Abstract
Background
Severe acute pancreatitis (SAP) is characterized by persistent organ failure and a high mortality rate, early diagnosis and disease assessment are crucial for improving patient prognosis.
Aims
To investigate the expression level, diagnostic value, and mechanism of action of serum miR-331-3p in patients with SAP.
Methods
One hundred eighty-five patients with AP (including 130 patients with mild-to-moderate acute pancreatitis (MAP) and 55 patients with SAP) and 100 healthy controls (HC) were enrolled. RT-qPCR was conducted to detect the expression of serum miR-331-3p, and its diagnostic efficacy was analyzed using receiver operating characteristic (ROC) curves. Correlation analysis was carried out using the Pearson correlation coefficient. The molecular mechanism was explored by combining dual-luciferase reporter gene assay and cell function experiments.
Results
The level of serum miR-331-3p expression in the SAP group was markedly lower than that in the MAP and HC groups (P < 0.05). The AUC for distinguishing MAP from SAP was 0.866 (95% CI: 0.810–0.921), with a sensitivity of 83.6% and a specificity of 73.1%. miR-331-3p was strongly negatively correlated with SAP routine indicators, severity, and inflammation (P < 0.001). Mechanistically, miR-331-3p directly targets bromodomain protein 4 (BRD4), and the overexpression of miR-331-3p alleviate the inflammatory response in the SAP cell model and promote cell proliferation, whereas BRD4 overexpression reversed the effects of miR-331-3p.
Conclusions
Serum miR-331-3p is notably downregulated in SAP and may attenuate pancreatic inflammation through BRD4 targeting and inhibition, proposing its candidacy as a diagnostic biomarker.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12876-026-04638-3.
Keywords: SAP, Diagnosis, miR-331-3p, BRD4
Introduction
Severe acute pancreatitis (SAP) the most severe subtype of acute pancreatitis (AP), progresses rapidly and often leads to multiple organ dysfunction syndrome, seriously threatening the life and health of patients [1–3]. SAP has a relatively high mortality rate, which poses significant challenges for clinical diagnosis and treatment [4]. With respect to pathogenesis, autodigestion of the pancreas caused by abnormal activation of pancreatic enzymes is considered to be the initial event. Subsequently, a massive release of inflammatory cytokines further exacerbates inflammatory damage to the pancreas and the entire body [5, 6]. The clinical detection of serum amylase and lipase, although widely used in the diagnosis of AP, is limited by insufficient specificity and sensitivity [7]. Therefore, there is a urgent need to find new and efficient biomarkers for the early diagnosis and disease assessment of SAP.
In the field of AP, multiple studies have confirmed that miRNA expression profiles significantly change in the serum and pancreatic tissues of patients, and are closely associated with the occurrence, development, and severity of the disease [8, 9]. For instance, miR-551b-5p is substantially upregulated in the serum of patients with AP, and its expression level is positively correlated with the levels of inflammatory factors [10]. Similarly, miR-155 is abnormally expressed in AP, influencing the inflammatory process by regulating the polarization of macrophages [11]. These findings provide new perspectives for studying AP and make it possible for miRNAs to serve as potential biomarkers and therapeutic targets [12]. This study focuses on miR-331-3p, and previous studies have suggested that it has a potential regulatory role in inflammatory diseases [13]. In a model of ulcerative colitis, miR-331-3p expression is downregulated, and overexpression of miR-331-3p can alleviate intestinal inflammation by inhibiting target genes [14]. A study found that miR-331-3p was expressed was reduced in SAP associated with acute lung injury [15]. Additionally, miR-331-3p was revealed to mediate NLRP6-mediated suppression of inflammation following intracerebral hemorrhage [16]. miR-331-3p alleviated neuropathic pain and inflammatory response after SCI by targeting RAP1A [17]. However, the expression pattern and clinical significance of miR-331-3p in the context of SAP have yet to be reported.
