Western blot
AR42J samples were lysed using RIPA lysis buffer (Beyotime) in low temperature. Total protein (20 µg) was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking, membranes were incubated overnight at 4 °C with primary antibodies against GPX4 (Cat. No. ab125066, 1:1000) and ACSL4 (Cat. No. ab155282, 1:1000), SLC7A11 (Cat. No. ab307601, 1:1000), and β-actin (Cat. No. ab8227, 1:5000)/GAPDH (Cat. No. ab181602, 1:5000) (Abcam, UK). After washing, membranes were incubated with HRP-conjugated secondary antibodies at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, USA). Protein expression levels were quantified by densitometry using ImageJ software and normalized to β-actin as the internal loading control. All experiments were independently repeated at least three times.
Dual-luciferase reporter assay
The wild-type (WT) and mutant (MUT) SLC7A11 sequences were cloned into the pmirGLO vector (Promega, USA). 1⋅105 cells per well were seeded in 24-well format plates and cultured to approximately 60–70% confluence before co-transfected with WT or MUT reporter plasmids (0.5 µg) and either miR-215-5p mimic or negative control (NC) (final concentration: 50 nM). Luciferase activity was measured after 48 h using a Dual-Luciferase Reporter Assay System (Promega).
RIP assay
RIP assay was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. Briefly, AR42J cells were lysed in RIP lysis buffer, and cell extracts were incubated with magnetic beads conjugated with anti-Ago2 antibody or normal IgG antibody (Millipore) overnight at 4 °C. After protein digestion, the co-precipitated RNA was isolated and purified. The enrichment levels of miR-215-5p and SLC7A11 were determined by qRT-PCR analysis. IgG immunoprecipitation served as the negative control.
Measurement of Fe2+, LDH, MDA, GSH, and ROS
Fe2+ and Lactate dehydrogenase (LDH) content were measured using commercial kit (Cat. No. S1068S and Cat. No. C0016; Beyotime, China). Malondialdehyde (MDA) and glutathione (GSH) levels were determined using assay kits (Cat. Nos. A003-4-1 and A006-2-1, respectively; Nanjing Jiancheng, China). ROS degrees were detected using a ROS assay kit (Cat. No. S0033S; Beyotime) based on DCFH-DA fluorescence.
Statistical analysis
All data analyses (mean ± SD) were performed using GraphPad Prism 9.0.0 and R 4.0.0. Group comparisons were conducted using Student’s t-test, chi-square/Fisher’s exact test or one-way ANOVA followed by Bonferroni post hoc tests. Pearson correlation analysis was used to assess associations. Logistic regression analysis was performed to identify independent risk factors. ROC curve analysis was used to evaluate diagnostic performance. A two-tailed P < 0.05 was considered statistically significant.
Results
Clinical presentation of AP patients
Baseline clinical features of all subjects are shown in Table S1. The clinical characteristics of AP patients are listed in Table 1. There were no significant differences in terms of age, BMI, sex distribution, alcohol consumption, or underlying comorbidities, indicating good baseline comparability. Compared with the MAP group, NMAP patients exhibited significantly elevated levels of AST, BUN, Cr, WBC, CRP, PCT, TG, and GLU. In contrast, ALB and Ca2+ levels were markedly decreased in NMAP patients. BISAP scores were higher in the NMAP group, consistent with increased disease severity.
Table 1.
