转录因子增强子结合蛋白-4基因沉默对子宫内膜癌细胞凋亡的影响

摘要

目的：观察内源性转录因子增强子结合蛋白-4（AP-4）沉默对子宫内膜癌细胞凋亡的影响，并探讨其可能的作用机制。方法将人子宫内膜癌细胞（HEC-1-A 和 RL95-2）分为 NC 组、si＃1组、si＃2组，si＃1组转染 si-TFAP4片段1，si＃2组转染 si-TFAP4片段2，NC 组转染对照 siRNA。采用 Hoechest 33258染色观察凋亡核形态，采用 Annexin V-FITC ／PI 染色流式细胞术测算细胞凋亡率，采用 real-time PCR 技术检测存活素（Survivin）mRNA 表达，采用生物信息学分析预测 Survivin 编码基因 BIRC5的启动子区域内是否存在 AP-4的结合位点，采用染色体免疫共沉淀技术验证 AP-4是否结合到 BIRC5的启动子区域，采用双荧光素酶报告系统检测 BIRC5的启动子转录激活情况。结果在 HEC-1-A、RL95-2细胞中，与 NC 组相比，si＃1组、si＃2组凋亡核型细胞数均增加，细胞凋亡率均增加，Survivin mRNA 相对表达量均减少（P 均＜0．05）。BIRC5的启动子区域内存在一个高置信度的 AP-4结合位点，沉默 HEC-1-A、RL95-2细胞内源性 AP-4后能降低 AP-4对 BIRC5启动子区域的结合并抑制其转录激活状态。结论沉默内源性 AP-4能诱导子宫内膜癌细胞凋亡，其机制可能为降低了 AP-4对 Survivin 的转录激活，从而诱导了肿瘤细胞的凋亡。%Objective To investigate the effect of silencing transcription factor activating enhancer-binding protein 4 (TFAP-4)in vitro on apoptosis of endometrial cancer cells and its mechanism.Methods The human endometrial cancer cells (HEC-1-A and RL95-2 cells)were divided into NC group,si#1 group and si#2 group.The si#1 group and si#2 group were transfected with si-TFAP4 fragment 1 and si-TFAP4 fragment 2,respectively,and the NC group was transfected with control siRNA.The apoptosis was detected by Hoechest 33258 staining and the apoptosis rate by Annexin V-FITC/PI assay. The survivin (BIRC5)mRNA was detected by real-time PCR.Bioinformatics software was used to detect whether there were the AP-4 binding sites on the promoter of BIRC5.Chromatin immunoprecipitation was used to detect the binding between AP-4 and the promoter of BIRC5,and the dual-luciferase reporter gene assay was applied to detect the transcriptional activity of AP-4 on promoter of BIRC5.Results The number of apoptosis and the apoptosis rate in HEC-1-A and RL95-2 cells of the si#1 group and si#2 group was significantly increased,while the survivin mRNA expression was decreased as compared with that of the control group (all P <0.05).A high-confidence AP-4 binding site existed in the BIRC5 promoter region,and si-lencing AP-4 suppressed the binding of AP-4 on the promoter region of BIRC5,and suppressed the transcriptional activity of AP-4 on promoter of BIRC5.Conclusions Silencing AP-4 could induce the apoptosis of endometrial cancer cells through re-ducing the AP-4 on survivin transcription activation,and thereby inducing apoptosis of tumor cells.