Therefore, this study aims to systematically investigate the expression level of serum miR-331-3p in SAP patients, evaluate its diagnostic value for SAP, and deeply explore the underlying molecular mechanism to provide a new theoretical basis and potential targets for the early diagnosis and precise treatment of SAP.
Materials and methods
Study subjects
A total of 185 patients with AP admitted to The First People’s Hospital of Lanzhou were enrolled, including 130 cases of MAP group and 55 cases of SAP group. All patients met the diagnostic criteria specified in the Revised Atlanta Classification (2012) [18]. During the same period, 100 healthy volunteers who underwent physical examinations in our hospital were included in the HC group. This study was approved by the Ethics Committee of our hospital, and all subjects signed the informed consent form.
Inclusion criteria: For patients with MAP group, patients include those with mild AP without signs of organ failure and no complications, as well as those with moderately SAP presenting transient organ failure (< 48 h) and/or local or systemic complications, but without persistent organ failure. For patients with SAP group, eligibility requires confirmation of AP alongside the presence of persistent organ failure (lasting beyond 48 h). For the HC group, individuals were required to have no documented history of diseases related to the digestive system, exhibit normal liver or kidney function, and be free from recent infections, inflammatory conditions, or other significant illnesses. Exclusion criteria: Complicated with other pancreatic diseases such as pancreatic cancer and chronic pancreatitis; severe hepatic and renal insufficiency, malignant tumors, or autoimmune diseases; a history of major surgery or infection within the past month; pregnancy or lactation; and incomplete clinical data.
A post hoc Power analysis was performed with G*Power statistical software, which showed a power of 92.68%, indicating that our study was sufficiently powered to detect the observed effect.
Clinical data and sample collection
Clinical data, including the patient’s gender, age, and etiological composition, were collected. The APACHE II score and Ranson score were calculated within 24 h of admission. Fasting venous blood was collected from all participants in the early morning following their admission or on the day of their comprehensive physical examination. Following centrifugation at 4°C, 3000×g for 10 min, the serum was subsequently isolated into RNA-free EP tubes and preserved in an ultralow temperature refrigerator at -80℃ for future analysis.
Serum amylase and lipase levels were estimated using an automated biochemical analyzer, whereas C-reactive protein (CRP) levels were assessed through immunoturbidimetry. Additionally, the serum concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by ELISA. All procedures were conducted strictly following the protocols outlined in the respective assay kits (R&D Systems, USA), and the results are summarized in Table 1.
Table 1.
Clinical data of the subjects
| Variable | HC (n = 100) | AP (n = 185) | P-value | |
|---|---|---|---|---|
| MAP (n = 130) | SAP (n = 55) | |||
| Age (years) | 48.41 ± 6.14 | 48.29 ± 7.06 | 49.76 ± 6.26 | 0.356 |
| Gender (male/female) | 55/45 | 71/59 | 31/24 | 0.976 |
| BMI (kg/m2) | 22.18 ± 1.79 | 21.85 ± 1.39 | 22.01 ± 1.53 | 0.290 |
| Amylase (U/L) | 89.39 ± 12.92 | 361.59 ± 23.31a | 498.09 ± 62.59ab | < 0.001 |
| Lipase (U/L) | 87.68 ± 15.48 | 800.86 ± 60.34a | 1484.09 ± 219.78ab | < 0.001 |
| CRP (mg/L) | 4.49 ± 0.88 | 47.06 ± 8.13a | 153.29 ± 18.58 ab | < 0.001 |
| TNF-α (pg/mL) | 10.05 ± 2.97 | 38.83 ± 15.93a | 119.24 ± 32.67 ab | < 0.001 |
| IL-6 (pg/mL) | 7.24 ± 1.35 | 79.60 ± 16.30 a | 248.55 ± 63.92ab | < 0.001 |
