J Huazhong Univ Sci Technol［Med Sci］ 36(2):221-225,2016 10.1007/s11596-016-1570-2 J DOI Huazhong Univ Sci Technol［Med Sci］ 36(2):2016
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Functional Role of MicroRNA-19b in Acinar Cell Necrosis in Acute Necrotizing Pancreatitis Ming-xing HU (胡明星)1, 2, Hong-wei ZHANG (张宏伟)2, Qiang FU (付 强)2, Tao QIN (秦 涛)2, Chuan-jiang LIU (刘传江)2, Yu-zhu WANG (王玉柱)2, Qiang TANG (唐 强)2, Yu-xin CHEN (陈雨信)1# 1 Department of Hepatobiliary Pancreatic Surgery, Shandong Qilu Hospital, Shandong University, Jinan 250000, China 2 Department of Hepatobiliary Pancreatic Surgery, People’s Hospital of Zhengzhou University, School of Medicine, Zhengzhou University, Zhengzhou 450003, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2016
Summary: The expression of microRNA-19b (miR-19b) in acute necrotizing pancreatitis (ANP) and its functional role in acinar cell necrosis of SD rats were investigated. Twelve SD rats were divided into two groups randomly, including control group and ANP group. The rat ANP models were established by intraperitoneal injection of L-arginine (2400 mg/kg body weight), and equal volume of 0.9% NaCl was injected in the control group. MiRNA chip assay was performed to examine the expression of miRNAs in the pancreas in two different groups. Besides, to further explore the role of miR-19b in ANP in vitro, taurolithocholic acid 3-sulfate disodium salt (TLC-S) (200 μmol/L) was administrated to treat the rat pancreatic acinar cell line, AR42J, for establishing the ANP cells model. The quantitative real-time PCR (qRT-PCR) was adopted to measure the miR-19b expression. Moreover, the mimic miRNA, miRNA antisense oligonucleotide (AMO) and control vector were used to transfect AR42J cells, the expression of miR-19b was confirmed by qRT-PCR and the necrotizing rate of AR42J cells was detected with AO/EB method. The expression of miR-19b was significantly higher in ANP group than in control group as displayed by the miRNA chip assay. Furthermore, after inducing necrosis of AR42J cells in vitro, the expression of miR-19b was significantly increased by 2.51±0.14 times in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-19b was 5.94±0.95 times higher in the mimic miRNA group than in the control vector group, companied with an obviously increased acinar cell necrotizing rate (50.3%±1.5% vs. 39.6%±2.3%, P<0.05). Moreover, the expression of miR-19b in the miRNA AMO group was 0.38±0.15 times lower than in the control vector group, and the cell necrosis rate was much lower accordingly (23.1%±3.3% vs. 39.6%±2.3%, P<0.05). Besides, there was no significant difference between the control vector cells and the cells without treatment (P>0.05). The expression of miR-19b was significantly induced in ANP. In addition, up-regulation of miR-19b could promote the necrosis of pancreatic acinar cells and miR-19b deficiency could decrease the rate of pancreatic acinar cell necrosis. Key words: miRNA-19b; acute pancreatitis; acinar cells; necrosis
Acute pancreatitis (AP), with an incidence rate of 5–10/10 000 persons per year, is usually caused by cholelithiasis and alcohol abuse[1], and 5%–10% progress to acute necrotizing pancreatitis (ANP). ANP, with a mortality rate of 20%–30%[2], is defined as a continuous organic failure more than 48 h, companied with peripancreatic fluid collection and acinar cell necrosis. The morbidity and mortality rates of ANP could be decreased after reducing the necrosis of acinar cells, which may be a potentially valuable way in treating ANP[3]. MicroRNAs (miRNAs) are small non-coding RNAs, and play an essential role in cell proliferation, apoptosis, necrosis and signal transduction[4, 5]. More and more researches have focused on the function of miRNAs in pancreatic diseases, such as pancreatic carcinoma, chronic pancreatic fibrosis and acinar cell necrosis in AP[6–8]. Based on this information, we postulated that miRNA may be associated with ANP development. In Ming-xing HU, E-mail:
[email protected] # Corresponding author, E-mail:
