E-cadherin表达下调致GSK-3β磷酸化失活的分子机制研究  

Down-regulation of E-cadherin upregulates the phosphorylation level of GSK-3β in human hepatoma cells

作　　者：马修平[1] 张海[1] 汪亦品[1] 张丽[1] 马娟[1] 彭韬[1] 冷静[1] 

机构地区：[1]南京医科大学肿瘤中心病理学系,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2012年第7期896-902,共7页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(30871015;81172003)

摘　　要：目的:探讨E-cadherin表达下调在肝细胞肝癌(hepatocellular carcinoma,HCC)中诱导GSK-3β磷酸化失活信号通路的调控机制。方法:运用RNA干扰技术建立E-cadherin表达缺失的稳定细胞株。Western blot实验检测稳定株细胞(HepG2-E-cadherin-shRNA)E-cadherin的表达水平以及GSK-3β与Akt的磷酸化水平;用10μmol/L PI3K抑制剂(LY294002)处理HepG2-E-cadherin-shRNA细胞,Western blot检测细胞GSK-3β磷酸化水平,RT-PCR检测HepG2-E-cadherin-shRNA细胞PTEN和Egr-1的mRNA水平,Western blot检测细胞PTEN的蛋白表达水平。结果:Western blot实验结果表明,HepG2-E-cadherin-shRNA稳定株细胞E-cadherin表达水平较空白对照组下调约82.54%,GSK-3β磷酸化水平较空白对照组上升约298.93%,Akt磷酸化水平较空白对照组上升约218.98%;LY294002作用4 h后,GSK-3β磷酸化水平与空白对照组相比下降至48.13%;RT-PCR结果显示,下调HepG2细胞E-cadherin表达水平后,细胞PTEN的mRNA水平下降至76.14%,Egr-1的mRNA水平下降至66.89%,PTEN的蛋白表达水平下降至53.77%。结论:人HCC细胞中E-cadherin抑制性表达导致细胞内GSK-3β磷酸化失活很可能是通过PI3K/Akt的信号通路实现的。Objective：To explore the molecular regulatory mechanism of down-regulation of E-cadhefin inducing GSK-3β phosphorylation deactivation in hepatocellular carcinoma （HCC）. Methods：The stable cell line without E-cadherin expression was constructed by RNA interference method. The expression levels of E-cadherin,Akt,p-Akt,GSK-3β and p-GSK-3β were examined by Western blot analysis;The expression levels of p-GSK-3β in HepG2-E-cadherin-shRNA stable cell line were examined by Western blot analysis after 10 p, mol/L LY294002 （PI3K inhibitors） treatment;The mRNA level of Egr-1 and PTEN in HepG2-E-cadherinshRNA cells were tested by RT-PCR;The expression of PTEN was also examined by Western blot analysis. Results： Compared with the blank,E-eadherin expression level down-regulated 82.54% ,and the phosphorylation level of GSK-3β and Akt upregulated 298.93% and 218.98%,respectively. With the stimulation of PI3K inhibitor （LY294002）,the phosphorylation level of GSK-3β downregulated to 48.13% when compared with the blank. The mRNA level of PTEN dropped to 76.14% ,and Egr-1 to 66.89% when compared with the blank. The expression of PTEN protein also down-regulated to 53.77% when compared with the blank. Conclusion： Down-regulation of E-cadherin deactivated GSK-3β via PI3K/Akt signaling in HCC cells.

关 键 词：E-钙黏蛋白 肝细胞肝癌 Akt信号转导通路 糖原合酶激酶3Β PTEN 

分 类 号：R730.231[医药卫生—肿瘤]