The Research Of EP3 Receptor Isoforms In Human Cholangiocarcinoma Cells Proliferation, Migration And Invasion Through The Upregulation Of ?-Catenin Expression

Background:Prostaglandin E2(PGE2),as a kind of inflammatory mediator, is the main product of arachidonic acid after cyclooxygenase-2(COX-2) catalytic reaction and has been implicated in cholangiocarcinoma cell proliferation, migration and invasion through E Prostanoid receptors, including EP1, EP2, EP 3 and EP4.There are 5 isoforms of EP3 receptor, which are EP3-4, EP3-5, EP3-6, EP3-7 and EP3-8 receptors. However, the functions and the mechanisms of those splice variants of EP3 receptors in promoting liver cancer cell growth and invasion are not yet fully understood. In our previous studies, four isoforms of EP3 receptors were detected, which were EP3-4, EP3-5, EP3-6 and EP3-7 receptors, in CCLP1 and Hu CCT1 cells. However, the functions of the receptors in those cells are still not clear.It was reported that ?-Catenin is an important protein in Cell adhesion, and is closely correlated with malignancy, including cholangiocarcinoma. This study describes the functions of EP3 receptor isoforms and the mechanisms through which PGE2 regulates ?-Catenin expression and promoting cholangiocarcinoma cell growth and invasion.Research Object: 1. Clarifying the effect of EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms in human cholangiocarcinoma cells proliferation, migration and invasion. 2. Clarifying the mechanisms and associated signal signaling pathways through which PGE2 upregulate the ?-Catenin protein expression level via EP3-4 receptor.Research Content: 1. Overexpression the EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms in cholangiocarcinoma cells and HEK 293 T cells through transfection experiments2. Clarifying the role of EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms in human cholangiocarcinoma cells proliferation, migration and invasion 3. Investigating the signaling pathway and associated mechanisms of PGE2 upregulate the ?-Catenin protein expression level via EP3-4 receptorResearch method: 1. The cholangiocarcinoma cell lines CCLP1?Hu CCT1 human embryo kidney cell line HEK 293 T were cultured in a conventional method. 2. The m RNA level of the EP3 R transfected cells and the associated proteins in the signal transduction pathway were investigated by using Real-time PCR method 3. The EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms transfection efficiency in cholangiocarcinoma cells and HEK 293 T cells were detected with the Western blot method. 4. The growth of EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms transfected cholangiocarcinoma cells and HEK 293 T cells induced by sulprostone was detected with the WST assay. 5. The migration and invasion of EP3-4, EP3-5, EP3-6 and EP3-7 receptors isoforms transfected cholangiocarcinoma cells and HEK 293 T cells induced by sulprostone were investigated by using the Transwell method. 6. Chemical inhibitors were usedfor some key points in the signaling pathway, and combined with the Western Blot method, to analyzed the signaling pathway.Research Results: 1. In pc DNA, EP3-4, EP3-5, EP3-6 and EP3-7 receptors transfected CCLP1 cells, sulprostone could increase the proliferation ability to 122%, 145%, 136%, 120 % and 97%, increase the migration ability to 149%, 224%, 193%, 138 and 101%,and increase the invasion ability to 129%, 204%, 193%, 120% and 96%. The same results were observed in Hu CCT1 and HEK 293 T cells. 2. In EP3-4, EP3-5, EP3-6 and EP3-7 receptors transfected CCLP1 cells, the expression levels of ?-Catenin protein were increased to 134%, 126%, 102% and 103%. In pc DNA and EP3-4 receptor transfected CCLP1 cells, the expression levels of ?-Catenin protein were increased to143% and 193% induced by PGE2, and wereincreased to 140% and 218% induced by sulprostone. The same results were observed in Hu CCT1 and HEK 293 T cells. When L-798106 was used to block the effect of EP3 receptor, the upregulation of the expression level of ?-Catenin protein induced by sulprostone was also blocked. 3. In EP3-4 receptor transfected CCLP1 cells, the expression levels of c-Myc and Snail m RNAs were increased to 252% and 206%, and the expression levels of c-Myc and Snail proteins were increased to 208% and 180% induced by sulprostone. 4. In EP3-4 receptor transfected CCLP1 cells, the expression levels of ?-Catenin m RNA was no significant change, and the phosphorylation of ?-Catenin protein was decline to 40% induced by sulprostone. 5. In EP3-4 receptor transfected CCLP1, Hu CCT1 and HEK 293 T cells, the phosphorylation of GSK-3? protein was increased to 343%, the phosphorylation of AKT protein was increased to 319%, and the phosphorylation of EGFR protein was increased to170% induced by sulprostone. When Li Cl was used to block the effect of GSK-3?, LY294002 was used to block the effect of PI3K/AKT, AG1478 was used to block the effect of EGFR, or PP2 was used to block the effect of Src, the upregulation of the expression level of ?-Catenin protein induced by sulprostone was also changed.Conclusion: 1. PGE2 could increase the proliferation, migration and invasion ability of cholangiocarcinoma through EP3-4 and EP3-5 receptors. 2. PGE2 could up regulate the level of ?-Catenin protein through EP3-4/Src/EGFR/PI3K/AKT/GSK3? and inhibits the degradation of ?-Catenin protein, then promote the expression of c-Myc and Snail protein.