上皮性卵巢癌中TFAP2A对hTERT表达的调节及其机制研究

TFAP2A Might Active hTERT in EOC Cells without through PI3K/AKT Signaling Pathway in Epithelial Ovarian Cancer

目的:探讨在上皮性卵巢癌中TFA2A对hTERT表达的调节和作用机制。方法:采用免疫组织化学方法检测TFAP2A和h TERT蛋白在卵巢正常、交界及上皮性卵巢癌组织中的表达,采用Western Blot和qRT-PCR技术检测hTERT在敲减TFAP2A基因的SKOV3、CAOV3细胞中的表达水平、检测hTERT在过表达TFAP2A基因的HO8910细胞中的表达水平。在干扰TFAP2A的CAOV3细胞中或过表达TFAP2A的HO8910细胞中分别加入PI3K/AKT信号通路激动剂740-YP或抑制剂LY294002,检测相关蛋白表达变化,探讨TFAP2A、hTERT与PI3K/AKT信号通路的关系。结果:TFAP2A在71.88%的上皮性卵巢癌组织中呈高表达,h TERT在78.12%的上皮性卵巢癌组织中呈高表达;将hTERT和TFAP2A的免疫组化评分行Pearson相关性分析,两者间相关系数r=0.78,P<0.001。Western Blot和qRT-PCR的结果均显示,在SKOV3和CAOV3卵巢癌细胞中,敲减TFAP2A后,h TERT的表达均明显下降,而在HO8910卵巢癌细胞中,增强TFAP2A基因表达后,hTERT的表达均明显上升。在CAOV3和HO8910处理细胞中,分别使用PI3K/AKT信号通路激动剂740-YP或阻滞剂LY294002处理后,Western Blot检验hTERT和PI3K/AKT通路蛋白的表达,发现激动剂740-YP或阻滞剂LY294002可以逆转敲减或过表达TFAP2A引发的PI3K/AKT通路蛋白表达下调或上调,但不能逆转hTERT蛋白表达下调或上调。结论:在卵巢肿瘤组织中,TFAP2A和hTERT在上皮性卵巢癌组织中均呈高表达,且hTERT的表达和TFAP2A成正相关,在上皮性卵巢癌细胞中TFAP2A可调节hTERT的表达,且TFAP2A对h TERT的表达的调节不经由PI3K/AKT通路。

Objective: To investigate the regulation and mechanism of TFA2 A on hTERT expression in epithelial ovarian cancer.Methods: We detected the expression level of TFAP2 A and hTERT in normal ovarian epithelial tissues, borderline ovarian tumor tissues as well as epithelial ovarian cancer tissue by an IHC assay. The expression of TFAP2 A gene in CAOV3 and SKOV3 cell lines were silenced by using small interfering RNA technique.The expression of TFAP2 A in HO8910 cell line were overexpressed by overexpression plasmid. Western Blot and qRT-PCR were used to test the expression of hTERT in TFAP2 A-silenced SKOV3 and CAOV3 cells or TFAP2 A-overexpressed HO8910 cells. The relationship between TFAP2 A, hTERT and PI3 K/AKT signaling pathways was explored by using the agonist 740-YP and inhibitor LY294002 of the PI3 K/AKT signalling pathway in TFAP2 A-silenced CAOV3 cells or in TFAP2 A-overexpressed HO8910. Results: By immunohistochemistry assays, we found that TFAP2 A was highly expressed in 71.88%epithelial ovarian cancer tissues, as well as hTERT was highly expressed in 78.12% of epithelial ovarian cancer tissues. By Pearson correlation analysis, We also found that the expression levels of hTERT were consistent with TFAP2 A in different ovarian tissues, and the correlation coefficient was 0.78. Western Blot and qRT-PCR showed that the expression levels of hTERT was significantly decreased in TFAP2 A-silenced CAOV3 and SKOV3 cells, while it was increased in TFAP2 A-overexpressed HO8910. Western Blot showed that the hTERT would not significantly increased or decreased as the PI3 K/AkT proteins after using PI3 K/AkT signaling pathway agonist 740-YP or blocker LY294002. Conclusions: TFAP2 A up-regulates hTERT expression in epithelial ovarian cancer cells do not by way of PI3 K/AKT signaling.