| APACHE II score | / | 4.41 ± 1.68 | 13.22 ± 3.29 b | / |
| Ranson score | / | 1.05 ± 0.84 | 5.25 ± 1.65 b | / |
| Etiology (n, %) | ||||
| biliary | / | 64 (49.2) | 25 (45.5) | / |
| hypertriglyceridemia | / | 42 (32.3) | 18 (32.7) | / |
| alcoholic | / | 24 (18.5) | 12 (21.8) | / |
| Organ Failure (n, %) | ||||
| Respiratory failure | / | / | 32 (58.2) | / |
| Renal failure | / | / | 17 (30.9) | / |
| Circulatory failure | / | / | 15 (27.3) | / |
| Local complications (n, %) | / | 46 (35.4) | 53 (96.4) | / |
HC Health control, AP acute pancreatitis, MAP Mild to moderate acute pancreatitis, SAP Severe acute pancreatitis, BMI body mass index, CRP C-reactive protein, TNF-α tumor necrosis factor-α, IL-6 interleukin-6, APACHE acute physiology and chronic health evaluation
Pa < 0.001, compared with the HC group
Pb < 0.001, compared with the MAP group
P-value, AP group was compared with the HC group
RT-qPCR
Following the instructions provided in the RT-qPCR kit (Thermo Fisher Scientific, USA), total RNA was extracted from the serum samples, after which complementary DNA (cDNA) was synthesized through TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific, USA). RT-qPCR analysis was performed using the 7300 Real-Time Fluorescent Quantitative PCR System (Applied Biosystems; Thermo Fisher Scientific, USA) in combination with the SYBR-Green Real-Time Fluorescent Quantitative PCR Kit (Thermo Fisher Scientific, USA). U6 served as the internal reference gene for the subsequent PCR amplification. Analysis using the NormFinder algorithm confirmed no significant difference in U6 expression between the SAP group and HC group (P > 0.05), thereby establishing the suitability of U6 as a standardization benchmark. The reaction conditions were as follows: pre-denaturation at 95℃ for 30 s; followed by 40 cycles of denaturation at 95℃ for 5 s, and annealing at 60℃ for 30 s. Each sample was set up in triplicate, with three replicates performed to ensure the reliability of the quantitative results. The relative expression level of miR-331-3p was calculated using the 2−ΔΔCt method. Primer sequences are as follows: miR-331-3p forward, 5’-GAGCTGAAAGCACTCCCAA-3’ and reverse 5’-CACACTCTTGATGTTCCAGGA-3’; U6 forward, 5’-GCTTCGAGGCAGGTTACATG-3’ and reverse 5’-GCAACACACAACATCTCCCA-3’.
Cell culture
HPDE6-C7 cells were purchased from ATCC and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen) and maintained in a cell culture incubator at 37℃ with 5% CO₂. STR analysis was performed before experiments to confirm the absence of mycoplasma contamination in the cells. All experiments were performed using cells with passages 3 to 5. Upon reaching the logarithmic growth phase, the cells were treated with 10 nM cerulein (Cer; Solarbio, USA) and 10 mg/L lipopolysaccharide (LPS; Solarbio, USA) for 24 h to establish a cell model of SAP [19].
Cell transfection
Cells were cultured in six-well plates, and transfection was conducted in accordance with the protocol provided by the Lipofectamine 3000 reagent (Invitrogen, USA) once the cell confluency reached 70–80%. The miR-331-3p mimic, miR-NC, oe-BRD4 and oe-NC involved in the experiment were purchased from Guangzhou RiboBio Co., Ltd. The cells were divided into the following groups: control group (without any treatment), Cer group (treated with caerulein) Cer-LPS group (treated with caerulein and LPS), Cer-LPS + miR-NC group (transfected with the miR-331-3p negative control), Cer-LPS + miR-331-3p mimic group (transfected with the miR-331-3p mimic), Cer-LPS + miR-331-3p mimic + oe-NC group (co-transfected with the miR-331-3p mimic and pcDNA3.1), and Cer-LPS + miR-331-3p mimic + oe-BRD4 group (co-transfected with the miR-331-3p mimic and pcDNA3.1-BRD4).
