EGF自分泌介导EMT对Morris Hepatoma 3924A肝癌细胞的影响

Experimental study on the effect of EGF autocrine mediated EMT on Morris Hepatoma 3924A liver cancer cells

目的通过观察上皮细胞-间充质转化(EMT)相关蛋白表达变化探讨表皮生长因子(EGF)自分泌对Morris Hepatoma 3924A肝癌细胞的影响。方法制作Morris Hepatoma 3924A肝癌细胞CoCl_(2)缺氧模型。通过Western Blot分析EGF自分泌对肿瘤EMT相关蛋白的表达变化,CCK-8法检测肝细胞癌细胞增殖能力变化,划痕实验检测细胞侵袭能力改变的情况。结果①CCK8实验显示:在轻度缺氧状态下(<150μmol/L CoCl_(2))肿瘤细胞增殖受限不明显,AG-1478的48h IC50为10μmol/L;②Western Blot显示:CoCl_(2)组的E-cadherin表达减低,而N-cadherin及EGF表达增加(P<0.05),CoCl_(2)+AG-1478组E-cadherin表达增加,N-cadherin及EGF表达减少(P<0.05);③划痕实验显示:CoCl_(2)组有更高的迁移能力,人为抑制EGF自分泌后迁移能力明显减弱(P<0.05)。结论肿瘤细胞在缺氧状态下EGF自分泌会对自身起正反馈效果,抑制EGF自分泌可有效抑制肿瘤细胞EMT过程,降低肿瘤细胞的迁移能力。

Objectivee To explore the effect of EGF autocrine on EMT after liver cancer intervention by observing changes in EMT related proteins.Methods A Morris Hepatoma 3924A liver cancer cell model was established by cobalt chloride hypoxia.The expression changes of tumor EMT related proteins caused by ECF autocrine were analyzed by Western blot.The proliferation ability of liver cancer cells under different treatment methods was detected by the CCK-8 method.The changes in cell invasion ability were detected by a scratch assay.Results①CCK8 experiment showed that under mild hypoxia(<150μmol/L),the proliferation of tumor cells is not significantly restricted with cobalt chloride,and the 48h ICso of AG-1478 is 10μmol/L;②Western blot showed that compared with the control group,the expression of E-cadherin was reduced in the cobalt chloride group,while the expression of N-cadherin and EGF was increased,with statistical significance(P<0.05);The expression of E-cadherin increased,while the expression of N-cadherin and EGF decreased in the cobalt chloride+AG-1478 group compared to the cobalt chloride group,and the difference was statistically significant(P<0. 05);③The scratch test showed that the cobalt chloride group had higher migrationability compared to the normal control group,and the migration ability was significantly reduced after inhibiting ECFautocrine(P<0. 05).Conclusionn Under hypoxic conditions,tumor cells exhibit a positive feedback effect on their ownsecretion of EGF;Inhibiting EGF autocrine can effectively inhibit the EMT process of tumor cells;Inhibiting EGF autocrinecan reduce the migration ability of tumor cells.