Baseline characteristics of MAP and NMAP patients
| Variable | MAP (n = 72) | SAP (n = 58) | P-value |
|---|---|---|---|
| Age, years | 44.47 ± 9.26 | 46.24 ± 11.14 | 0.334 |
| BMI | 24.66 ± 1.81 | 24.55 ± 1.45 | 0.701 |
| Male, n | 44 | 38 | 0.737 |
| Drinking, n | 40 | 34 | 0.499 |
| Underlying conditions | |||
| Hypertension, n | 15 | 16 | 0.489 |
| Diabetes, n | 18 | 17 | 0.724 |
| Hyperlipidemia, n | 24 | 25 | 0.336 |
| Cardiovascular and Cerebrovascular Diseases, n | 4 | 2 | 0.691 |
| Causes | 0.305 | ||
| Gallstone, n | 31 | 17 | |
| Alcoholic, n | 18 | 14 | |
| Hypertriglyceridemic, n | 14 | 18 | |
| Other, n | 9 | 9 | |
| ALT, U/L | 29.29 ± 10.09 | 32.20 ± 15.68 | 0.225 |
| AST, U/L | 29.80 ± 10.21 | 34.90 ± 13.40 | 0.018 |
| ALB, g/L | 36.90 ± 4.01 | 34.54 ± 3.64 | < 0.001 |
| BUN, mmol/L | 4.92 ± 0.69 | 7.05 ± 2.06 | < 0.001 |
| Cr, µmol/L | 61.02 ± 5.95 | 70.85 ± 24.05 | 0.003 |
| WBC, 109/L | 8.08 ± 2.60 | 11.04 ± 4.14 | < 0.001 |
| CRP, mg/L | 53.06 ± 37.04 | 87.04 ± 45.70 | < 0.001 |
| PCT, ng/mL | 0.44 ± 0.23 | 1.01 ± 1.09 | < 0.001 |
| K+, mmol/L | 3.87 ± 0.22 | 3.80 ± 0.19 | 0.075 |
| Ca2+, mmol/L | 2.23 ± 0.09 | 2.08 ± 0.11 | < 0.001 |
| TC, mmol/L | 4.83 ± 0.69 | 5.18 ± 1.84 | 0.169 |
| TG, mmol/L | 3.74 ± 1.40 | 4.50 ± 2.12 | 0.020 |
| GLU, mmol/L | 7.77 ± 0.94 | 9.81 ± 1.97 | < 0.001 |
| AMY, U/L | 342.28 ± 197.27 | 401.03 ± 230.24 | 0.126 |
| LPS, U/L | 660.23 ± 319.76 | 741.14 ± 356.62 | 0.180 |
| BISAP | < 0.001 | ||
| 0 | 20 | - | |
| 1 | 40 | 22 | |
| 2 | 12 | 31 | |
| 3 | - | 5 |
Abbreviations: ALB, Albumin; ALT, Alanine aminotransferase; AMY, Amylase; AST, Aspartate aminotransferase; BISAP, Bedside index for severity in acute pancreatitis; BMI, Body mass index; BUN, blood urea nitrogen; Ca2+, Calcium; Cr, Creatinine; CRP, C-Reactive Protein; LPS, Lipase; K+, Potassium; GLU, Glucose; MAP, mild acute pancreatitis; NMAP, non-mild acute pancreatitis; TC, Total Cholesterol; TG, Triglycerides; PCT, Procalcitonin; WBC, White Blood Cell Count
Association between miR-215-5p expression and predicts disease severity
Patients were categorized into low (n = 66) and high (n = 64) groups based on median serum miR-215-5p levels (Table 2). Patients with high miR-215-5p levels exhibited significantly increased levels of BUN, WBC, CRP, GLU, and LPS. Conversely, Ca2+ levels were reduced. No significant variation was noted in demographic characteristics or most liver function indicators. Multivariate analysis revealed that miR-215-5p (OR = 7.77, 95% CI: 2.34–30.18), along with BUN (OR = 6.17, 95% CI: 1.82–24.20), CRP (OR = 4.28, 95% CI: 1.18–18.11), Ca2+ (OR = 0.14, 95% CI: 0.03–0.47), BISAP score (OR = 8.25, 95% CI: 3.04–30.52), and were independent risk factors for severe AP (Table 3).
Table 2.
