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

Author’s Accepted Manuscript Circulating microRNA-216 as a marker for the early identification of severe acute pancreatitis Xiao-Xin Zhang, Li-Hui Deng, Wei-Wei Chen, Na Shi, Tao Jin, Zi-Qi Lin, Yun Ma, Kun Jiang, XiaoNan Yang, Qing Xia www.elsevier.com

PII: DOI: Reference:

S0002-9629(16)30650-4 http://dx.doi.org/10.1016/j.amjms.2016.12.007 AMJMS352

To appear in: The American Journal of the Medical Sciences Received date: 14 May 2016 Revised date: 4 December 2016 Accepted date: 8 December 2016 Cite this article as: Xiao-Xin Zhang, Li-Hui Deng, Wei-Wei Chen, Na Shi, Tao Jin, Zi-Qi Lin, Yun Ma, Kun Jiang, Xiao-Nan Yang and Qing Xia, Circulating microRNA-216 as a marker for the early identification of severe acute pancreatitis, The American Journal of the Medical Sciences, http://dx.doi.org/10.1016/j.amjms.2016.12.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Circulating microRNA-216 as a marker for the early identification of severe acute pancreatitis Xiao-Xin Zhang, MD, Li-Hui Deng, MD, PhD, Wei-Wei Chen, MD, PhD, Na Shi, MD, Tao Jin, MD, PhD, Zi-Qi Lin, MD, PhD, Yun Ma, BD, Kun Jiang, MD, Xiao-Nan Yang, MD and Qing Xia, MD From the Department of Integrated Traditional Chinese and Western Medicine, West China Hospital of Sichuan University, Chengdu, China. E-mails: [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected], [email protected],

[email protected]

and

[email protected],

respectively Xiao-Xin Zhang and Li-Hui Deng contributed equally to this work. Correspondence: Qing Xia and Li-Hui Deng, Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, 37# Guoxue Street, Chengdu 610041, China. Tel: (+86) 28-8542-3373, E-mail: [email protected] (QX); [email protected] (LHD) Short Title: Circulating miR-216 for severity of acute pancreatitis There are no financial or other conflicts of interest involved in the article. Funded by National Nature Science Foundation of China (No. 81300358) and China Postdoctoral Science Foundation (2014T70878). Key Indexing Terms: acute pancreatitis; biomarker; microRNA-216; miR-216a; 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: AP mice were induced by intraperitoneal injection of 50 g/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 (qRT-PCR) 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 AP mice 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 vs MAP, P = 0.04; SAP vs MSAP, P = 0.00), but no difference was seen between MAP and MSAP patients (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 complications [1] 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 last two decades have demonstrated that the first 24 hours after symptom onset are critical for identifying those patients who are at risk of developing complications or death [3-10]. 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 plays a definitive role, has widespread applicable value, or is consistently accurate [3, 11-13]. 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 post-transcriptional level [14, 15]. To date, more than 2500 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 over- or under-expressed 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 [19-29]. 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 [30-33]. 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 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 [19-22, 26, 29]. These promising results, however, were demonstrated in animal experiments. The role of circulating miR-216 in patients with AP has been reported in two studies [26, 35]. One exploratory study [26] 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 Classification [34] instead of the Revised Atlanta Classification [1]. Another observational study [35] 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 is not 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.

MATERIALS AND METHODS Animals Male BALB/c mice (weight, 25–30 g; age, 8–10weeks) were purchased from the Experiment Animal Center of Sichuan University, maintained at 22 ± 2°C under a 12-hour day-night cycle and fed standard mouse chow and tap water ad libitum over 1 week of acclimation before the experiment. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocols and experiments were approved by the Ethics Committee for Animal Experiments of Sichuan University. Experimental Groups Mice were fasted for 12 hours and were given free access to tap water until 2 hours prior to experiments, at which point they were randomized into the AP and control groups. AP models were induced by cerulean injection as follows: mice were given an intraperitoneal injection (IP) of 50 μg/kg/hour cerulean either 7 [36, 37] or 12 [38, 39] times; normal saline (NS) was administered to the controls. Mice that received 7 cerulean injections were sacrificed at 5 different time points (8, 9, 10, 11 or 12 hours after the first cerulean injection, which were termed the AP8, 9, 10, 11 and 12h groups, with n = 4 for each group), whereas mice that received 12 injections were sacrificed 24 hours (AP24h group) after the first cerulean injection (n = 4). Mice in the control group were sacrificed after 8 hours (n = 4). Serum samples were collected and stored at -80°C for the biochemical

