PGE_2上调AREG表达促进胆管癌细胞生长的机制研究

PGE_2 promotes the proliferation of cholangiocellular carcinoma cells by up-regulating AREG protein expression

目的:探讨AREG蛋白在前列腺素E2(prostaglandin E2,PGE2)促进人胆管上皮癌细胞CCLP1增殖能力中的作用和机制。方法:用PGE2、4种EP受体激动剂(17-phenyltrinor Prostaglandin E2、Butaprost、Sulprostone和Prostaglandin E1 Alcohol)、EP2受体拮抗剂AH6809、PKA抑制剂H89、AC抑制剂SQ22536处理CCLP1细胞,通过Western blot、WST等实验检测AREG蛋白表达水平以及CCLP1细胞增殖能力的变化。结果:10μmol/L PGE2处理CCLP1细胞后,AREG蛋白的表达水平与对照组相比升高了44.2%(P<0.05);20μmol/L AREG中和抗体Mab262和SAB1402118分别处理1 h后的WST实验结果显示,PGE2诱导的CCLP1细胞增殖可被AREG中和抗体抑制,分别下降了18%、20%(P<0.01)。10μmol/L 4种EP受体激动剂处理CCLP1细胞的结果表明,EP2受体激动剂具有明显促进AREG蛋白表达的作用(表达水平上调了20.1%,P<0.05),而EP1、EP3、EP4无明显促进表达作用;10μmol/L EP2受体拮抗剂AH6809处理CCLP1细胞后,AREG蛋白的表达水平较EP2受体激动剂处理组下调了20%(P<0.05)。用PKA抑制剂H89处理后,AREG蛋白的表达水平和CREB蛋白的磷酸化水平较PGE2处理组分别下降了54.4%、45%(P<0.05);用CMV500-DN-CREB质粒转染CCLP1细胞抑制了CREB的表达和磷酸化后,AREG的表达量较对照组明显下降约65%(P<0.01)。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AREG的表达,从而促进CCLP1细胞的增殖。

Objective：To investigate the role of AREG in PGE2-induced proliferation of cholangiocarcinoma cells（CCLP1 cells）.Methods：CCLP1 cells were treated with PGE2,EP1-4 receptor agonist,EP2 receptor antagonist H89,PKA inhibitor H89,cAMP inhibitor SQ22536.Western blot and WST were used to examine the expression of AREG protein and the cell proliferation ability in CCLP1 cells.Results：The level of AREG protein increased by 44.2%（P 0.05） after treated with 10 μmol/L PGE2.It was previously proved that PGE2 could promote the proliferation of CCLP1 cells,however,this effect was obviously inhibited by 20%（P 0.01） when CCLP1 cells were pretreated with the 20 μmol/L neutralizing-antibody（Mab262 and SAB1402118） of AREG.Then four selective EP receptor agonists（17-phenyltrinor Prostaglandin E2,Butaprost,Sulprostone and Prostaglandin E1 Alcohol） 10 μmol/L were used to stimulate CCLP1 cells,it were found that only EP2 agonist could induce the expression of AREG protein,increased by 20.1%（P 0.05）,whereas the other three agonists downregulated the protein expression of AREG.In agreement with this result,the expression of AREG protein improved by EP2 agonist was indeed suppressed by 20%,when CCLP1 cells were pretreated with 10 μmol/L AH6809,the EP2 antagonist.After treated with EP2 receptor agonist（10 μmol/L） for 24 h,the proliferation ability of CCLP1 cells increased by 26.5%.PKA inhibitor H89 and cAMP inhibitor SQ22536 can restrict the AREG protein increase induced by PGE2,and the AREG protein expression was downregulated by 54.4% and 46.6% respectively,compared with PGE2 treated group.It was reported that PGE2 could induce the phosphorylation of CREB in CCLP1 cells,which could bind and activate the CRE element of AREG promoter.However,H89 and SQ22536 could strikingly inhibit the phosphorylation of CREB induced by PGE2.At the same time,CMV500-DN-CREB plasmid was transfected into CCLP1 cells,resulting in a striking restriction of AREG protein expression induced by PGE2.Conclusion：Prostaglandin E2 might promote the cell proliferation of CCLP1 cells through EP2 receptor,which consequently upregulates the expression of AREG by activation of cAMP-PKA-CREB pathway.