Relationship between miR-215-5p expression and clinical indicators in AP
| Variable | Low expression (n = 66) | High expression (n = 64) | P-value |
|---|---|---|---|
| Age, years | 44.89 ± 10.06 | 45.65 ± 10.28 | 0.676 |
| BMI | 24.66 ± 1.62 | 24.57 ± 1.70 | 0.748 |
| Male, n | 40 | 42 | 1.000 |
| Drinking, n | 34 | 40 | 0.276 |
| ALT, U/L | 29.67 ± 9.76 | 31.96 ± 15.47 | 0.239 |
| AST, U/L | 30.63 ± 11.20 | 33.57 ± 12.63 | 0.163 |
| ALB, g/L | 36.44 ± 3.79 | 35.24 ± 4.17 | 0.088 |
| BUN, mmol/L | 4.29 ± 1.20 | 6.46 ± 2.12 | < 0.001 |
| Cr, µmol/L | 64.34 ± 14.34 | 66.50 ± 19.96 | 0.480 |
| WBC, 109/L | 8.59 ± 3.25 | 10.24 ± 3.91 | 0.010 |
| CRP, mg/L | 55.80 ± 37.87 | 81.03 ± 47.11 | 0.001 |
| PCT, ng/mL | 0.58 ± 0.50 | 0.81 ± 1.00 | 0.112 |
| K+, mmol/L | 3.87 ± 0.21 | 3.80 ± 0.21 | 0.095 |
| Ca2+, mmol/L | 2.20 ± 0.11 | 2.12 ± 0.12 | < 0.001 |
| TC, mmol/L | 4.96 ± 0.95 | 5.01 ± 1.65 | 0.841 |
| TG, mmol/L | 3.85 ± 1.56 | 4.31 ± 1.99 | 0.140 |
| GLU, mmol/L | 8.17 ± 1.47 | 9.21 ± 1.96 | 0.001 |
| AMY, U/L | 345.550 ± 182.55 | 392.21 ± 240.97 | 0.216 |
| LPS, U/L | 634.60 ± 246.32 | 759.98 ± 403.65 | 0.035 |
Abbreviations: ALB, Albumin; ALT, Alanine aminotransferase; AMY, Amylase; AP, Acute pancreatitis; AST, Aspartate aminotransferase; BMI, Body mass index; BUN, Blood urea nitrogen; Ca2+, Calcium; Cr, Creatinine; CRP, C-reactive protein; LPS, Lipase; K+, Potassium; GLU, Glucose; TC, Total cholesterol; TG, Triglycerides; PCT, Procalcitonin; WBC, White blood cell count
Table 3.
Logistic regression analysis of risk factors for NMAP
| Parameters | Univariate | Multiple | |||
|---|---|---|---|---|---|
| OR value (95% CI) | P-value | OR value (95% CI) | P-value | ||
| Age | 1.47 (0.73, 2.97) | 0.273 | |||
| BMI | 0.77 (0.38, 1.55) | 0.480 | |||
| Sex | 1.20 (0.59, 2.50) | 0.605 | |||
| Drinking | 1.58 (0.79, 3.20) | 0.196 | |||
| ALT | 1.00 (0.49, 2.00) | 1.000 | |||
| AST | 1.65 (0.82, 3.33) | 0.159 | |||
| ALB | 0.46 (0.22, 0.94) | 0.035 | 0.32 (0.08, 1.05) | 0.069 | |
| BUN | 4.12 (1.98, 8.87) | < 0.001 | 6.17 (1.82, 24.20) | 0.005 | |
| Cr | 1.77 (0.88, 3.59) | 0.109 | |||
| WBC | 2.13 (1.05, 4.35) | 0.035 | 1.17 (0.33, 4.12) | 0.798 | |
| CRP | 3.89 (1.87, 8.36) | < 0.001 | 4.28 (1.18, 18.11) | 0.032 | |
| PCT | 1.37 (0.68, 2.76) | 0.367 | |||
| K+ | 0.58 (0.28, 1.16) | 0.126 | |||
| Ca2+ | 0.15 (0.06, 0.32) | < 0.001 | 0.14 (0.03, 0.47) | 0.002 | |
| TC | 0.60 (0.29, 1.21) | 0.159 | |||
| TG | 2.42 (1.20, 4.98) | 0.014 | 0.66 (0.16, 2.35) | 0.534 | |
| GLU | 3.85 (1.87, 8.16) | < 0.001 | 3.42 (0.99, 12.81) | 0.054 | |
| AMY | 1.74 (0.81, 3.53) | 0.117 | |||
| LPS | 1.45 (0.72, 2.93) | 0.290 | |||
| BISAP | 6.96 (3.58, 14.99) | < 0.001 | 8.25 (3.04, 30.52) | < 0.001 | |
| miR-215-5p | 6.27 (2.97, 13.80) | < 0.001 | 7.77 (2.34, 30.18) | 0.001 | |
Abbreviations: ALB, Albumin; ALT, Alanine aminotransferase; AMY, Amylase; AST, Aspartate aminotransferase; BISAP, Bedside index for severity in acute pancreatitis; BMI, Body mass index; BUN, Blood urea nitrogen; Ca2+, Calcium; Cr, Creatinine; CRP, C-reactive protein; LPS, Lipase; K+, Potassium; GLU, Glucose; NMAP, non-mild acute pancreatitis; TC, Total cholesterol; TG, Triglycerides; PCT, Procalcitonin; WBC, White blood cell count
miR-215-5p is elevated in NMAP and exhibits diagnostic value
Serum miR-215-5p expression was elevated in AP volunteers compared with healthy controls (Fig. 1A). miR-215-5p was markedly higher in NMAP than in MAP patients (Fig. 1B). miR-215-5p exhibited strong diagnostic performance for distinguishing NMAP from MAP (AUC = 0.851, Fig. 1C). miR-215-5p expression increased progressively with higher BISAP scores (Fig. 1D), indicating a close association with disease severity.