determination and evaluation of the miR-216a and miR-216b levels. One half of the pancreas was placed in 10% buffered formalin solution for histopathological grading of the severity of pancreatitis, and the other half was submitted for miR-216a and miR-216b analysis. A blinded histological analysis was performed as reported by Schmidt et al [40]. For each pathological section, edema, inflammatory infiltration and acinar cell necrosis were evaluated in 5 random visual fields under a 200x microscope. Serum miR-216a and miR-216b levels were quantified using RT-PCR, and the expression between different time points was compared. Subjects Plasma samples and data from patients with AP and healthy volunteers were obtained from a prospective observational clinical study (chictr-DOD-15005864). The study protocol and informed consent were approved by the Clinical Trials and Biomedical Ethics Committee of West China Hospital, Sichuan University. All procedures in studies with human participants were performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki as well as its later amendments or comparable ethical standards. All subjects provided written informed consent before enrolling. According to the Revised Atlanta Classification in 2012 [1], the diagnosis of AP requires two of the following three features: (1) abdominal pain consistent with AP (acute onset of a persistent, severe, epigastric pain that often radiates to the back); (2) serum lipase activity (or amylase activity) at least three times greater than the

upper limit of normal; and (3) characteristic findings of AP on contrast-enhanced computed tomography (CECT), magnetic resonance imaging (MRI) or transabdominal ultrasonography. MAP was defined as the absence of both organ failure (OF) and local or systemic complications, MSAP as the presence of transient OF or local or systemic complications in the absence of persistent OF, and SAP as persistent OF. OF is defined as a score of 2 or more for one of three organ systems using the modified Marshall scoring system [41]. Persistent OF is defined as OF for more than 48h. AP patients between 18 and 70 years of age who were admitted to West China Hospital, China, within 48 hours after the onset of disease were enrolled. Patients were excluded if they 1) were pregnant, 2) had any malignancy, or 3) had serious primary diseases of any other system. Healthy volunteers were enrolled as controls. Collection of Human Plasma Peripheral blood samples were collected from patients with AP using BD Vacutainer EDTA tubes within 24 hours after admission. The processing of all blood samples began within 30 minutes after collection and was performed according to the following procedures: maintained upright for 20-25 minutes, centrifugation at room temperature at 600 g for 30 minutes, further centrifugation of the supernatant at 24°C and 1500 g for 10 minutes, and storage at -80°C. Quantitative Reverse Transcription-Polymerase Chain Reaction Total RNA from 100 µl serum from mice or 400 µl plasma from human subjects was isolated using the mirVana™ PARIS™ Kit (Ambion, USA) according to the manufacturer’s protocol. Total RNA from the pancreas was extracted using TRIzol

reagent. A Taqman miRNA real-time RT-PCR kit (Applied Biosystems, USA) was used to detect and quantify the levels of mature miRNA in the total RNA from serum or plasma. Briefly, total RNA was reverse-transcribed to cDNA using a Taqman microRNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer’s instructions. qRT-PCR of the target miRNA was performed with Taqman Universal PCR Master Mix (Applied Biosystems, USA) in an 7900HT Sequence Detection System, which automatically analyzed the data using SDS software 2.4. Each reaction was performed in triplicate and contained 0.75 µl cDNA, 0.5 µl 20×Taqman microRNA Assay Mix, 5 µl 2×Taqman Universal PCR buffer and 3.75 µl ddH2O. The amplification was performed as follows: denaturation at 50°C for 2 minutes and at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. The miRNA levels that were not detected after 45 cycles of real-time PCR were considered to have a Ct equivalent to 45. U6 snRNA was used as the internal control. Total RNA of rodent pancreas were reverse-transcribed to cDNA using the Revert Aid™ Frist Strand cDNA Synthesis Kit (Fermentas, Canada) according to the manufacturer’s instructions. qRT-PCR was conducted with a 30-µl qRT-PCR reaction buffer: 5 µl cDNA, 3 µl 10×buffer (Mg2+ free), 2 µl MgCl2 (25 mM), 0.36 µl dNTP (25 mM), 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 1 µl SYBR Green I (10 µM), 0.3µl Taq DNA polymerase (5 u/µl) and 15.34 µl deionized double distilled water. An FTC-3000 real-time PCR detection system (Funglyn, Canada) was used with the following program: pre-denaturation at 94°C for 2 minutes, followed by 45 cycles of