CCK-8 assay
Transfected cells were initially seeded at a density of 100 µL/well in 96-well plates, with three replicate wells designated for each experimental group. Following incubation for 24, 48, and 72 h, 10 µL of CCK-8 solution (Promega, USA) was introduced to each well, and the cultures were subsequently maintained for an additional 2 h. The OD values were measured at a wavelength of 450 nm using a microplate reader, with these OD values served as indicators of the ability of the cells to proliferate.
Detection of inflammatory factors
The supernatants from each cellular group were collected, and subsequent quantification of IL-6 (Human IL-6 Quantikine ELISA Kit, D6050; Sensitivity: 0.7 pg/mL; Assay Range: 3.1–300 pg/mL) and TNF-α (Human TNF-α Quantikine ELISA Kit, DTA00D; Sensitivity: 0.049 pg/mL, Assay range: 0.2–10 pg/mL) levels was performed using ELISA. This procedure was conducted rigorously following the manufacturer’s guidelines provided with the kit (R&D Systems, USA).
Apoptosis of cells
Following cell transfection or treatment, cells were digested by trypsin and washed with PBS to remove residual serum. Subsequently, the cells were resuspended in 100 µL Annexin V Binding Buffer, and 5 µL Annexin V-FITC and 5 µL PI solution (BD Biosciences, CA, USA) were added. Cell suspensions were incubated for 15 min in the dark. Finally, it was detected by flow cytometry.
Dual-luciferase reporter gene assay
Binding sites between miR-331-3p and BRD4 were predicted, and wild-type (wt) and mutant (mut) BRD4 3’UTR luciferase reporter gene vectors were constructed. BRD4-wt or BRD4-mut was co-transfected with the miR-331-3p mimic or negative control into HPDE6-C7 cells. After 48 h, the ratio of firefly luciferase activity to Renilla luciferase activity (relative activity) was estimated using a dual-luciferase reporter gene kit (Solarbio, Beijing, China).
Statistical analysis
Analyses were performed using SPSS 26.0 software, and graphs were generated with GraphPad Prism 9. The normality of the data distribution was assessed by the Shapiro-Wilk test. The quantitative data were expressed as mean ± standard deviation (SD). Comparisons among multiple groups were conducted by ANOVA, followed by Tukey’s HSD post-hoc test, with pairwise comparisons performed using the least significant difference t-test (LSD-t test). The diagnostic performance was evaluated by ROC curve and AUC, and the optimal cut-off value was determined by Youden index. The 5-fold cross-validation analysis provided a more rigorous internal diagnostic assessment, and the results are shown in Supplementary Table 1.
Results
Baseline data of the study subjects
No significant differences in the distributions of gender or age were found among the SAP group, MAP group, and HC group (P > 0.05). Additionally, the etiological composition did not differ significantly between the SAP group and the MAP group (P > 0.05). In terms of serological indicators, the levels of serum amylase, lipase, CRP, TNF-α, and IL-6 in the SAP group were significantly higher than those in the MAP group and the HC group (P < 0.001). Furthermore, the concentrations of these indicators in the MAP group were significantly greater than those in the HC group (P < 0.001) (Table 1).
Expression and diagnostic value of serum miR-331-3p in SAP
The relative expression levels of serum miR-331-3p in the HC, MAP, and SAP groups were 1.01 ± 0.17, 0.78 ± 0.16, and 0.51 ± 0.17, respectively. Notably, the MAP group presented significantly lower expression than the HC group (P < 0.001), while the SAP group demonstrated a significantly lower expression than both the MAP and HC groups (P < 0.001) (Fig. 1A). The ROC curve analysis showed that the AUC of miR-331-3p for differentiating healthy individuals from MAP patients was 0.822 (95% CI: 0.768–0.876), with an optimal cutoff value of 0.87, a sensitivity of 73.1%, and a specificity of 81.0% (Fig. 1B). The AUC for differentiating MAP from SAP patients was 0.866 (95% CI: 0.810–0.921), with an optimal cutoff value of 0.66, a sensitivity of 83.6%, and a specificity of 73.1% (Fig. 1C). Additionally, patients were divided into good and poor prognosis group according to their prognostic outcome, and the results showed that serum miR-331-3p was down-regulated in the poor prognosis group (Fig. 1D). ROC analysis revealed an AUC of 0.816 to differentiate the two groups of patients (Fig. 1E).
