LYN通过Wnt/β-catenin信号通路对胃癌细胞迁移、侵袭能力的影响

LYN's effects on the migration and invasion of gastric cancer cells through Wnt/β-catenin signaling pathway

目的探讨LYN对胃癌细胞迁移、侵袭等生物学行为的影响,并分析其对Wnt/β-catenin信号通路的调控机制。方法﹑选取2017年10月-2018年5月承德医学院附属医院收治的胃癌患者的胃癌组织和癌旁组织样本,采用qRT-PCR检测在临床收集的胃癌和癌旁组织样本中的LYN表达水平。以胃癌AGS细胞为实验对象,利用脂质体Lipolectamine2000将shRNA-LYN转染至胃癌ACS细胞中作为实验组(sh-LYN组),未转染细胞为对照组(NC组)。qRT-PCR检测转染前后AGS细胞中LYN mRNA表达情况。Transwell检测细胞的迁移和侵袭;TUNEL检测细胞凋亡;蛋白印迹分析(Western Blot)检测Wnt/β-catenin信号通路相关蛋白,对其调控机制进行研究。计量资料以均值±标准差(Meun±SD)表示,两组间比较采用独立样本t检验。P<0.05为差异具有统计学意义。结果LYN mRNA表达在胃癌组织中被显著上调(t=11.04,P<0.001);sh-LYN转染胃癌细胞可使LYN蛋白的表达量低于正常对照组(F=961.26,P=0.016);转染后,sh-LYN组细胞的迁移、侵袭受到抑制(t=3.437,4.450,P=0.011,0.026),转染后sh-LYN组细胞的凋亡率显著高于正常对照组(t=6.482,P=0.003);Westerm Blot检测结果:相比于正常对照组,sh-LYN薇低LYN的表达之后,可显著降低Wnt3和β-catenin的表达。下游蛋白的检测发现LYN敲低后,EMT上皮标记E-钙黏蛋白(E-calherin)的表达增加,EMT间充质标记N-钙黏蛋白(N-cadherin),波形蛋白(vimentin)表达则下调,相关蛋白锌指蛋白1(Snaill)和锌指蛋白2(Snail2)表达也下调。结论敲低LYN可抑制胃癌细胞的迁移和侵袭,并诱导胃癌细胞凋亡,其过程与Wnt/β-catenin信号通路有关。

Objective To explore the effects of LYN on the biological behaviors of gastric cancer cells,such as migration and invasion,and analyze the regulatory mechanism of Wnt/β-catenin signaling.Methods Gastric and paracancerous tissue samples from gastric cancer patients admitted to the Affiliated Hospital of Chengde Medical University from Oct.2017 to May 2018 were selected to detect the expression level of LYN in clinically collected gastric cancer and paracancerous tissue samples by qRT-PCR.Gastric cancer AGS cells were used as experimental subjects,and shRNA-LYN was transfected into gastric cancer AGS cells using Lipofectamine 2000 as experimental group(Sh-LYN group),and untransfected cells as control group(NC group).LYN mRNA expression in AGS cells before and after transfection was detected by qRT-PCR.Transwell detected cell migration and invasion.Cell apoptosis was detected by TUNEL.Western Blot analysis was performed to detect proteins related to the Wnt/β-catenin signaling pathway and to study its regulatory mechanism.Measurement data were expressed as(Mean±SD),and independent sample t test was used for comparison between the two groups.P<0.05 was considered statistically significant.Results LYN mRNA expression was significantly up-regulated in gastric cancer tissues(t=11.04,P<0.001).Sli-LYN transfected gastric cancer cells could make LYN protein expression lower than that of the normal control group(F=961.26,P=0.016).After transfection,migration and invasion of cells in the sh-LYN group were inhibited,and the apoptosis rate of cells in the sh-LYN group was significantly higher than that in the normal control group(t=3.437,4.450,P=0.011,0.026).Western Blot results:sh-LYN reduced the expression of Wnt3 andβ-catenin significantly when compared with the normal control group.The detection of downstream proteins showed that after LYN knockdown,the expression of EMT epithelial labeled E-cadherin was increased,and EMT mesenchymal labeled N-cardherin and vimentin were down-regulated.The other related proteins snail 1 and snail 2 were also down-regulated.Conclusion LYN knockdown inhibits migration and invasion of gastric cancer cells and induces apoptosis in gastric cancer cells,a process associated with the Wnt/Rcatenin signaling pathway.