94°C for 20 seconds, 62°C for 20 seconds and 72°C for 30 seconds. All reactions were run in triplicate. U6 snRNA was used to normalize the miRNA expression levels. Statistical Analysis Statistical analysis was performed using SPSS 20.0. The threshold cycle (Ct) was defined as the fractional cycle number of fluorescence that passed through a given threshold. The target miRNA expression was analyzed by the 2-△△Ct method (△Ct = Ct target miRNA

- Ct

internal control).

Continuous variables were expressed as the mean ± the

standard error, and categorical variables were expressed as proportions. A one-way ANOVA, Kruskal-Wallis test, Chi-square test or Fisher's exact test was used, when appropriate, to determine significant differences between the groups. The Pearson correlation test was used to analyze the correlations between the expression of miRNAs and the histopathological severity scores. A two-sided P‐value less than 0.05 was considered statistically significant.

RESULTS Serum Amylase Levels Serum amylase levels in all AP groups were significantly higher than in the control group (all P < 0.05) (Figure 1). The amylase level began to increase 8 hours after the first cerulean injection and peaked at 12 hours. The serum amylase level was significantly higher in the AP12h group than in the AP8, 9, 10, 11 and 24h groups (all P < 0.05). No significant difference was observed in the serum amylase level among the other time points in the 7-injection groups and the AP24h group (all P > 0.05). Histopathological Changes The histopathological changes that were seen in the pancreatic tissues are shown in Figure 2. The pancreatic tissues in the control group lacked obvious changes that are indicative of AP (Figure 2A). Interstitial edema, granulocyte infiltration, and acinar cell necrosis were found by microscopy (Figure 2B-G). The average scores for edema, inflammation infiltration, and acinar cell necrosis were increased in the AP groups (Figure 2H-J). The average scores for inflammation and acinar cell necrosis were similar in mice that were euthanized at 8, 9, 10, and 11 hours (all P > 0.05) and were significantly higher in animals that were euthanized at 12 and 24 hours after the first cerulean injection. As shown by the total severity score in Figure 2K, the 12-injection group (AP24h group) exhibited more severe pancreatic injury than the 7-injection group at each time point (all P < 0.05).

qRT-PCR to Detect the MiR-216a and MiR-216b Levels in Murine Serum The upregulation of serum miR-216a was induced by cerulean injection in both the 7- and 12-injection groups (Figure 3A). In mice in the 7-injection groups, miR-216a expression was significantly higher at all time points than that in mice in the control group (all P < 0.05). The increase in the serum miR-216a expression in the 12-injection AP group was significantly greater than that in the control group and the 7-injection AP groups at 9, 10, 11, and 12 hours after the first cerulean injection (all P < 0.05). Serum miR-216b expression was significantly upregulated in the AP8, 9 and 10h groups compared with the control group (all P < 0.05) (Figure 3B). The serum miR-216b level was also significantly higher in the AP8h and AP10h groups compared with the AP11, 12 and 24h groups (all P < 0.05). No difference was observed in the expression level of serum U6 in any of the groups (P > 0.05). qRT-PCR to Determine the MiR-216a and MiR-216b Levels in the Murine Pancreas The pancreatic miR-216a level was significantly lower in the AP groups than in the control group (all P < 0.05) (Figure 3C). The pancreatic miR-216a level in the AP8h group was higher than that in the AP10, 12 and 24h groups (all P < 0.05) and was also higher in the AP11h group than those in the AP12 and 24h groups (both P < 0.05). In contrast, the relative expression of pancreatic miR-216b was similar among all groups (P > 0.05) (Figure 3D). The groups exhibited insignificant differences with respect to the expression of pancreatic U6 (P > 0.05).

