Prostaglandin E₂ Promotes CCLP1 Cell Proliferation By Upregulation Express Of SnoN Through CAMP-PKA-CREB Signaling Pathway

Background:Human hepatocellular carcinoma (HCC) is one of the most devastating malignancies in the China,also has a low five-year survival rate, and seriously threat human's health and life, Studies has been confirmed that PGE₂ could induce the development of tumor throught promoting the tumor cell proliferation, invasion and metastasis, tumor blood vessel formation. Currently , PGE₂ is considered to play its roles by binding the cell surface PGE₂ receptors (EP receptors), EP receptors are membrane G protein-coupled receptors, there are four kinds, respectively, referred as EP1, EP2, EP3 and EP4 receptors.SnoN(ski-related novel gene)is one of the Ski oncogene family's members, Current research indicates that SnoN protein in many human tumor cells was high expression in many human tumor cells, But the reports about SnoN protein was less in human liver groups,and the roles of SnoN in CCLP1 cell was unclear when we used PGE₂ deals with the cell and Observed it's proliferation capacity. In this study, PGE₂, four kinds of EP receptor agonists, adenylate cyclase agonist Forskolin, the Fitting seems content of cAMP dbcAMPand the PKA inhibitor H89 are used to deal with human Bile duct epithelial cells CCLP1, to observe the role of SnoN when we used PGE₂ regulates the cell and the possible signal transduction pathways.Objective:To explore the role of SnoN when we used PGE₂ regulates the cell and the possible signal transduction pathways.Methods:1. Cell culture: human Bile duct epithelial cells (CCLP1) were cultured in vitro as routine. 2. CCLP1 cells were Treated with PGE₂, SnoN mRNA expression was examined by RT-PCR.3. CCLP1 cells were Treated with PGE₂ , SnoN protein expression was examined by Western blot in Cytoplasm of CCLP1 cells.4. CCLP1 cells were Treated with EP1-4 receptor agonist (EP1: 17-phenyltrinor Prostaglandin E₂;EP2:Butaprost;EP3:Sulprostone;EP4: Prostaglandin E1 Alcohol), the cell's ability of proliferation was examined by WST. CCLP1 cells were Treated with EP2 receptor agonist, the cell's ability of proliferation was examined by WST.5. CCLP1 cells were Treated with EP1-4 receptor agonist (EP1:17-phenyltrinor Prostaglandin E₂ ; EP2:Butaprost ; EP3:Sulprostone ; EP4:Prostaglandin E1 Alcohol), SnoN protein expression was examined by Western blot in Cytoplasm of CCLP1 cells.6. CCLP1 cells were Treated with Forskolin,dbcAMP,Forskolin+H89,SnoN protein expression was examined by Western blot respectively in Cytoplasm of CCLP1 cells.7. CCLP1 cells were Treated with Forskolin, Forskolin+H89, the cell's ability of proliferation was examined by WST.8. CCLP1 cells were Treated with Forskolin, Forskolin+H89, the levels of CREB phosphorylation in CCLP1 cells were examined by Western blot.Results:1. The expression of SnoN mRNA in CCLP1 cells were increased by 22.5% after treated with PGE₂ (10μmol/L) for 24 h(P<0.01).2. The expression of SnoN in cytoplasm of CCLP1 cells were increased by 13.4%,24.5%,35.6%,23.5% after treated with PGE₂ of 1μM,5μM,10μM,20μM respectively. (P<0.05) 3. the cell's ability of proliferation increased more obviously than the control group after treated with 10μM EP1-4 receptor agonist (EP1: 17-phenyltrinor Prostaglandin E₂;EP2:Butaprost;EP3:Sulprostone;EP4: Prostaglandin E1 Alcohol) respectively for 24 h. the cell's ability of proliferation were increased by 13.3%,15.4%,16.1%,18.2% after treated with1μM,5μM,10μM,20μM EP2 receptor agonist respectively for 24 h.4. The expression of SnoN in cytoplasm of CCLP1 cells were increased by 64.9% after treated with 10μM EP2 receptor agonist,while increased by35.6%,29.6%,24.9%, after treated with 10μM EP1, EP3, EP4 receptor agonist respectively for 24 h.5. The expression of SnoN in cytoplasm of CCLP1 cells were increased by 25.1% after treated with 10μM Forskolin, while decreased by 9.1% compared with the Forskolin treated group after treated with 10μM Forskolin +H89 for 24 h.6. The expression of SnoN in cytoplasm of CCLP1 cells were increased by 90.1% after treated with 500μM dbcAMP for 24 h.7. the cell's ability of proliferation were increased by 2.7%,3.6%,4.4% (P<0.05)respectively after treated with1μM,5μM,10μM Forskolin, while decreased by2.7%,9.1%,27.1%,45.7%(P<0.05)respectively after treated with1μM,5μM,10μM,20μM H89 for 24 h.8. The levels of CREB phosphorylation in CCLP1 cells were increased by 71.3%( P<0.05 ) compared with the control group after treated with Forskolin(10μmol/L),while decreased by 14.1% after treated with H89+Forskolin(10μmol/L) for 24h.Conclusions:1. PGE₂ might up-regulate the expression of SnoN of CCLP1 cells.2. PGE₂ might up-regulate the expression level of SnoN through EP2 receptor of CCLP1 cells which could be partly related to the cAMP-PKA-CREB Signaling Pathway, and promotes the cell proliferation.