Fig. 1.
Expression level, diagnostic efficacy of serum miR-331-3p and its correlation with clinical indicators. A. The expression of serum miR-331-3p in the SAP group was significantly lower than that in the MAP group and HC group (P < 0.001); B-C. ROC curves showed the diagnostic efficacy of miR-331-3p in distinguishing HC from MAP (AUC=0.822) and MAP from SAP (AUC=0.866); D. Serum miR-331-3p was down-regulated in the poor prognosis group; E. The ROC curve reflected the diagnostic effect of miR-331-3p for good prognosis and poor prognosis (AUC=0.816). Experiments were performed in triplicate biological replicates, and data are presented as mean ± SD. ***P < 0.001, Compared with the HC group/good prognosis; ###P < 0.001, Compared with the MAP group
Correlation analysis between serum miR-331-3p and clinical indicators
Pearson correlation analysis demonstrated that the serum miR-331-3p level was significantly negatively correlated with the levels of amylase (r=-0.757, P < 0.001, Fig. 2A) and lipase (r=-0.739, P < 0.001, Fig. 2B). Meanwhile, a notable inverse relationship between the serum miR-331-3p level and both the APACHE II score (r=-0.742, P < 0.001, Fig. 2C) and the Ranson score (r=-0.719, P < 0.001, Fig. 2D). Furthermore, a notable inverse relationship was observed between the serum miR-331-3p level and inflammatory markers, including CRP, TNF-α, and IL-6, with correlation coefficients of r=-0.716, r=-0.687, and r=-0.641 (P < 0.001, Figs. 2E-G).
Fig. 2.
Correlation between serum miR-331-3p and clinical indicators. A-B. miR-331-3p showed a negative correlation with amylase (r=-0.757) and lipase (r=-0.739) (P < 0.001); C-D. miR-331-3p was significantly negatively correlated with APACHE II score (r=-0.742) and Ranson score (r=-0.719) (P < 0.001); E-G. miR-331-3p was significantly negatively correlated with inflammatory indicators including CRP (r=-0.716), TNF-α (r=-0.687), and IL-6 (r=-0.641) (P < 0.001)
Validation of the SAP cell model
Cellular experimental results revealed that, in comparison to the control group, the expression level of miR-331-3p in the Cer-treated group was markedly decreased (P < 0.01). Furthermore, the miR-331-3p level in the Cer-LPS-treated group was significantly lower than that in the Cer-treated group (P < 0.01) (Fig. 3A). The cell proliferation assay indicated that the OD value in the Cer-treated group was markedly reduced relative to the control group (P < 0.01), with the OD value in the Cer-LPS-treated group exhibiting a further notable decline (P < 0.001) (Fig. 3B). Concurrently, inflammatory factor assays demonstrated that the levels of TNF-α and IL-6 in the Cer-treated group were substantially elevated compared to the control group (P < 0.001), and these inflammatory factors were further augmented in the Cer-LPS-treated group (P < 0.01) (Figs. 3C-D).
Fig. 3.