The Correlation between MiR-216 and the Histological Severity Scores A Pearson correlation analysis showed that the serum miR-216a level was positively correlated with pancreatic edema, infiltration and total scores, but no correlation was found with the acinar cell necrosis score (Table 1). The level of pancreatic miR-216a demonstrated a significant negative correlation with pancreatic edema, infiltration, acinar cell necrosis and total scores. No significant correlations were observed between serum or pancreatic miR-216b and the histopathology severity scores (all P > 0.05, Table 1). The serum miR-216a level was negatively correlated with pancreatic miR-216a (r = -0.483, P = 0.009), and the serum miR-216b level was positively correlated with the pancreatic miR-216b level (r = 0.513, P = 0.005). Subject Demographics and MiR-216a Expression In all, 32 subjects were selected, including 24 patients with AP (8 MAP, 8 MSAP and 8 SAP) and 8 healthy volunteers. The demographic characteristics of the patients with AP are shown in Table 2. Plasma miR-216a levels were scarcely detectable in 8 healthy volunteers (all nearly Ct = 45), whereas the levels were detectable and were measured in all AP patients. Compared with the MAP group, the plasma miR-216a level was 1.7-fold higher in the MSAP group and 2.7-fold higher in the SAP group. The plasma miR-216a level was significantly upregulated in patients with SAP compared with patients in the MAP and MSAP groups (SAP vs MAP, P = .04; SAP vs MSAP, P = .00), and the plasma miR-216a levels were similar between patients in the MAP

and MSAP groups (MAP vs MSAP, P = .73) (Figure 4). No detectable difference was seen in terms of plasma U6 expression between healthy volunteers and AP patients (P = .246).

DISCUSSION MiRNAs have been identified in a wide array of biological fluids [42]. Due to their stability and insensitivity to external variables, circulating miRNAs have been recognized as promising non-invasive diagnostic or prognostic biomarkers of pathophysiological processes [43-46]. In 2010, Kong et al. [21] speculated that plasma miR-216a might be a specific biomarker of pancreatic injury, with a higher specificity than amylase and lipase. Similar results were reported in subsequent studies [19, 20, 22, 28, 29]. Recently, more evidence has revealed that miRNAs are potential biomarkers of pancreatitis, as reported in AP models induced by the intraperitoneal injection of L-arginine [19, 21, 23], retrograde infusion of sodium taurocholate into the pancreatic duct [26], injection of cerulean [20, 22, 28, 29] or cyanohydroxybutene

[19],

or

a

combined

injection

of

cerulean

and

lipopolysaccharide[25]. The cerulean model is widely accepted by investigators of pancreatic diseases. This model is non-invasive and easy to establish, has a highly reproducible nature, and exhibits the closest parallel with clinically observed AP induced by hyperstimulation. Various degrees of severity can be achieved by adjustments of

either the dosage or the number of cerulean injections [47]. The current study differs from previous studies [20, 22, 28, 29], in which histopathological responses induced by cerulean were merely mild or moderate, in that various severity of histopathological changes were generated in this study by two different patterns of cerulean injections. Cerulean injections evoked a dose- and time-dependent pattern of changes in terms of histopathological processes. Histopathological changes in mice in the 12-injection group demonstrated the successful induction of more severe AP. It would be better to represent pancreatic injury conditions from mild edematous pancreatitis to severe necrotizing pancreatitis through models that involve 7 and 12 cerulean injections, which might be used to mimic the complicated processes of AP. In this study, pancreatic miR-216a expression was significantly decreased in the AP group compared with the control group, and presented a significant negative correlation with pancreatic edema, infiltration, acinar cell necrosis and total scores. Therefore, these results suggested that pancreatic miR-216a might reflect pancreatic tissue injury. As for serum miR-216a, the level of this miRNA was significantly increased in AP mice and it was positively correlated with pancreatic edema, infiltration and total scores, but no correlation was observed with the acinar cell necrosis score. These results suggested that the serum miR-216a level is associated with the severity of edema and inflammation in pancreatitis but that it does not completely reflect the degree of necrotic injury as demonstrated by histopathology. The serum miR-216a level was negatively correlated with the pancreatic miR-216a level. We initially supposed that circulating miRNAs may be a result of the

