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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 study the effect of TFAP4 on malignant proliferation of endometrial carcinoma cells and to explore its possible mechanism by using in vitro cell model. Methods Endometrial cancer cells (HEC-1-A and RL95-2) were silenced by small interfering RNA (RNAi), and then endogenously stimulated with AP-4 expression by plate-cloning and soft agar cloning The changes of tumor cell monoclonal formation and non-anchored growth were detected by experiments. The change of tumor cell cycle was analyzed by pyridine iodide staining flow cytometry, and the transcription level of CDKN1A was detected by fluorescence quantitative PCR. Bioinformatics analysis was used to predict whether AP-4 and Pokemon binding sites existed in the promoter region of CDKN1A. The co-localization of AP-4 and Pokemon to the promoter region of CDKN1A was further verified by chromosome coimmunoprecipitation. Three replicate wells were set up for each experiment, and each experiment was repeated more than 3 times. The data of two groups were compared by t-test. One-way ANOVA was used to compare the data between groups. The test level was α = 0.05. Results After silenced the expression of endogenous AP-4, the clonogenic capacity of two endometrial cancer cells (HEC-1-A and RL95-2) was reduced from 0.38 times to 0.55 times and the soft agar colony formation ability was reduced to 0.21 Times to 0.27 times. It was further found that silencing endogenous AP-4 expression induced G0 / G1 phase arrest and increased CDKN1A transcriptional levels from 7.34-fold to 9.22-fold in both endometrial cancer cells. Public database analysis showed that there are AP-4 and Pokemon adjacent binding sites in the promoter region of CDKN1A. The silencing of endogenous AP-4 can reduce the binding of AP-4 and Pokemon to CDKN1A promoter region simultaneously The enrichment rate of AP-4 dropped to 0.07-fold to 0.21-fold and the Pokemon enrichment rate decreased to 0.04-fold to 0.18-fold. Conclusion Silencing endogenous AP-4 can inhibit the malignant proliferation of endometrial cancer cells in vitro. The mechanism may be that it inhibits the transcriptional repression of CDKN1A by AP-4 / Pokemon transcription complex and thus suppresses tumor cells Cycle of operation.