Fig. 1.
miR-215-5p is upregulated, as measured by qRT-PCR, in AP patients and correlates with disease severity. A Relative expression of miR-215-5p in healthy controls (n = 100) and AP patients (n = 130). B Relative expression of miR-215-5p in MAP (n = 72) and NMAP (n = 58) groups. C Receiver operating characteristic (ROC) curve analysis evaluating the diagnostic performance of miR-215-5p in distinguishing NMAP from MAP. D Relative expression of miR-215-5p in patients stratified by BISAP score. Data are presented as mean ± SD. ***P < 0.001, *P < 0.05
CAE induces pancreatic acinar cell injury and upregulates miR-215-5p
CAE treatment significantly reduced cell viability (Fig. 2A) and increased LDH release (Fig. 2B), confirming successful induction of cellular injury. miR-215-5p was significantly upregulated in CAE-treated cells, whereas transfection with a miR-215-5p inhibitor effectively suppressed its expression (Fig. 2C). To evaluate the specificity of the miR-215-5p inhibitor, the expression levels of several unrelated miRNAs were assessed following transfection. The results showed that inhibition of miR-215-5p did not significantly affect the expression of other tested miRNAs (Figure S2).
Fig. 2.
CAE induces pancreatic acinar cell injury and upregulates miR-215-5p expression. A Cell viability of AR42J cells following CAE treatment, assessed by CCK-8 assay. B LDH release in AR42J cells after CAE treatment. C Relative expression of miR-215-5p in AR42J cells under different conditions measured by qRT-PCR. Data are presented as mean ± SD from at least three independent experiments. **P < 0.01, ***P < 0.001
miR-215-5p inhibition attenuates ferroptosis and inflammatory injury
To investigate the function of miR-215-5p, ferroptosis-related markers were assessed. Inhibition of miR-215-5p reversed the CAE-induced increases in intracellular Fe2+ (Fig. 3A), ROS (Fig. 3B), and MDA levels (Fig. 3C), as well as the reduction in GSH levels (Fig. 3D). miR-215-5p inhibition restored GPX4 expression and suppressed ACSL4 expression, which had been altered by CAE treatment (Fig. 3E). Similarly, Fer-1 reversed the CAE-induced alterations in Fe2+, MDA, GSH, and GPX4 levels, indicating that ferroptosis is involved in CAE-induced cellular injury (Figure S1). These findings suggest that downregulated miR-215-5p protected cells against CAE-caused injury, at least in part, by suppressing ferroptosis. CAE-induced elevations in IL-6 and TNF-α were attenuated following miR-215-5p inhibition (Fig. 3F).
