The identification and roles of microRNA targeting protein kinase D1 in rat with acute pancreatitis

Abstract

Objective To predict and verify the upstream regulatory microRNA (miRNA) of protein kinase D1 (PKD1), and to investigate its role in cerulein induced acute pancreatitis (AP) in rats. Methods Potential upstream regulatory miRNA of PKD1 was predicted by using bioinformatics software.Dual luciferase reporter gene system and Western blot were applied to verify the regulation of PKD1 by the selected miRNA.Experimental AP was induced by 6 intraperitoneal injection of cerulein (20 μg/kg) at hourly intervals after administration of the CY5-labeled notarget control (AP group, n=20) or selected miRNA (treatment group, n=20), respectively by intraperitoneal injection into rats.Other rats were divided randomly into a normal control group (n=10) without any treatment.Besides 10 rats in either AP or treatment group were sacrificed 6 hours after the first injection of cerulein, and the rats were all sacrificed 24 hours after the first injection.The blood samples and pancreatic tissues of each rat were collected to test serum amylase and lipase activities, or to make hematoxylin-eosin stain for AP pathological scores as well as PKD1 immunohistochemical staining, respectively. Results TargetScan 7.1 software analysis showed that miR-128-3p was the potential upstream regulatory miRNA of PKD1, which was verified by dual luciferase reporter gene system and Western blot detection.Compared to the normal control group, serum amylase and lipase activities after 6 h exposure to cerulein increased in both AP group and the treatment group[13 313.00(9 424.00-15 995.00) U/L, 13 552.00(10 399.50-18 408.25) U/L vs.1 430.50(1 214.25-1 543.25) U/L; 547.00 (515.00-627.00) U/L, 857.50(522.00-1 222.25) U/L vs.34.00(32.50-34.75) U/L], and the differences were significant(χ2=8.715, P<0.05; χ2=9.115, P<0.05), which indicated that the rat models of AP were successfully established.The immunohistochemical scores of PKD1 after 24 h exposure to cerulein decreased in the treatment group[0.50(0-2.75) scores], compared with the normal control group [4.00(4.00-8.00) scores]and the AP group [4.00(3.75-8.00) scores], and difference was significant(χ2=18.302, P<0.05). Accordingly, the total pathological scores of HE staining decreased significantly in the treatment group, as compared to the AP group (3.80±0.85 vs.6.90±1.15, t=4.481, P<0.01). The results showed that the inflammatory cell infiltration and tissue necrosis were significantly improved after miR-128-3p treatment. Conclusions miR-128-3p is the upstream regulatory microRNA of PKD1 which protects pancreata from necrotic injury and inflammatory cell infiltration in PKD1-mediated acute pancreatitis. Key words: Acute pancreatitis; miR-128-3p; Protein kinase D1; Necrosis