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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 investigate the regulatory mechanism of E-cadherin expression down-regulation in GSK-3β inactivation signal pathway in hepatocellular carcinoma (HCC). METHODS: Stable cell lines with loss of E-cadherin expression were established by RNAi technique. The expression of E-cadherin and the phosphorylation of GSK-3β and Akt in HepG2-E-cadherin-shRNA cells were detected by Western blot. HepG2-E-cadherin- The phosphorylation of GSK-3β was detected by Western blot. The mRNA levels of PTEN and Egr-1 in HepG2-E-cadherin-shRNA cells were detected by RT-PCR. The protein expression of PTEN was detected by Western blot. Results: The results of Western blot showed that the E-cadherin expression in HepG2-E-cadherin-shRNA cells was down-regulated by 82.54% compared with the blank control group. The phosphorylation of GSK-3β was increased by 298.93% Compared with the blank control group, the level of GSK-3βphosphorylation was reduced by 48.13% after LY294002 treatment for 4 h, and the level of E-cadherin in HepG2 cells was down-regulated by RT-PCR The mRNA level of PTEN decreased to 76.14%, the mRNA level of Egr-1 decreased to 66.89%, and the protein expression level of PTEN decreased to 53.77%. Conclusion: Inhibition of E-cadherin expression in human HCC cells leads to the inactivation of GSK-3β phosphorylation in human HCC cells probably through PI3K / Akt signaling pathway.