leakage of miRNAs into the blood from damaged tissues [19]. However, serum miR-216a expression exhibited no correlation with the necrosis score and could not completely reflect the degree of necrotic injury as observed by histopathology. Therefore, the concept of miR-216a leakage could not entirely justify the increased expression of serum miR-216a in cerulean-induced murine AP. Other hypotheses on the transfer and secretory mechanisms of circulating miRNAs from injured tissues [48, 49] are still in their infancy, and thus further studies are imperative to elucidate the nature of circulating miRNAs. A significant increase was observed in the serum miR-216a level in mice from both the 7- and 12-time injection groups, which signified that the values behaved as a biomarker of AP [19-22, 29]. The miR-216a expression was higher in the 7-injection group than in the control group, with a maximal expression of serum miR-216a at 8 hours after the first cerulean injection, which is consistent with the results of Goodwin et al [20]. Our result showed a declining trend in the expression of serum miR-216a in mice that received 7 injections of cerulean. The study by Goodwin et al. [20] did not investigate the differences among different time points groups using the same dosage and number of cerulean injections, but the average expression of miR-216a at 6 hours after the last cerulean treatment (equivalent to the AP11 or 12h groups in this study) was not much lower than that at 3h after the last treatment (equivalent to AP8h in this study). The discrepancy between the two studies may have been caused by a wider dynamic range of responses to pancreatic injury in the study by Goodwin et al., where an increase of up to 100 times over the control levels

or assay lower limits were observed. The serum miR-216a level in the 12-injection group was significantly higher than that in most of the 7-injection groups. This result suggested that circulating miR-216a might have a potential value in the identification of the severe form of pancreatitis. The difference in the pancreatic miR-216b level between the control and AP groups was insignificant, which suggested that this miRNA might not reflect pancreatic tissue injury. Serum miR-216b was significantly upregulated as it nearly reached a peak at 8 hours after the first cerulean injection, and then declined, but returned to an approximately normal level at 11 hours and did not rise at all in the 12-injection group. Previously, although both Endo et al. [19] and Calvano et al. [28] failed to detect baseline levels of miR-216b in serum or plasma in control rats, both of these investigators found that miR-216b expression increased in response to pancreatic injury. Smith et al. [29] also confirmed that the serum miR-216b level was increased at 1 hour after 3 times cerulean treatment in rats and dogs models of pancreatic injury, but that the level then decreased at 8 hours. No correlation was found between miR-216b level in the serum or pancreas and histopathological severity. It can be inferred that, compared with miR-216a, miR-216b may not serve as an acceptable, potential biomarker of AP severity. To verify the values of the plasma miR-216a level in the early identification of SAP, we determined its expression in patients with early-stage AP. Similar to the results reported by Blenkiron et al. [26] and Kusnierz-Cabala et al. [35], the plasma miR-216a level was detectable in all AP patients in this study. Although the plasma

miR-216a level in patients with MSAP was 1.7 times higher than that in patients with MAP, no significant difference was observed in the level of miR-216a between patients with MAP and those with MSAP, which is consistent with the findings of Blenkiron et al. [26]. A dramatic upregulation of miR-216a was seen in patients with SAP compared with patients with MAP and MSAP in this study, whereas no significant difference was observed between those with MAP and SAP according to the study by Kusnierz-Cabala et al. [35]. Consequently, the expression of miR-216a might be considered a prognostic biomarker that could be used to differentiate SAP patients in an initial disease stage from those with MAP and MSAP, despite the great difficulty in the distinction of MAP and MSAP patients according to plasma miR-216a levels. The following discrepancies in the study methods should be taken into account in the assessment of the observations reported by Blenkiron et al. [26] and Kusnierz-Cabala et al. [35] and those reported in this study. First, the SAP patients that were included in the study by Kusnierz-Cabala et al. [35] actually comprised moderate and severe pancreatitis patients, but the mortality of patients with MSAP was far lower than that of patients with SAP [1], which exposed a difference between true MSAP and SAP patients. Therefore, their study possibly concealed intrinsic values of miR-216a in SAP by combining the values from patients with MSAP. Second, in the current study, blood samples were collected within 24 hours after hospital admission (within 48 hours after the onset of symptoms), whereas blood was collected within 48 hours after hospital admission in the study by Kusnierz-Cabala et al. [35] and within 96 hours from the onset of disease in the study by Blenkiron et al.

