| Background:Prostaglandin E2(PGE2) is one of the predominant products of arachidonic acid catalyted by cyclooxygenase-2(COX-2). It is found that PGE2mediates its biological effects through binding and activating4different G rprotein-coupled receptors (EP1, EP2, EP3, EP4), including tumor proliferation, migration, and invasion.Amphiregulin (AREG) is one of the members of epithelium growth factor (EGF) family and is synthetized as a252-amino acid transmember glycoprotein,which binds epithelium growth factor receptor (EGFR) and activates a variety of downstream signal transduction pathway.after cleaved by member-anchored metaproteinase (ADAM-17). It plays important roles in proliferation, survival of cells and resistence to chemotherapy in many tumors. It is reported that AREG expression is not detected in the normal liver tissues, however, highly increased AREG expression is detected in human chronic liver cirrhosis and liver cancer. Rescent evidences suggest that AREG can play a unique role in tumorgenesis and in the maintenance of the neoplastic phenotype of hepatocarcinoma cells.Previously, we have that PGE2could promote the proliferation of cholangiocarcinoma cells CCLP1. Neverthless, it is still unclear wether PGE2could regulate the expression of AREG and what is the role of AREG in PGE2-induced proliferation of CCLP1cells in hepatocarcinoma. In this study, cholagiocarcinoma cells CCLP1were treated with PGE2, four EP receptor agonists, adenylyl cyclase inhibitor SQ22536, the PKA inhibitor H89and etc, and hence, the expression level of AREG and the relationship with the proliferation ability of CCLP1has been investigated, aiming to clarify the role of AREG in PGE2induced proliferation of CCLP1cells and the possible mechanisms of signal transduction pathway.Objectives:1. To investigate the effects of PGE2on the expression of AREG in CCLP1cells and the possible mechanisms involved.2. To explore the role of AREG in PGE2induced proliferation of CCLP1cells.Methods:1. The cholagiocarcinoma cells CCLP1cells were cultured in vitro as routine.2. CCLP1cells were treated with PGE2or EP1-4receptors agonists and the level of AREG protein was examined by western bolt.3. CCLP1cells were treated with PGE2for24h,36h,48h and cell proliferation was estimated using the cell proliferation reagent WST.4. After CCLP1cells were treated with PGE2and PGE2+Mab262/SAB140218, the WST assay was used to detect the cell proliferation in CCLP1cells.5. Western blot analysis was performed to investigate AREG protein expression in CCLP1cells with PGE2and PGE2+H89treatment.6. CCLP1cells were treated with PGE2, AH6809, H89as well as treated with PGE2after tansfection with either CMV500-DN-CREB or CMV500, and then western blot was performed to detect AREG protein expression.Results:1. The level of AREG protein in CCLP1cells was increased by13.4%,28.5%,44.2%(P<0.05) repectively compared with control group, after CCLP1were cells treated with1μM,5μM,10μM PGE2.2. CCLP1cells were Treated with5μM PGE2for Oh,24h,36h,48h, the WST assay indicated that the proliferation of CCLP1cells was increased by13.9%,34.3%.15.1%(P<0.05) respectively. However, preatreated with the20μM neutralizing-antibody (Mab262or SAB140218) for1h and then treated with5μM PGE2for35h,the proliferation was surpressed by18%and20%respectively compared with PGE2treatment ((P<0.05).3. With four selective EP receptor agonists stimulating CCLP1cells, we found that only EP2agonist could induce the expression of AREG protein (increased by20.1%, P<0.05), while the other three EP receptors have no significant changes. Treated with1μM,5μM,10μM EP2receptor agonist, CCLP1cells were assayed with western blot and WST assay. Western blot showed that the expression of AREG protein was improved by30.5%,31.2%,37.6%(P<0.05). The proliferation of cells was increased by10.5%,14.9%,16.1%(P<0.05), however, the proliferation improved by EP2agonist was significantly inhibited after pretreated with EP2antagonist AH6809(downregulated by20%)(P<0.05).4. The expression of AREG protein improved by PGE2was suppressd by46.8%,54.4%(P<0.05) when preteated with AC inhibitor AQ22536and PKA inhibitor H89.5. H89and SQ22536could strikingly inhibit the phosphorylation of CREB induced by PG.E2((downregulated by45.5%compared with PGE2treatment).6. Afer transfected with vector CMV500-DN-CREB or CMV500, CCLP1cells were treated with PGE2, and then western blot assay was performed.This research found that PGE2could obviously induce the expression of AREG in CMV500transfected cells, whereas there was no significant change in CMV500-DN-CREB transfected cells.Conclusion:Prostaglandin E2might promote the cell proliferation of CCLP1cells through EP2receptor, which consequently upregulates the expression of AREG by activating of cAMP-PKA-CREB pathway. |
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