Fig. 3.
miR-215-5p inhibition attenuates CAE-induced ferroptosis and inflammatory responses in AR42J cells. A-D Levels of ferroptosis-related indicators, including Fe2+ A, ROS B, MDA C, and GSH D, measured using commercial biochemical assay kits. E Relative protein expression of GPX4 and ACSL4 detected by Western blot. F Levels of inflammatory cytokines IL-6 and TNF-α measured by ELISA. Data are presented as mean ± SD from at least three independent experiments. **P < 0.01, ***P < 0.001
SLC7A11 mediates the action of miR-215-5p on AR42J cells
SLC7A11 expression was significantly decreased in serum of NMAP patients (Fig. 4A) and in CAE-treated cells (Fig. 4B). Correlation analysis revealed an opposing association between miR-215-5p and SLC7A11 abundance (R = -0.650, Fig. 4C). Bioinformatics prediction identified a putative binding site. miR-215-5p mimic inhibited luciferase activity of the WT SLC7A11 reporter but failed to affect the MUT construct (Fig. 4D). RIP assay showed that both miR-215-5p and SLC7A11 were significantly enriched in the Ago2 immunoprecipitation group compared with the IgG control (Fig. 4E), indicating that miR-215-5p and SLC7A11 coexist in the same RNA-induced silencing complex. To exclude off-target effects, two siRNAs targeting SLC7A11 (si-RNA 1# and 2#) was used. Both siRNAs showed consistent effects on SLC7A11 expression and ferroptosis-related indicators (Fe2+ and MDA), confirming specificity (Figure S3). si-SLC7A11 #1 exhibited a more pronounced knockdown efficiency and biological effect and was selected for subsequent experiments. SLC7A11 was decreased in CAE-treated cells, whereas miR-215-5p inhibitor effectively promoted its expression (Fig. 5A). Co-transfection with si-SLC7A11 abolished the protective effects of miR-215-5p inhibition, as evidenced by increased Fe2+ (Fig. 5B), ROS (Fig. 5C), and MDA levels (Fig. 5D) and decreased GSH content (Fig. 5E). Silencing of SLC7A11 reversed the miR-215-5p inhibitor-induced upregulation of GPX4 and downregulation of ACSL4 (Fig. 5F). The reduction in IL-6 and TNF-α content induced by miR-215-5p inhibition was also negated by SLC7A11 knockdown (Fig. 5G).
Fig. 4.
miR-215-5p directly targets SLC7A11. A Relative serum expression of SLC7A11 in MAP (n = 72) and NMAP (n = 58) patients measured by qRT-PCR. B Relative expression of SLC7A11 in AR42J cells treated with CAE measured by qRT-PCR. C Pearson correlation analysis between miR-215-5p and SLC7A11 expression levels. D Dual-luciferase reporter assay showing the effect of miR-215-5p mimic on luciferase activity of wild-type (WT) and mutant (MUT) SLC7A11 3′UTR constructs. E RIP assay showing the enrichment levels of miR-215-5p and SLC7A11 in Ago2 and IgG immunoprecipitation complexes. Data are presented as mean ± SD from at least three independent experiments. **P < 0.01, ***P < 0.001
Fig. 5.
SLC7A11 mediates the regulatory effects of miR-215-5p on ferroptosis in AR42J cells. A Relative protein of SLC7A11 detected by Western blotting in AR42J cells under different treatments. B-E Effects of miR-215-5p inhibition and SLC7A11 silencing on ferroptosis-related indicators, including Fe2+B, ROS C, MDA D, and GSH E levels. F Relative protein levels of GPX4 and ACSL4 detected by Western blotting. G Levels of IL-6 and TNF-α detected by ELISA. Data are presented as mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Discussion
AP is a common inflammatory disorder of the pancreas that can lead to severe abdominal pain and multi-organ dysfunction [1]. miRNAs have been extensively investigated in AP, as they regulate disease pathogenesis and hold promise as diagnostic biomarkers and indicators of disease severity [11, 12]. In this study, miR-215-5p was upregulated in AP patients, suggesting a potential role in promoting inflammation and tissue injury. Notably, its expression was further elevated in NMAP, indicating a close association with disease severity. From a clinical perspective, patients with severe AP exhibit pronounced systemic inflammation accompanied by metabolic and electrolyte disturbances, including AST, BUN, Cr, WBC, CRP, PCT, TG, and GLU, alongside reduced ALB and Ca2+ levels. These findings are consistent with previous reports [17, 18], indicating that severe AP is characterized by systemic inflammation, metabolic disturbances, and organ dysfunction. Elevated BUN often reflects hypovolemia and hemodynamic instability and is considered an early predictor of severe AP severity [19]. Increased CRP and WBC levels indicate an amplified inflammatory response, which