[email protected]
the present study, we examined the relationship between miRNA expression and acinar cell necrosis by miRNA chip assay both in vivo and in vitro. Furthermore, quantitative real-time PCR (qRT-PCR) was performed to confirm the expression of the targeting miRNA, miRNA-19b (miR-19b), discovered on miRNA chip assay. Moreover, the expression of miR-19b was both down-regulated and up-regulated to investigate its effect on acinar cell necrosis during ANP, which is essential in exploring the regulatory mechanisms of acinar cell necrosis. 1 MATERIALS AND METHODS 1.1 Animals and Cell Line SD rats were purchased from the Experimental Center of Zhengzhou University, China. The rat pancreatic acinar cell line, AR42J, was from Cell Bank of the Chinese Academy of Sciences, China. 1.2 In Vivo Assay Twelve SD rats were assigned to two groups randomly (n=6 each), and anaesthetized with pentobarbital sodium (30 mg/kg body weight) through intraperitoneal
222 J Huazhong Univ Sci Technol［Med Sci］ 36(2):2016 injection. The ANP group was injected with L-arginine (2400 mg/kg body weight) intraperitoneally, while the control group was treated with equal volume of 0.9% NaCl. The rats were sacrificed 12 h later. Part of the pancreatic tissue was frozen in liquid nitrogen and the left tissue was fixed with 10% formaldehyde, followed by dehydration and paraffin imbedding. 1.3 Detection of Pancreatic Necrosis Hematoxylin-eosin (H&E) staining was carried out on the prepared pancreatic tissue slides. And pancreatic cell necrosis was detected by invert microscopy. 1.4 MiRNA Assay Pancreatic tissue of 3 rats in each group was used to perform the miRNA chip assay to analyze the expression of miRNA, which was conducted by the Minxin Technological Company in Shanghai, China. The original data were further analyzed through deleting the signal background, followed by statistical analysis to investigate the miRNA expression difference of the two groups. The targeting miRNA in association with pancreatic acinar cell necrosis was selected for further investigation. 1.5 Cell Culture and ANP Model Establishment The cell line Ar42J was cultured in DMEM-F12 (Hyclong, USA) containing 20% fetal calf serum (Invitrogen, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, and maintained in a 5% CO2 humidified incubator at 37ºC. The medium was changed and the cells were passaged (1:4) every 48 h. To establish the ANP model in vitro, the AR42J cells were cultured in a 24-well plate (1×105/well) and treated with taurolithocholic acid 3-sulfate disodium salt (TLC-S) (200 μmol/L) (Sigma, USA). The medium was collected 1 h later to examine the amylase level with the detection kit (Tiancheng Biotechnical Company, China). 1.6 Cell Transfection The mimic miRNA, miRNA antisense oligonucleotide (AMO) and control vector were designed and synthesized by Jikai Genetic Company (China). AR42J cells were cultured in a 24-well plate (1×105/well). According to the instruction of the kit, transfection was performed in a certain MOI (1:50). The medium containing virus was discarded 48 h later and the fluorescent reagent (Ruibo Biotechnical Company, China) was added. Then the transfection efficiency was detected with fluorescent microscope. Finally, TLC-S was used to establish the ANP model. 1.7 qRT-PCR Total RNA of the AR42J cells, harvested with Trizol method, was used for the reverse transcription to synthesize cDNA according to the instruction of the RT-PCR kit (Takara, Japan). The primers of miR-19b were synthesized by the Ruibo Biotechnical Company (China). The qPCR assay was performed using the recommended thermal cycling conditions of the kit: 40 cycles of 5 s at 95ºC, 30 s at 60ºC after initial denaturation at 95ºC for 30 s. The results were balanced with β-actin before cal– culating the relative expression level with 2 ΔΔCT method. 1.8 Cell Necrosis Detection The AR42J cells were suspended in the medium at a concentration of 8×105/mL. The cell slide was made with 25 μL suspension medium and 1 μL fluorescent reagent.
