The Role And Mechanism Of IL-6 In Epithelial-mesenchymal Transition In Barrett Esophagus

Background and ObjectiveIL-6 is a pluripotent proinflammatory cytokines, mainly involved in regulating immune defense reaction. Its release is triggered by tissue damage or infection. Recent studies have found that IL-6 plays an important role in tumor growth and metastasis. Its overexpression has been implicated in the pathogenesis and prognosis of different tumors including ovary, prostate, breast, kidney, lung and so on. IL-6 can play important roal in inducing EMT in breast and prostate tumor. IL-6 controls tumor formation and growth in early colitis-associated colon cancer.The studie found that the expression of IL-6 and IL-6 mRNA was elevated in BE mucosa compared with duodenum and adjacent and nonadjacent squamous epithelium, using immunohistochemistry, protein array, and real-time reverse transcription-PCR methods. Other reports suggested that IL-6 had effect on promoting tumor in Barrett esophagus. However, the role and mechanism of IL-6 in Barrett esophageal in epithelial mesenchymal transition and carcinogenesis is still unclear. In our present study we used different concentrations of IL-6 to stimulate the Barrett’s esophagus cells, and then observed the cell morphology, EMT-associated molecules, and detected the metastasis and invasion abilities. Furthermore, we explored the signaling pathways mechanism invilved in EMT. We fed rats with high fat diet to make non-alcoholic fatty liver model, measured serum liver function and cytokines such as IL-6 and observed the change of esophagus to explore whether high-fat diet feeding could induce esophagitis or precancerous lesion.Methods(1) In vitro, we applied 0,10,20,50,100 ng/mL of IL-6 to stimulate CP-D cells. The optical microscope morphological was used to observate changes of the cell.Western blot, RT-PCR and immunofluorescence were performed to evaluate the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin. Cell migration was examined by transwell and wound-healing assay.(2)Western blot technique was applied to determine the changes of MAPK/ERK, PI3K/AKT signaling pathway as well as E-cadherin and Vimentin. Then we used the PI3K inhibitor LY294002 and ERK1/2 inhibitor U0126 to explore the possible mechanism of IL-6 induction of EMT.(3) 40 male rats were randomly divided into control and experimental groups, with a normal diet and high-fat diet, respectively. Weight and body length were measured at 0,3,6,9,12,15 week, and the liver function, the serum lipids and inflammatory factors indicators interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-a) was determinated at 15 week. The liver and esophagus of rats was removed, recording the liver and esophagus gross morphology and HE staining, the lower esophageal mucosa infiltration of inflammatory cells. Western blot technique was applied to determine the expression of IL-6 protein esophageal tissue.Results(1) The CP-D cell morphology was changed and cell adhesion connection was reduced after exposed to the different concentration of IL-6 in 24 hours. The mRNA and protein expression levels of epithelial marker E-cadherin was decreased, while the mRNA and protein expression levels of mesenchymal cell marker Vimentin was elevated,50 ng/mL of IL-6 was the optimal concentration inducing CP-D cell occuring EMT. Transwell and wound-healing assay proved that IL-6 can cause CP-D cell migration and invasion capacity enhancement.(2)After IL-6 stimulation of CP-D cells, the expression of phosphorylation-AKT was increased. The expression of phosphorylation ERK1/2 had the same trend. We pretreated CP-D cells with ERK1/2, AKT signaling pathway inhibitor 10μmol/L LY294002 and U0126, respectively,and then used 50 ng/mL IL-6 stimulation of CP-D cells, found that AKT and ERK signaling pathway was inhibited. Western Blot results showed that the expression of E-cadherin was increased and Vimentin expression levels was reduced after the pretreatment of CP-D cells with LY294002 and U0126 in the presence of IL-6 stimulation indicating that signaling pathway inhibitors can effectively prevent the CP-D EMT change with IL-6 stimulation. Transwell experiments showed that CP-D cell migration was decreased with the addition of the signal pathway inhibitor.(3) High-fat diet leading to weight gain in the experimental group was significantly faster than the control group; experimental fatty liver could be established within the first 15 weeks, serum lipids and inflammatory factor indicators IL-1, IL-6, TNF-a were higher than control group. However, there was no significant difference in liver function. Inflammatory cell infiltration was found in esophageal mucosa through HE staining and IL-6 protein in esophageal tissue was significantly higher by semi-quantitative detection.Conclusion:(1) IL-6 induced EMT of CP-D cell, while the CP-D cell invasion and migration capacity was enhanced.(2) IL-6 induced EMT through activation of PI3K/Akt and MEK/Erk signaling pathway.(3) High-fat diet for 15 weeks could cause rats fatty liver, inflammatory cells were infiltrated in the lower esophageal mucosal, IL-6 protein expression was increased in the esophageal tissue, suggesting that high-fat diet may cause esophagitis and IL-6 may play an important role.