plays a central role in disease progression from mild to severe forms [20]. Elevated blood glucose reflects impaired pancreatic endocrine function and systemic metabolic disturbances [21]. Hypocalcemia, a common attribute of severe AP, is linked to fat saponification and increased disease severity [22]. miR-215-5p is elevated in NMAP patients and correlates with the aforementioned clinical markers, implying that miR-215-5p may be involved in the pathological process of AP and could serve as a biomarker for assessing disease severity. Previous studies have demonstrated elevated levels of inflammatory markers such as IL-6, TNF-α, IL-10, CRP, and leukocyte-associated inflammatory responses in AP patients, particularly in severe disease [23, 24]. In the present study, patients with elevated miR-215-5p expression exhibited increased WBC and CRP levels, further supporting the association between miR-215-5p and systemic inflammatory activation in AP. The BISAP, which combines blood biochemistry, clinical parameters, and imaging assessments to identify high-risk patients at an early stage, is commonly used for initial screening in emergency departments [25]. miR-215-5p levels increased progressively with higher BISAP scores. This association further supports the close connection between miR-215-5p expression and the systemic burden and clinical severity of AP. miR-215-5p remained an independent predictor for severe AP after controlling for conventional clinical variables, underscoring its robustness in predicting disease progression. Notably, high miR-215-5p expression group exhibited elevated lipase levels, a pancreas-specific marker of injury, further supporting its relevance in AP. miR-215-5p possesses good diagnostic performance in distinguishing NMAP from MAP, indicating its potential as a noninvasive molecular marker for clinical application.
AR42J cells, which retain key characteristics of pancreatic acinar cells and are widely used to model AP-related cellular injury was employed [26]. CAE, a cholecystokinin analog, is commonly used to induce acinar cell hyperstimulation due to its high reproducibility and controllability [27]. Consistent with clinical findings, miR-215-5p was upregulated in CAE-exposed cells. Inhibition of miR-215-5p alleviated CAE-induced cellular injury, suggesting that its aberrant upregulation contributes to pancreatic acinar cell damage. Ferroptosis, a regulated cell death process driven by iron-dependent lipid peroxidation, induces pancreatic acinar cell loss in AP and represents a key mechanism driving pancreatic injury and inflammatory responses [8]. Fe²⁺ catalyzes the generation of extremely reactive hydroxyl radicals via Fenton reactions, which insult polyunsaturated fatty acids (PUFAs) and trigger lipid peroxidation [28]. ROS act as key mediators in this process. MDA, a byproduct of lipid peroxidation indicative of oxidative damage [29]. GSH, in conjunction with GPX4, constitutes a major antioxidant defense system that suppresses ferroptosis, whereas ACSL4 acts as a positive regulator by promoting PUFA incorporation into membrane phospholipids [16]. CAE treatment led to increased intracellular Fe²⁺, ROS, and MDA levels, accompanied by decreased GSH content and reduced GPX4 expression, which are hallmark features of ferroptosis. These alterations were partially reversed by treatment with the ferroptosis inhibitor, confirming the involvement of ferroptosis in CAE-induced cellular injury. Inhibition of miR-215-5p attenuated these ferroptotic changes, indicating that miR-215-5p promotes pancreatic acinar cell injury, at least in part, through the ferroptosis pathway. Inflammatory signaling play a crucial role in AP advancement. IL-6 and TNF-α were selected as representative pro-inflammatory cytokines in the present cellular experiments to evaluate inflammatory activation associated with ferroptosis-mediated pancreatic acinar cell injury. Inhibition of miR-215-5p reduced CAE-mediated elevations in IL-6 and TNF-α levels, suggesting that the miR-215-5p/SLC7A11 axis contributes to inflammatory signaling during AP progression. TNF-α is one of the earliest pro-inflammatory factors to be elevated. It triggers the NF-κB, leading to the release of chemokines and recruitment of inflammatory cells [30]. IL-6 further amplifies and sustains the inflammatory cascade [31]. Inhibition of miR-215-5p reduced CAE-mediated elevations in IL-6 and TNF-α amounts, suggesting that miR-215-5p acts as an activator of inflammatory responses in pancreatic acinar cells.