Fluorescent microscope was used to count the number of necrotizing cells and the necrotizing rate was calculated. 1.9 Statistical Analysis The statistical analysis was performed with SPSS 17.0 software. The measurement data were displayed as ±s and its significant difference was analyzed by Student’s t-test. The χ2 test was used to analyze the significant difference of enumeration data. P<0.05 was regarded as statistically significant difference. 2 RESULTS 2.1 Pancreatic Acinar Cell Necrosis In Vivo In the ANP group, necrosis of numerous pancreatic acinar cells was found on H&E staining of the rat pancreatic tissue slide, while the pancreatic tissue was normal in the control group (fig. 1).
Fig. 1 The H&E staining of the rat pancreatic tissue (×400) A: control group; B: ANP group 2.2 MiRNA Chip Assay The result of the miRNA chip assay showed that the expression of miR-19b in the ANP group was higher than that in the control group (fig. 2, P<0.05). 2.3 Amylase Level Measurement The amylase level of medium in the ANP group was obviously increased as compared with the control group (P<0.05). The amylase levels in the mimic miRNA group, miRNA AMO group and vector group were all higher than those in the control group, suggesting the successful establishment of ANP model in vitro (table 1).
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Table 1 Amylase levels of the cell culture medium in the in vitro assay Groups
Before transfection
ANP Control * P<0.05 vs. control group
1084.5±64.9* 290.3±6.8
After transfection Mimic group
AMO group
Vector group
1304.7±99.1* 355.4±28.7
1130.9±92.2* 362.1±27.0
1184.7±136.4* 326.3±73.4
creased by 2.51±0.14 times in comparison with the control group (P<0.05). As revealed by the qRT-PCR assay (fig. 3), the expression of miR-19b was 5.94±0.95 times higher in the mimic group than in the control group (P<0.05). Moreover, the expression of miR-19b in the AMO group was 0.38±0.15 times lower than in the control vector group (P<0.05). Besides, there was no significant difference between the control vector cells and the cells without treatment (P>0.05).
Fig. 3 Lentivirus transfection and miRNA expression A and B: quantitative real-time RT-PCR analysis for miR-19b in AR42J cell line. The expression of miR-19b was normalized to U6 expression using 2-ΔΔCT. Data are representative of three independent experiments. * P<0.05 vs. control group; C: Cells were infected with 50 MOI of vehicle, miR-19b mimic or miR-19b AMO, and then imaged at 72 h post-transfection (×40). Left, bright field filter view; right, FITC filter view
Fig. 2 This hierarchically clustered heat map illustrates the changes in miRNA expression profiles between the ANP groups and normal pancreas groups. The significantly expressed miRNA cluster was identified using the Student’s t-test. The red and green sections represent increases and decreases in miRNA expression respectively between normal pancreas groups and ANP groups. MiR-19b was up-regulated in ANP groups as compared with normal pancreas groups significantly.
2.4 qRT-PCR Results After treatment with TLC-S for one h, the expression of miR-19b in the ANP group was significantly in-
2.5 Cell Necrotizing Rate The cell necrotizing rate in the ANP group was statistically higher than that in the blank group before transfection (33.13%±1.6% vs. 4.93%±0.98%, P<0.05). To further investigate the role of miR-19b in ANP, the AR42J cells were transfected with mimic miRNA, miRNA AMO and control vector adenovirusly, then the cells in different groups were further treated with TLC-S. As shown in fig. 4 and 5, the necrotizing rate in the control vector group was significantly higher than in the non-treated group (39.6%±2.3% vs. 6.1%±1.5%, P<0.05). There was an obviously increased acinar cell necrotizing rate in the mimic miRNA group relative to the control vector group (50.3%±6.3% vs. 39.6%±2.3%, P<0.05). Besides, the cell necrotizing rate was significantly lower
224 J Huazhong Univ Sci Technol［Med Sci］ 36(2):2016 in the miRNA AMO group than in the control vector
group (23.1%±3.3% vs. 39.6%±2.3%, P<0.05).
