CAJ | 학술논문

目的：探讨ERK、GSK3β和Snail在Fas诱导的EMT中的作用。方法选用结肠癌SW480细胞及胃癌AGS细胞作为研究对象，使用U0126以阻断其ERK/MAPK通路的激活，稳定转染GSK3βS9A以抑制其GSK3βSer9位点的磷酸化及信号转导，稳定转染Snail shRNA以抑制其Snail活性，并对接受上述处理的细胞分别给予低剂量FasL刺激后，再利用侵袭试验、免疫荧光、免疫印迹、RT-PCR、qRT-PCR、免疫共沉淀及荧光素酶报告基因等方式检测细胞的形态、功能变化。结果 Fas信号通路的激活可抑制E-cadherin的转录表达，且这一过程依赖于ERK/MAPK通路。通过转染稳定敲除Snail表达后，Fas对E-cadherin的转录抑制作用显著减弱。在细胞内过表达突变型GSK3βS9A可显著降低Fas通路激活后Snail的表达上调水平、E-cadherin的转录抑制水平及细胞的侵袭能力增加程度。免疫共沉淀提示， GSK3β与ERK、Snail在细胞核存在相互作用。结论在消化道肿瘤细胞中，Fas诱导EMT的调控机制包括ERK/MAPK通路的激活、ERK对GSK3β的磷酸化（Ser9位点）失活、GSK3β失活导致的Snail表达上调及核易位，以及Snail作用下的E-cadherin转录水平下调。
목적：탐토ERK、GSK3β화Snail재Fas유도적EMT중적작용。방법선용결장암SW480세포급위암AGS세포작위연구대상，사용U0126이조단기ERK/MAPK통로적격활，은정전염GSK3βS9A이억제기GSK3βSer9위점적린산화급신호전도，은정전염Snail shRNA이억제기Snail활성，병대접수상술처리적세포분별급여저제량FasL자격후，재이용침습시험、면역형광、면역인적、RT-PCR、qRT-PCR、면역공침정급형광소매보고기인등방식검측세포적형태、공능변화。결과 Fas신호통로적격활가억제E-cadherin적전록표체，차저일과정의뢰우ERK/MAPK통로。통과전염은정고제Snail표체후，Fas대E-cadherin적전록억제작용현저감약。재세포내과표체돌변형GSK3βS9A가현저강저Fas통로격활후Snail적표체상조수평、E-cadherin적전록억제수평급세포적침습능력증가정도。면역공침정제시， GSK3β여ERK、Snail재세포핵존재상호작용。결론재소화도종류세포중，Fas유도EMT적조공궤제포괄ERK/MAPK통로적격활、ERK대GSK3β적린산화（Ser9위점）실활、GSK3β실활도치적Snail표체상조급핵역위，이급Snail작용하적E-cadherin전록수평하조。
Aims To explore the role of ERK, GSK3βand Snail in Fas-induced EMT process. Methods Fas-signaling-induced changes of cellular morphology and functions in SW480 or AGS cells with blockage of ERK/MAPK pathway, inhibition of GSK3βtranscriptional activity or knock-down of Snail were detected by invasion assay, immunofluorescence, Western blot, RT-PCR , qRT-PCR, immunoprecipitation and luciferase reporter gene assay. Results The activation of the Fas signaling pathway could inhibit the transcription of E-cadherin in an ERK/MAPK pathway dependent manner. The inhibition of E-cadherin transcription by Fas signaling was significantly reduced after stable transfection of Snail shRNA. The overexpression of mutant GSK3βS9A significantly reduced Fas-induced Snail upregulation, E-cadherin downregulation, and cellular motility enhancement. The result of coimmunoprecipitation demonstrated the interaction between GSK3β, ERK and Snail in nucleus. Conclusion In gastrointestinal cancer cells, Fas signaling can induce EMT through the activation of ERK/MAPK pathway, the phosphorylation and inactivation of GSK3βby ERK, the upregula-tion and nuclear translocation of Snail resulting from GSK3βinactivation, as well as Snail-induced downregu-lation of E-cadherin.