SLC7A11 was identified as a downstream target of miR-215-5p. SLC7A11 is a key component of the cystine/glutamate antiporter system Xc⁻, which is essential for maintaining intracellular redox homeostasis by regulating glutathione synthesis [32]. Downregulation of SLC7A11 impairs cystine uptake, leading to loss of GSH contributes to ferroptosis sensitization [16]. SLC7A11 was reduced in both NMAP patients and CAE-treated cells and was negatively correlated with miR-215-5p levels. Rescue experiments demonstrated that silencing SLC7A11 prevented the protective impact of miR-215-5p inhibition, thereby confirming that SLC7A11 mediates the pro-ferroptotic effects of miR-215-5p. When SLC7A11 was suppressed, the anti-inflammatory function of miR-215-5p downregulation were abolished, suggesting that the miR-215-5p/SLC7A11 axis may regulate inflammatory signaling pathways, potentially via ferroptosis-related mechanisms.
Several limitations should be acknowledged. This study was conducted at a single center with a relatively limited sample size, which may introduce selection bias. Patients were classified according to the 2012 Revised Atlanta Classification criteria; MSAP and SAP cases were combined into a single NMAP group for statistical analysis because of the limited number of SAP patients. Although this strategy improved statistical power, potential biological and clinical heterogeneity between MSAP and SAP patients may not have been fully captured. Although the present study demonstrated the regulatory role of miR-215-5p in pancreatic acinar cell ferroptosis using a caerulein-induced in vitro model, in vivo validation was not performed. Whether modulation of miR-215-5p can influence pancreatic injury severity, systemic inflammation, and organ dysfunction in acute pancreatitis animal models remains to be determined. Elevated serum miR-215-5p levels were detected in NMAP patients. However, the present study could not distinguish whether miR-215-5p primarily exerts local effects within pancreatic tissue or functions systemically as a circulating regulatory molecule. Future investigations employing animal models, tissue-specific expression analysis, and in vivo gain- and loss-of-function approaches are warranted to further clarify the biological role and therapeutic potential of miR-215-5p in severe AP. An additional consideration concerns the biological interpretation of circulating SLC7A11 mRNA. In the present study, SLC7A11 expression was measured from total serum RNA by qRT-PCR; therefore, the detected transcripts cannot be assumed to originate directly from pancreatic tissue. Circulating SLC7A11 mRNA may be present as cell-free extracellular RNA, associated with extracellular vesicles, or released into the circulation secondary to cellular injury and tissue damage. Thus, the serum SLC7A11 measurements in this study should be interpreted primarily as an indicator of circulating RNA abundance rather than a pancreas-specific readout of SLC7A11 expression. The biological origin of circulating SLC7A11 mRNA remains unresolved in the present study. Future studies incorporating tissue-specific analyses and characterization of extracellular RNA compartments will be required to clarify the source and biological significance of circulating SLC7A11 in acute pancreatitis.
Conclusions
miR-215-5p is upregulated in severe AP patients and is closely associated with disease severity. miR-215-5p may promotes pancreatic acinar cell injury by targeting SLC7A11 and inducing ferroptosis. These findings provide novel insights into the molecular mechanisms underlying acute pancreatitis progression. However, given the observational nature of the clinical data, the association between miR-215-5p and disease severity should be further explored.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
Not applicable.
Author contributions
Conceptualization, JYL, DQS and CL; Data curation, JYL and DQS; Formal analysis, QG, CYL, JYL and CL; Investigation, QG, CYL, JYL and DQS; Methodology, QG, CYL, DQS and CL; Project administration, DQS and CL; Resources, JYL; Software, QG, CYL and JYL; Supervision, DQS and CL; Validation, QG, CYL, JYL and CL; Visualization, QG, CYL, JYL, DQS and CL; Writing - original draft, JYL; Writing - review & edit-ing, QG, CYL, DQS and CL.
Funding
No funding was received to assist with the preparation of this manuscript.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki. This study was approved by the Ethics Committee of Rudong People’s Hospital. Written informed consent was obtained from all participants.
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.
Qian Gao and Chunyan Li contributed equally to this work.
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Supplementary Materials
Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.