title: Overexpressed miRNA-155 dysregulates intestinal epithelial apical junctional complex in severe acute pancreatitis.
abstract: OBJECTIVE
  To investigate whether miRNA-155 (miR-155) dysregulates apical junctional complex (AJC) protein expression in experimental severe acute pancreatitis (SAP).
  
  
  METHODS
  Twenty-four male BALB/c mice were randomly assigned to two groups: the SAP group (n = 12) receiving sequential intraperitoneal injection of 50 µg/kg caerulein and 10 mg/kg lipopolysaccharide over 6 h, and the control group (n = 12) receiving intraperitoneal injection of normal saline. Animals were sacrificed 3 h following the last injection for collection of blood samples and pancreas and distal ileal segment specimens. Routine pancreas and intestine histology was used to assess SAP pathology and intestinal epithelial barrier damage. Levels of serum amylase, diamine oxidase (DAO), and tumor necrosis factor (TNF)-α were determined using commercial kits. Total RNA samples were isolated from intestinal epithelial specimens and reversely transcribed into cDNA. miR-155 and RhoA mRNA expression profiles were determined using quantitative real-time polymerase chain reaction. Target genes for miR-155 were predicted using the miRTarBase database, RNA22 and PicTar computational methods. Western blotting was performed to quantitate the protein expression levels of the target gene RhoA, as well as zonula occludens (ZO)-1 and E-cadherin, two AJC component proteins.
  
  
  RESULTS
  Intraperitoneal injection of caerulein and lipopolysaccharide successfully induced experimental acute pancreatic damage (SAP vs control, 10.0 ± 2.0 vs 3.2 ± 1.2, P < 0.01) and intestinal epithelial barrier damage (3.2 ± 0.7 vs 1.4 ± 0.7, P < 0.01). Levels of serum amylase (21.6 ± 5.1 U/mL vs 14.3 ± 4.2 U/mL, P < 0.01), DAO (21.4 ± 4.1 mg/mL vs 2.6 ± 0.8 mg/mL, P < 0.01), and TNF-α (61.0 ± 15.1 ng/mL vs 42.9 ± 13.9 ng/mL, P < 0.01) increased significantly in SAP mice compared to those in control mice. miR-155 was significantly overexpressed in SAP intestinal epithelia (1.94 ± 0.50 fold vs 1.03 ± 0.23 fold, P < 0.01), and RhoA gene containing three miR-155-specific binding sites in the three prime untranslated regions was one of the target genes for miR-155. RhoA (22.7 ± 5.8 folds vs 59.6 ± 11.6 folds, P < 0.01), ZO-1 (46 ± 18 folds vs 68 ± 19 folds, P < 0.01), and E-cadherin proteins (48 ± 15 folds vs 77 ± 18 folds, P < 0.01) were underexpressed in SAP intestinal epithelia although RhoA mRNA expression was not significantly changed in SAP (0.97 ± 0.18 folds vs 1.01 ± 0.17 folds, P > 0.05).
  
  
  CONCLUSIONS
  TNF-α-regulated miR-155 overexpression inhibits AJC component protein syntheses of ZO-1, and E-cadherin by downregulating post-transcriptional RhoA expression, and disrupts intestinal epithelial barrier in experimental SAP.
authors.affiliation: Rui Tian, Rui-Lan Wang, Hui Xie, Wei Jin, Kang-Long Yu, Intensive Care Unit, First People's Hospital, Shanghai Jiaotong University, Shanghai 201620, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Tian
authors.affiliation: Rui Tian, Rui-Lan Wang, Hui Xie, Wei Jin, Kang-Long Yu, Intensive Care Unit, First People's Hospital, Shanghai Jiaotong University, Shanghai 201620, China.
authors.forename: Rui-Lan
authors.initials: RL
authors.lastname: Wang
authors.affiliation: Rui Tian, Rui-Lan Wang, Hui Xie, Wei Jin, Kang-Long Yu, Intensive Care Unit, First People's Hospital, Shanghai Jiaotong University, Shanghai 201620, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Xie
authors.affiliation: Rui Tian, Rui-Lan Wang, Hui Xie, Wei Jin, Kang-Long Yu, Intensive Care Unit, First People's Hospital, Shanghai Jiaotong University, Shanghai 201620, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Jin
authors.affiliation: Rui Tian, Rui-Lan Wang, Hui Xie, Wei Jin, Kang-Long Yu, Intensive Care Unit, First People's Hospital, Shanghai Jiaotong University, Shanghai 201620, China.
authors.forename: Kang-Long
authors.initials: KL
authors.lastname: Yu
mesh_terms: D000208:Acute Disease; D006631:Amine Oxidase (Copper-Containing) / Q000097:blood; D000681:Amylases / Q000097:blood; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002108:Ceruletide; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D007082:Ileum / Q000378:metabolism* / Q000473:pathology; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D008070:Lipopolysaccharides; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000378:metabolism*; D010195:Pancreatitis / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010539:Permeability; D012333:RNA, Messenger / Q000378:metabolism; D012720:Severity of Illness Index; D019108:Tight Junctions / Q000378:metabolism* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000097:blood; D015854:Up-Regulation; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein
chemical_list: D015820:Cadherins; D008070:Lipopolysaccharides; D035683:MicroRNAs; C532623:Mirn155 microRNA, mouse; D012333:RNA, Messenger; C569043:Tjp1 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D062826:Zonula Occludens-1 Protein; D002108:Ceruletide; D006631:Amine Oxidase (Copper-Containing); D000681:Amylases; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
keywords: Apical junctional complex; Intestinal barrier dysfunction; Severe acute pancreatitis; miRNA-155