目的:利用体外细胞模型,研究转录因子增强激活子结合蛋白-4(TFAP4)对子宫内膜癌细胞恶性增殖的影响及探讨其可能的作用机制。方法利用小干扰RNA技术沉默子宫内膜癌细胞(HEC-1-A和RL95-2)内源性增强激活子结合蛋白-4(AP-4)的表达后,通过平板克隆及软琼脂克隆形成实验检测肿瘤细胞单克隆形成及非锚定生长能力的变化。利用碘化吡啶染色流式细胞术分析肿瘤细胞周期的变化,并利用荧光定量PCR技术检测CDKN1A的转录水平。利用生物信息学分析预测CDKN1A的启动子区域内是否存在AP-4及锌指蛋白Pokemon的结合位点,进一步利用染色体免疫共沉淀技术验证AP-4及Pokemon是否共同结合到CDKN1A的启动子区域。每次实验设3个复孔,每个实验重复3次以上,两组间数据比较采用 t 检验,多组间数据比较采用单因素方差分析,检验水平α=0.05。结果沉默内源性 AP-4的表达后,两种子宫内膜癌细胞(HEC-1-A 和RL95-2)的克隆形成能力降为0.38倍至0.55倍,软琼脂克隆形成能力降为0.21倍至0.27倍。进一步发现,沉默内源性AP-4的表达能诱导两种子宫内膜癌细胞发生G0/G1期阻滞并增加CDKN1A的转录水平为7.34倍至9.22倍。公共数据库分析显示,CDKN1A的启动子区域内存在AP-4及Pokemon相邻的结合位点,沉默内源性AP-4后能同时降低AP-4及Pokemon与CDKN1A的启动子区域的结合,其中AP-4的富集率降至0.07倍至0.21倍,Pokemon的富集率降至0.04倍至0.18倍。结论沉默内源性AP-4能在体外模型中,抑制子宫内膜癌细胞的恶性增殖,其机制可能与破坏了AP-4/Pokemon转录复合物对CDKN1A的转录抑制,从而阻抑了肿瘤细胞的周期运行。
Objective To investigate the effect and mechanism on malignant proliferation of endometrial cancer cells by silencing transcription factor AP-4in vitro.MethodsThe endometrial cancer cells (HEC-1-A and RL95-2) were treated by small interference RNA targeting by AP-4, and malignant proliferation capability was detected by clone formation analysis and soft agar colony assay. The cell cycle was detected by propidium iodide staining and flow cytometry analyzing, and CDKN1A mRNA level was measured by real-time quantitive polymerase chain reaction (real-time PCR). The AP-4 and Pokemon binding sites was predicted by bioinformatics software, and chromatin immunoprecipitation was used to detect the binding between AP-4 and Pokemon to promoter of CDKN1A. All values were presented as means±standard deviation (SD). Student's t-test was used to determine statistical differences between two groups, and One-Way ANOVA analysis was used to determine statistical differences among more than two groups.ResultsThe proliferation of endometrial cancer cells was remarkable suppressed by silencing AP-4 both in HEC-1-A and RL95-2. The siRNA targeting AP-4 treatment induced a block in the G0/G1 phase and increased mRNA level of CDKN1A. The bioinformatics software and publication datasets showed that AP-4 and Pokemon could directly bind on same region of CDKN1Apromoter, and silencing AP-4 suppressed the binding between AP-4 and Pokemon to promoter ofCDKN1A. ConclusionsSilenced AP-4 could inhibit endometrial cancer cells malignant proliferation and binding between AP-4/Pokemon complex and promoter ofCDKN1A in vitro.