[Abstract] Objective: To explore the Mechanism of Prostaglandin E2 affects the cell prolifelation ability of human Bile duct carcinoma cells CCLP1. Methods: CCLP1 cells were treated with PGE2, EP1-4 receptor agonist , AC agonist Forskolin ,PKA antagonist H89, cAMP analogues dbcAMP. The levels of SnoN mRNA，SnoN protein expression and the cell prolifelation ability were examined by RT-PCR, Western blot and WST in CCLP1 cells. Results:The expression of SnoN in CCLP1 cells were increased by 13.4%、24.5%、35.6%、23.5%（P<0.05）after treated with 1μM、5μM、10μM、20μM PGE2 for 24h respectively, and the expression of SnoN mRNA in CCLP1 cells were increased by 22.5%（P<0.01）after treated with PGE2（10μmol/L）for 24 h；The expression of SnoN in CCLP1 cells were increased by 64.9%（P<0.05）after treated with EP2 receptor agonist (10μmol/L) for 24h , and the cell prolifelation ability of CCLP1 were increased by 26.5%， but just increased by 35.6% 、29.6%、24.9%（P<0.05）after treated with EP1、EP3、EP4(10μmol/L) receptor agonist for 24h， and the cell prolifelation ability of CCLP1 were increased by 12.5%、16.9%、30.2% respectively；The levels of SnoN expression and CREB phosphorylation in CCLP1 cells were increased by 25.1%、71.3%（P<0.05）compared with the control group after treated with AC agonist Forskolin(10μmol/L) for 24h, and the cell prolifelation ability of CCLP1 were increased by 2.7%、3.6%、4.4%（P<0.05）after treated with 1μM、5μM、10μM Forskolin respectively；The expression of SnoN in CCLP1 cells were increased by 90.1%（P<0.05）when we treated with cAMP analogues dbcAMP (500μmol/L) for 24h, but when we treated the cell with PKA antagonist H89 (10μmol/L) for 24h, the levels of SnoN expression and CREB phosphorylation in CCLP1 cells were decreased by 9.1%、14.1%（P<0.05）compared with the Forskolin treated group. and the cell prolifelation ability of CCLP1 were decreased by 2.7%、9.1%、27.1%、45.7%（P<0.05）after treated with 1μM、5μM、10μM、20μM H89 respectively. Conclusion: Our findings suggested that 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 Signal Conduction Pathway,and then promotes the cell prolifelation ability of CCLP1.
[Key words] Bile duct carcinoma; PGE2; EP prostanoid receptor; SnoN; cAMP-PKA-CREB
[摘要] 目的：探讨PGE2对人胆管上皮癌细胞CCLP1增殖能力影响的作用机制。方法：用PGE2、EP1-4四种受体激动剂、AC激动剂Forskolin、PKA抑制剂H89 、cAMP拟似物dbcAMP处理CCLP1细胞，通过RT-PCR、Western blot、 WST等实验检测SnoN mRNA、SnoN蛋白表达量以及CCLP1细胞增殖能力的变化。结果：1μM、5μM、10μM、20μM PGE2处理CCLP1细胞24h后，细胞中SnoN蛋白的表达水平分别上升了13.4%、24.5%、35.6%、23.5%（P<0.05），10μM PGE2处理CCLP1细胞24h后，SnoN mRNA的表达水平与对照组相比上升了22.5%（P<0.01）；10μM EP2受体激动剂处理CCLP1细胞24h后，细胞中SnoN蛋白的表达水平与对照组相比上升了64.9%（P<0.05），细胞增殖能力上升了26.5%，而10μM EP1、EP3、EP4受体激动剂处理CCLP1细胞24h后，SnoN蛋白的表达水平与对照组相比分别上升了35.6%、29.6%、24.9%（P<0.05），细胞增殖能力也分别上升了12.5%、16.9%、30.2%；10μM AC激动剂Forskolin处理CCLP1细胞24h后，SnoN蛋白的表达水平及CREB蛋白的磷酸化水平较对照组分别上升了25.1%、71.3%， 1μM、5μM、10μM Forskolin处理CCLP1细胞24小时后细胞增殖能力也分别上升了2.7%、3.6%、4.4%；用500μM cAMP拟似物dbcAMP处理CCLP1细胞24小时后SnoN蛋白的表达水平较对照组上升了90.1%（P<0.05）；而用PKA抑制剂H89阻断Forskolin的作用后，SnoN蛋白的表达水平和CREB蛋白的磷酸化水平较Forskolin处理组分别下降了9.1%、14.1%，1μM、5μM、10μM、20μM H89分别处理CCLP1细胞后，细胞增殖能力较对照组分别下降了2.7%、9.1%、27.1%、45.7%，差异均有统计学意义。结论：PGE2可通过EP2受体激活cAMP-PKA-CREB信号转导通路上调CCLP1细胞SnoN的表达，从而促进CCLP1细胞的增殖。
[关键词] 胆管上皮癌；PGE2；EP2受体；SnoN；cAMP-PKA-CREB
肝癌是我国的高发肿瘤之一，五年生存率低，严重危及人类的健康和生命。研究证实PGE2可以通过促进肿瘤细胞的增殖[1]、侵袭转移[2]、肿瘤血管的形成[3]而促进肿瘤的发生发展。目前认为PGE2是通过细胞膜表面的PGE2受体(EP受体)发挥作用, EP受体为细胞膜G蛋白偶联受体, 有4种亚型, 分别称为EP1、EP2、EP3 和EP4 受体[4]。
SnoN（ski-related novel gene）是Ski原癌基因家族的成员之一，目前研究表明，SnoN蛋白在人类许多肿瘤细胞中高表达[5]，但在人肝癌组织中关于SnoN蛋白的报道较少，有关SnoN在PGE2对CCLP1细胞增殖能力调控中的作用目前还不清楚。本研究应用PGE2、四种EP受体激动剂、腺苷酸环化酶（AC）激动剂Forskolin、cAMP拟似物dbcAMP及PKA抑制剂H89处理人胆管上皮癌细胞CCLP1，观察其SnoN 表达水平的变化及与CCLP1细胞增殖能力变化的关系，意在阐明SnoN蛋白在PGE2调控CCLP1细胞生长中的作用及其可能的信号转导通路。
1 材料和方法
1.1 材料
人肝癌胆管上皮癌细胞CCLP1细胞株（美国匹兹堡大学医学院 Wu Tong教授赠），DMEM培养基（美国Invitrogen公司），小牛血清（杭州四季青公司），PGE2 (美国Cayman公司)，EP1受体激动剂（17-phenyltrinor Prostaglandin E2）、EP2受体激动剂（Butaprost）（美国Sigma
