Circulating microRNA 216 as a Marker for the Early Identification of Severe Acute Pancreatitis

Abstract

Background

To study the value of circulating microRNA 216 (miR-216) as a marker for the severity of acute pancreatitis (AP) in both murine models and patients.

Materials and Methods

Mice with AP were induced by intraperitoneal injection of 50 μg/kg/hour cerulean either 7 times, sacrificed at 8, 9, 10, 11 or 12 hours after the first injection, or 12 times, sacrificed at 24 hours after the first injection. Plasma samples and data from patients with AP were obtained from a prospective cohort. Quantitative reverse transcription polymerase chain reaction was used to determine the miR-216a and miR-216b level.

Results

The upregulation of miR-216a and miR-216b in the serum of mice was induced by cerulean injection in both the 7- and 12-injection groups (P < 0.05). The downregulation of miR-216a in pancreatic tissues of mice with AP was detected (P < 0.05), but no difference was observed in pancreatic miR-216b levels among any of the groups (all P > 0.05). The serum miR-216a level was positively correlated with pancreatic histopathology severity scores, and was negatively correlated with pancreatic miR-216a (r = −0.483, P = 0.009). The plasma miR-216a level was significantly upregulated in patients with severe AP (SAP) compared with patients with mild AP (MAP) or moderate severe AP (MSAP) (SAP versus MAP, P = 0.04; SAP versus MSAP, P = 0.00), but no difference was seen between patients with MAP and those with MSAP (P = 0.73).

Conclusions

Circulating miR-216a might be a potential biomarker for the early identification of SAP.

Introduction

Acute pancreatitis (AP), which is inflammation of the pancreas, can have a complicated clinical course with severe local and systemic complications¹ that lead to tremendous emotional, physical and financial burdens.² The overall mortality rate is 10%, but patients with the severe form of the disease are at an increased risk of death with a mortality rate of 36-50%.¹ Extensive studies conducted in the past 2 decades have demonstrated that the first 24 hours after symptom onset are critical for identifying those patients at risk of developing complications or death.3, 4, 5, 6, 7, 8, 9, 10 It is of paramount importance to identify the at-risk group of patients as soon as possible and initiate aggressive treatment without delay. Nevertheless, none of the current clinical scoring systems or biochemical markers play a definitive role, have widespread applicable value or are consistently accurate.3, 11, 12, 13 Therefore, the early identification of the development of severe acute pancreatitis (SAP) remains a great challenge.

MicroRNAs (miRNAs) are a class of noncoding single-stranded RNAs that are 18-22 nucleotides long. They are present in eukaryotic cells and regulate target gene expression at the posttranscriptional level.14, 15 To date, more than 2,500 human microRNA sequences have been discovered,¹⁶ and these sequences target approximately 60% of all protein-coding genes. MiRNAs play diverse role in many cellular processes.¹⁷ The discovery of overexpressed or underexpressed miRNAs not only broadens our biological understanding of various diseases but also opens new avenues for the development of novel diagnostic and prognostic strategies. Due to their high stability, even in poorly preserved specimens, miRNAs are valuable in clinical research and biomarker discovery.¹⁸ Accumulating evidence suggests that miRNAs may act as potential biomarkers for pancreatic tissue injury, and substantial attention has been focused on the miRNAs involved in AP.19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 An improvement in our understanding of the role that miRNAs play in AP could lead to the development of new diagnostic and prognostic tools for use in future clinical applications.

The expression of miRNA-216 (miR-216) is relatively specific to the pancreas.30, 31, 32, 33 Because the pancreas consists of approximately 90% acinar cells, miR-216 is suggested to be primarily expressed in acinar cells and may play an important role in maintaining the differentiation status of acinar cells and in promoting the progression of pancreatic injury. In recent years, miR-216, including both the miR-216a and miR-216b subtypes, has been explored in several studies as a circulating biomarker of acute pancreatic injury.19, 20, 21, 22, 26, 29 These promising results were demonstrated in animal experiments.

The role of circulating miR-216 in patients with AP has been reported in 2 studies.26, 35 One exploratory study²⁶ evaluated the expression of miR-216a in the plasma of patients with AP. However, the authors only enrolled patients with mild acute pancreatitis (MAP) and moderately severe acute pancreatitis (MSAP) and did not include those with SAP. Moreover, these patients were classified according to the Determinant-Based Classification³⁴ instead of the Revised Atlanta Classification.¹ Another observational study³⁵ was based on the Revised Atlanta Classification but did not demonstrate the significance of circulating miR-216 in the distinction of SAP from MSAP and MAP.

Because the role of miR-216 in pancreatitis has not been firmly established, especially in the severe form of pancreatitis, we sought to determine the expression of serum miR-216 and its correlation with histopathological severity in cerulean-induced murine models of mild edematous pancreatitis to severe necrotizing pancreatitis. We also sought to preliminarily verify plasma miR-216a as a clinical biomarker for the severity stratification of patients with AP, as categorized by the Revised Atlanta Classification.