Validation of the SAP cell model. A Cer-LPS treatment significantly decreased cellular miR-331-3p expression (P < 0.001); (B) Cer-LPS inhibited cell proliferation (P < 0.01); C-D. Cer-LPS significantly increased the levels of TNF-α and IL-6 (P < 0.001). Experiments were performed in triplicate biological replicates, and data are presented as mean ± SD. **P < 0.01, ***P < 0.001, Compared with the control group; #P < 0.05, ##P < 0.01, Compared with the Cer group
Validation of the targeting relationship between miR-331-3p and BRD4
Further transfection experiments were conducted to modulate the expression of miR-331-3p in SAP model cells. The findings revealed a significant reduction in miR-331-3p levels in these model cells. Subsequent transfection with the miR-331-3p mimic resulted in a notable increase in miR-331-3p expression, whereas transfection with the miR-331-3p inhibitor yielded the opposite effect (P < 0.001, Fig. 4A). Bioinformatics analyses indicated the presence of complementary binding sites between miR-331-3p and the 3’ UTR of BRD4 (Fig. 4B). The dual luciferase reporter gene assay revealed a significant decrease in luciferase activity in the BRD4-wt + miR-331-3p mimic group than in the BRD4-wt + miR-NC group (P < 0.001), whereas no significant differences were detected among the BRD4-mut groups (P > 0.05, Fig. 4C), which confirmed that miR-331-3p directly targets BRD4. Additionally, we found that the expression level of BRD4 mRNA was significantly elevated in SAP model cells and that the upregulation of miR-331-3p significantly inhibited the expression of BRD4 mRNA in model cells (P < 0.01), whereas knocking down miR-331-3p had the opposite effect (P < 0.01, Fig. 4D).
Fig. 4.
Validation of the targeting relationship between miR-331-3p and BRD4. A. Transfection with miR-331-3p mimic/inhibitor effectively regulated its expression in model cells (P < 0.01); B-C. Prediction of binding sites between miR-331-3p and BRD4 3’UTR, and Dual-luciferase assay confirmed their targeted binding (P < 0.001); D. The expression of BRD4 mRNA was increased in SAP model cells; upregulation of miR-331-3p inhibited its expression, while knockdown promoted it (P < 0.01); **P < 0.01, ***P < 0.001, Compared with the control group; ##P < 0.01, ###P < 0.001, Compared with the Cer-LPS group. E-F. The expression of BRD4 mRNA in the SAP group was significantly higher than that in the MAP group and HC group (P < 0.001), and its level was negative correlation with the expression of miR-331-3p (r=-0.824, P < 0.001). Experiments were performed in triplicate biological replicates, and data are presented as mean ± SD. ***P < 0.001, Compared with the HC group; ###P < 0.001, Compared with the MAP group
Analysiss of clinical samples revealed that the expression level of BRD4 mRNA in the MAP group was elevated compared to that in the HC group. Moreover, a significant increase in BRD4 mRNA expression was observed in the SAP group, surpassing levels found in the HC group and the MAP group (P < 0.001, Fig. 4E). Moreover, a significant negative correlation was identified between the expression levels of miR-331-3p and BRD4 mRNA in SAP patients (r=-0.824, P < 0.001, Fig. 4F).
Effects of miR-331-3p targeting BRD4 on SAP model cells
To determine the role of miR-331-3p in SAP model cells through the targeting of BRD4, we conducted co-transfection experiments to modulate the expression levels of BRD4 mRNA in these cells (Fig. 5A). RT-qPCR detection revealed that Cer-LPS treatment increased BRD4 mRNA content in cells, while transfection with miR-331-3p mimic downregulated its expression. Following co-transfection with BRD4 under these conditions, its levels were substantially restored (P < 0.01). The results from the cell proliferation assays indicated that, in comparison to the model group, the upregulation of miR-331-3p markedly enhanced the proliferation of SAP model cells (P < 0.01). Notably, the overexpression of BRD4 reversed this effect (P < 0.05) (Fig. 5B). The results of cytokine detection showed that the concentrations of IL-6 and TNF-α in the cell supernatant of the SAP model group were markedly higher than those in the control group. Intergroup comparisons revealed that, compared to those in the model + miR-NC group, the concentrations of IL-6 and TNF-α in the cell supernatant of the model + miR-331-3p mimic group were significantly lower (P < 0.001); compared to those in the model + miR-331-3p mimic + oe-NC group, the concentrations of the aforementioned cytokines in the model + miR-331-3p mimic + oe-BRD4 group were significantly increased (P < 0.01) (Fig. 5C). Additionally, cell apoptosis rate was increased in Cer-LPS-treated group, while miR-331-3p mimic reduced cell apoptosis. Co-transfection of over-expressing miR-331-3p and BRD4 counteracted the effect of miR-331-3p (P < 0.001) (Fig. 5D).