A
B
D
E
Fig. 4 The necrosis of AR42J cells (AO/EB staining, ×40) A: control group; B: TLC-s group; C: miR-19b AMO+TLC-S group; D: miR-19b mimic+TLC-S group; E: vehicle+TLC-S group
Fig. 5 The necrosis of AR42J cells A: before transfection. B: after transfection. Data are representative of ±s from three independent experiments performed in triplicate. *P<0.05 vs. blank group or vehicle group; #P<0.05 vs. vehicle+TLC-S group
3 DISCUSSION Edematous pancreatitis is characterized by cell apoptosis, while cell necrosis is a hallmark of ANP[9]. Acinar cell necrosis and following inflammation are the leading causes of the mortality of ANP patients[10]. However, the detailed mechanism has not been elucidated yet. Hu et al reported that the expression of Bcl-2 and Bcl-xL could be up-regulated by Reg4 mRNA through EGFR/Akt signaling pathway, which could inhibit pancreatic acinar cell necrosis induced by arginine and caerulein[11]. To our knowledge, the functional role of miRNA in acinar cell necrosis during ANP has not been reported at present. MiRNAs are endogenous, small non-codingRNA molecules consisting of 21–25 nucleotides, which can form RNA induced silencing complex by binding with Argonaute, and inhibit targeting protein expression through specific combination with 3'-UTR (untranslated region) of the corresponding gene[12, 13]. Many literatures have focused on the relationship between inflammation and miRNA, such as miRNA-155 is correlated with in-
flammation regulation during the early phase of ANP[14]. Besides, miR-216a and miR-217 are specially expressed in mouse serum during AP[15]. Furthermore, miR-19b can mediate cell proliferation, migration, senescence and apoptosis through regulating p53 expression[16]. In previous study, we have established the ANP rat model and found miR-19b was obviously up-regulated on miRNA chip assay. In the present study, ANP model in vitro was established, and qRT-PCR assay revealed that the expression of miR-19b in ANP model was 2.51±0.29 times higher than in the control group, which indicated miR-19b was statistically up-regulated in ANP (P<0.05). In addition, we up-regulated and down-regulated the expression of miR-19b with mimic miRNA (5.94±0.95) and miRNA AMO (0.38±0.15), and found that miR-19b overexpression could increase AR42J cells necrosis, while the necrotizing rate was decreased with miR-19b deficiency. In conclusion, overexpression of miRNA-19b in ANP could promote pancreatic acinar cells necrosis by mediating the coding of targeting genes, which needs to be further elucidated.
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Conflict of Interest Statement We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Functional Role of MicroRNA-19b in Acinar Cell Necrosis During Acute Necrotizing Pancreatitis”. REFERENCES 1 Booth DM, Mukherjee R, Sutton R, et al. Calcium and reactive oxygen species in acute pancreatitis: Friend or Foe? Antioxid Redox Signal, 2011,15(10):2683-2698 2 Banks PA, Banks TL, Dervenis C, et al. Classification of acute pancreatitis—2012: revision of the Atlanta classification and definitions by international consensus. Gut, 2013,62(1):102-111 3 Xu M, Wang KN, Wu K, et al. Pyrrolidine dithiocarbamate inhibits nuclear factor κB and Toll-like receptor 4 expression in rats with acute necrotizing pancreatitis. Gut Liver, 2015,9(3):411-416 4 Lakner AM, Steuerwald NM, Walling TL, et al. Inhibitory effects of miRNA-19b in hepatic stellate cell-mediated fibrogenesis. Hepatology, 2012,56(1):300-310 5 Popov A, Szabo A, Mandys V. Small nucleolar RNA U91 is a new internal control for accurate microRNAs quantification in pancreatic cancer. BMC Cancer, 2015,15(1): 774 6 Paik WH, Song BJ, Kim HW, et al. MicroRNA-200c as a prognostic biomarker for pancreatic cancer. Korean J Gastroenterol, 2015,66(4):215-220 7 Masamune A, Nakano E, Hamada S, et al. Alteration of
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