食管癌细胞中沉默Survivin基因对ATG16L1表达的影响

Effeet of Survivin Gene Silencing on Expression of ATG16L1 in Esophageal Carcinoma Cells

目的探讨食管癌Eca109细胞中沉默生存素(Survivin,SVV)基因的表达对自噬相关蛋白ATG16L1表达的影响。方法使用雷帕霉素(rapamycin,RAPA)在Eca109细胞中诱导建立自噬模型;使用Survivin抑制剂YM155和Survivin-siRNA作用于自噬模型,采用免疫荧光法观察自噬体数量变化,采用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)检测Survivin和ATG16L1的mRNA表达水平,Western blot法检测LC3-Ⅱ、p62、Survivin、ATG16L1的蛋白表达水平,双荧光素酶报告基因检测Survivin与p62的作用关系。结果RAPA诱导Eca109细胞后,自噬标志蛋白LC3-Ⅱ蛋白的表达水平升高,p62蛋白的表达水平降低,同时细胞内自噬体数目增多。在RAPA组中ATG16L1mRNA和蛋白表达水平均升高。在细胞自噬模型中沉默Survivin表达后,Survivin和ATG16L1的mRNA表达水平均降低。过表达Survivin能够显著抑制p62的启动子活性,下调p62的蛋白表达。结论沉默食管癌Eca109细胞中Survivin基因表达可以下调ATG16L1的表达,可能与Survivin靶向抑制p62有关。

Objective To investigate the effect of silencing the expression of survivin gene on the expression of autophagy related protein ATG16L1 in esophageal carcinoma Eca109 cells.MethodssRapamycin(RAPA)was used to induce Eca109 cells to establish autophagy model,Survivin inhibitor YM155 and Survivin-siRNA were used to treat the autophagy model,the number of autophagosomes after cell induction was observed by immunofluorescence.The mRNA expression levels of Survivin and ATG16L1 were detected by real-time quantitative polymerase chain reaction(RT-qPCR).The protein expression levels of LC3-Ⅱ,p62,Survivin and ATG16L1 were detected by Western blot,and double luciferase reporter gene assay was used to detect the interaction between Survivin and p62.Results Eca109 cells were induced by RAPA,the expression levels of autophagy marker protein LC3-Ⅱwas incresed and the protein expression of p62 was decreased,and the number of autophagosomes were incresed.In RAPA group,the ATG16LlmRNA and protein expression levels were increased.The mRNA expression and protein expression of Survivin and ATG16LIdecreased after silencing of Survivin in the cellular autophagy model.Survivin overexpression could significantly inhibit the promoter activity and down-regulate the protein expression of p62.Conclusion Silencing Survivin gene expression in esophageal cancer Eca109 cells downregulates ATG16LI expression,which may be associated with Survivin-targeted p62 inhibition.