Fig. 5.
Effects of miR-331-3p targeting BRD4 on cells. A Co-transfection effectively regulated BRD4 expression; (B) Overexpression of miR-331-3p promoted cell proliferation, and overexpression of BRD4 reversed this effect (P < 0.01); (C) Overexpression of miR-331-3p decreased the levels of IL-6 and TNF-α, and overexpression of BRD4 reversed this effect (P < 0.001); (D) Overexpression of miR-331-3p suppressed apoptosis, while overexpression of BRD4 counteracted this effect (P < 0.001). Experiments were performed in triplicate biological replicates, and data are presented as mean ± SD. **P < 0.01, ***P < 0.001, Compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001, Compared with the Cer-LPS group
Discussion
SAP is a critical disease characterized by uncontrolled local pancreatic inflammation, systemic inflammatory response syndrome, and multiple organ dysfunction, and early diagnosis and intervention are crucial for improving prognosis [20, 21]. This was the first study to demonstrate that serum miR-331-3p is significantly downregulated in SAP and regulates pancreatic inflammatory responses by targeting BRD4, providing new evidence for the diagnosis and treatment of SAP.
Recent studies have indicated that serum miR-4695-5p is upregulated in SAP and exacerbates pathological damage by targeting SESN2 [22]. Moreover, miR-216 may serve as a biomarker for the early identification of SAP [23]. Our clinical data revealed that miR-331-3p was significantly downregulated in SAP patients. The AUC of miR-331-3p for distinguishing SAP from MAP was 0.866, and it was significantly negatively correlated with routine markers (amylase, lipase), suggesting that it may serve as an diagnostic biomarker for SAP. Meanwhile, miR-331-3p also had the ability to predict the poor prognosis of patients. Furthermore, the progression of AP is closely related to an uncontrolled inflammatory response [24]. The miR-217-5p/YAF2 axis is involved in the pancreatic injury and inflammation caused by SAP [25]. Our results showed that miR-331-3p is closely related to the severity of the disease and the levels of inflammatory factors, suggesting that it may be involved in the pathological process of SAP.
New research indicates that miR-486-5p predicts the progression of SAP by mediating the inflammatory response and the ATG7/p38 MAPK pathway [26]. Moreover, miR-20b-5p regulates inflammation in SAP by targeting AKT3 [27]. At the molecular level, we found through dual-luciferase assays and cell function experiments that miR-331-3p directly targets BRD4 and inhibits its expression, thereby alleviating pancreatic inflammatory responses. This finding agrees with the known role of BRD4 in inflammatory regulation: as a member of the bromodomain protein family, BRD4 can initiate the transcription of pro-inflammatory genes by binding to acetylated histones [28, 29]. Existing studies have reported that BRD4 is highly expressed in inflammatory diseases such as acute lung injury and colitis, whereas inhibiting BRD4 can effectively alleviate inflammatory damage [30, 31]. Cell experiments showed that miR-331-3p may exert a protective effect by targeted inhibition of BRD4, thereby alleviating inflammatory damage and apoptosis of pancreatic cells. Previous studies have shown that BRD4 is upregulated in AP [32]. Similarly, our results indicated that BRD4 expression is notably upregulated in SAP and exhibits a significant inverse correlation with the miR-331-3p levels. These findings provide new insights into targeted therapy for SAP that block the inflammatory cascade by upregulating miR-331-3p or inhibiting the activity of BRD4.