[26]. Hence, the time point may be a variable that ultimately affects the expression of miR-216a. Third, the variances in samples of plasma and serum could not be completely excluded, as the difference in the miRNA levels in the serum and corresponding plasma samples from the same individuals could be measured [50-52]. Nevertheless, in this study, the results of miR-216a expression in human subjects were consistent with those in an animal model of pancreatitis, which demonstrated its value in the early diagnosis of pancreatitis and the identification of severe disease. Some inconsistency was present in the measurement of circulating miR-216a in healthy volunteers in the results of the studies by Blenkiron et al. [26] and Kusnierz-Cabala et al. [35] and those in this study. In this study, as all Ct values were higher than or nearly equivalent to 45, the plasma miR-216a values in healthy volunteers were considered scarcely detectable; in contrast, the levels of this miRNA were measureable in the studies by Blenkiron et al. [26] and Kusnierz-Cabala et al. [35]. In the study by Blenkiron et al., however, four out of five healthy volunteers had raw Ct values over 30, which indicated internally low miR-216a expression in the plasma of healthy volunteers. This discrepancy might be attributed to ethnic differences in the subjects or differences in assay protocols. Despite the potential interest in our findings, our study still had insufficiencies and expectations: 1) additional animal models and more frequent time points can provide an accurate expression and allow more convincing comparison; 2) an appropriate adjustment in protocols and reagents, the use of enriched samples, or the use of more advanced technologies such as digital PCR may improve the

detection sensitivity to enable a better understanding of miR-216a behavior in AP; 3) as a pilot study, only 8 subjects were managed in each patient group and no differences were found in the plasma miR-216a levels between MAP and MSAP patients. Although similar to the study by Blenkiron et al. [26], which enrolled 5 subjects in each group, it is possible that small sample size resulted in the inability to distinguish MSAP from MAP. In conclusion, circulating miR-216a may serve as a potential biomarker for SAP, but validations in a larger sample size of patients with AP are required in the future.

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Figure Legends Figure 1: The activities of serum amylase (u/dl). *vs Control, P < 0.05; # AP12h vs AP8, 9, 10, 11 and 24h, all P < 0.05 Figure 2: Histopathological changes and Histopathological severity scores. A-G: Histopathological changes of pancreatic tissue in different groups (HE stain, × 200); H: edema scores, I: inflammatory infiltration scores, J: acinar cell necrosis scores, K: total scores; *vs Control, P < 0.05; #vs AP8 and 9h, P < 0.05; $vs AP10, 11 and 12h, P < 0.05; & vs AP10h, P < 0.05 Figure 3: The expression of miR-216 in mice serum and pancreas A: serum miR-216a expression, B: serum miR-216b expression; C: pancreatic miR-216a expression, D: pancreatic miR-216b expression; *vs control, P < 0.05; ∑ vs AP10, P < 0.05; # vs AP11 and 12h, P < 0.05; Φ vs AP12 and 24h, P < 0.05; ∆ vs 24h, P < 0.05 Figure 4: The expression of miR-216a in AP patient plasma. *vs MAP, P < 0.05; # vs MSAP, P < 0.05; MAP mild acute pancreatitis, MSAP moderately severe acute pancreatitis, SAP severe acute pancreatitis

Table 1: The correlation of miR-216 expression and histological severity scores Serum miR-216a

Serum miR-216b

Pancreas miR-216a

Pancreas miR-216b

r

P

r

P

r

P

r

P

Edema

.44

.019

.051

.798

-.687

.000

.138

.483

Inflammatory infiltration

.393

.039

-.220

.261

-.390

.040

-.155

.431

Acinar cellular necrosis

.351

.067

-.185

.345

-.532

.004

-.181

.358

Total score

.447

.017

-.127

.520

-.614

.001

-.067

.736

Histopathologic scores

Table 2: Characteristics of the patients

Age, year ( x

 se )

Gender, male (%) BMI ( x

 se )

HV

MAP

MSAP

SAP

P value

(n=8)

(n = 8)

(n = 8)

44.50±3.6

44.7 ± 4.9

45.2 ± 2.5

50.4 ± 4.3

.761

6 (75)

7 (87.5)

5 (62.5)

7 (87.5)

.570

25.6±0.8

26.6 ± 1.4

24.9 ± 1.1

27.4 ± 1.6

.681

(n = 8)

HV healthy volunteers, MAP mild acute pancreatitis, MSAP moderately severe acute pancreatitis, SAP severe acute pancreatitis, BMI body Mass Index