Although our results reveal that the miR-331-3p/BRD4 axis is a key regulator of SAP, the exact downstream signaling pathways mediating these effects require further investigation. It has been reported that BRD4 has been shown to regulate autophagy through SIRT1 [32]. Therefore, we speculate that the miR-331-3p/BRD4 axis may affect disease progression by altering autophagy, and this hypothesis can be tested in future studies by evaluating markers such as LC3 and p62. Furthermore, activation of the NF-κB pathway leads to the substantial production and release of key inflammatory mediators such as IL-6 and TNF-α [33]. The onset of AP can induce pancreatic gland cells to release pro-inflammatory cytokines and produce a large number of inflammatory mediators by activating nuclear transcription factor NF-kappaB, which leads to multiple organ dysfunction [34, 35]. Therefore, the reduction in inflammatory factors observed following miR-331-3p overexpression is likely a direct consequence of BRD4 inhibition suppressing NF-κB activity. Nevertheless, the exact downstream signaling pathways mediating these effects require further investigation. To fully elucidate and validate this, high-throughput methods such as RNA-seq sequencing will be essential in future studies. This study holds important clinical significance. As a potential diagnostic biomarker, miR-331-3p provides a new detection target for the early diagnosis of SAP. Determination of its mechanism of action has laid a theoretical foundation for new therapeutic strategies, which are expected to improve the inflammatory response and prognosis of SAP through precise regulation. In clinical nursing, integrating dynamic monitoring of serum miR-331-3p and inflammatory factors can guide interventions: for patients with very low miR-331-3p levels and high levels of inflammatory factors, it is necessary to strengthen disease observation, enhance fluid resuscitation and organ function support, optimize the timing of nutritional support to reduce inflammatory stimulation, and thus improve prognosis.
This study preliminarily reveals the diagnostic potential and mechanisms of miR-331-3p in SAP, but certain limitations remain: Firstly, clinical samples were sourced from a single institution with limited sample size, which may lead to bias in the results and affect the universality of the conclusions. Additionally, the study lacks external validation cohorts and longitudinal follow-up data, failing to provide dynamic changes in miR-331-3p during intervention. It is also worth noting that the MAP group included patients with mild and moderately severe AP, which may have introduced heterogeneity. Although this binary distinction meets the need to identify organ failure, it limits the ability to distinguish between mild and moderate severity. Future studies will further conduct multi-center, large-sample clinical research to more comprehensively and accurately evaluate the diagnostic value and clinical application potential of miR-331-3p. Secondly, the study primarily focused on a class of in vitro cell models. Although our in vitro data show a plausible mechanistic link, a significant inverse association in patient sera does not equate to causality. Meanwhile, the study lacks a sufficient comparison of the diagnostic performance of miR-331-3p with existing biomarkers. The overall regulatory network and therapeutic effects of miR-331-3p in complex in vivo environments remain to be confirmed. Future work should conduct assays in additional cell lines and establish animal models of pancreatitis to functionally validate the causal role of the miR-331-3p/BRD4 axis and determine its feasibility in clinical treatment. Finally, the use of U6 as a reference gene for miR-331-3p may pose a limitation on stability, and the need for control via exogenous peaks or multiple reference miRNAs will improve the reliability of normalization.
In conclusion, serum miR-331-3p is significantly downregulated in patients with SAP and is closely related to the severity of SAP. miR-331-3p alleviates pancreatic inflammatory responses by targeted inhibition of BRD4 expression, which is expected to be a potential biomarker for assessing the severity of SAP. Whether miR-331-3p/BRD4 axis has therapeutic potential still needs to be further confirmed by in vivo assays and preclinical studies.
Supplementary Information
Acknowledgements
Not applicable.
Authors’ contributions
Conceptualization, R.X., J.K., D.S., W.C., X.W.; Data curation, R.X., J.K., D.S., W.C., X.W.; Formal analysis, D.S., W.C.; Funding acquisition, W.C.; Investigation, D.S.; Methodology, R.X., J.K., D.S., W.C., R.X., J.K.; Project administration, W.C.; Resources, D.S., W.C.; Software, D.S., W.C.; Supervision, W.C.; Validation, D.S.; Visualization, D.S.; Roles/Writing - original draft, D.S.; Writing - review & editing, R.X., J.K., W.C., X.W.
Funding
No funding was received to assist with the preparation of this work.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
The study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The First People’s Hospital of Lanzhou before the study began (No. 2017034). The written informed consent has been obtained from the participants involved.
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.
Rong Xiong and Jingwei Kou contributed equally to this work.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.