TFAP4低表达对胃癌细胞恶性生物学行为的影响及机制

Effect and mechanism of low expression of TFAP4 on malignant biological behavior of gastric cancer cells

目的探讨TFAP4低表达对胃癌细胞恶性生物学行为的影响及机制。方法用RT-PCR、Westernblot⁃ting法检测细胞中TFAP4表达,选取TFAP4 mRNA和蛋白表达最高的MGC-803细胞作为实验细胞。将MGC-803细胞随机分为空白对照组(Control组)、阴性对照组(NC组)、TFAP沉默组(siTFAP组)、TFAP沉默+空载转染组(siT⁃FAP4+Vector组)、TFAP沉默+HMGB1过表达组(siTFAP4+HMGB1组)并进行转染。用RT-PCR技术检测各组细胞中TFAP4 mRNA表达,确认转染效率。用RT-PCR技术检测细胞中HMGB1 mRNA表达,用Westernblotting法检测细胞中TFAP4、HMGB1蛋白表达及胞质、胞核YAP蛋白表达,用划痕实验检测细胞迁移能力,用Transwell小室实验检测细胞侵袭能力,用体外血管生成实验检测内皮细胞成管能力,用双荧光素酶报告基因实验验证TFAP4对HMGB1的转录调控作用。结果与NC组比较,siTFAP4组细胞迁移率低,细胞侵袭数目和管腔形成数目少,TFAP4、HMGB1及胞核YAP表达低,胞质YAP表达高(P均<0.05)。与siTFAP4+Vector组比较,siTFAP4+HMGB1组细胞迁移率高,细胞侵袭数目和管腔形成数目多,HMGB1、胞核YAP表达高,胞质YAP表达低(P均<0.05)。双荧光素酶实验结果显示,与pGL3-Basic+TFAP4+pRL-TKVector转染细胞比较,pGL3-Basic-Promoter+TFAP4+pRL-TKVector转染细胞荧光素酶相对活性高(P<0.05)。结论TFAP4低表达可抑制胃癌细胞侵袭、迁移,促进内皮成管,其机制可能与TFAP转录调控HMGB1激活YAP信号有关。

Objective To investigate the effect and mechanism of transcription factor activating enhancer-binding protein 4(TFAP4)on malignant biological behavior of gastric cancer cells.Methods RT-PCR and Western blotting were used to detect the expression of TFAP4,and MGC-803 cells with the highest expression of TFAP4 mRNA and protein were selected as experimental cells.MGC-803 cells were randomly divided into the blank control group(Control group),negative control group(NC group),TFAP silencing group(siTFAP group),TFAP silencing+no-load transfection group(siTFAP4+Vector group),and TFAP silencing+HMGB1 overexpression group(siTFAP4+HMGB1 group),and then cells in all groups were transfected.The mRNA expression of TFAP4 in each group was detected by RT-PCR to confirm the transfection efficiency.The mRNA expression levels of TFAP4 and HMGB1 in the cells were detected by RT-PCR,the protein expression of TFAP4 and HMGB1 and the protein expression of YAP in cytoplasm and nucleus were detected by Western blotting,the cell migration ability was detected by Scratch test,and the cell invasion ability was detected by Transwell chamber test.In vitro angiogenesis assay was used to test the tube forming ability of endothelial cells,and dual luciferase reporter assay was used to verify the transcriptional regulation of TFAP4 on HMGB1.Results Compared with NC group,the cell migration rate,the number of invasion and lumen formation significantly decreased,the expression of TFAP4,HMGB1 and YAP in cell nucleus decreased,and the expression of YAP in cytoplasm increased in the siTFAP4 group(all P<0.05).Compared with the siTFAP4+Vector group,the cell migration rate,number of invasion and lumen formation increased,the expression of HMGB1 and YAP in cell nucleus increased,and the expression of YAP in cytoplasm decreased in siTFAP4+HMGB1 group(all P<0.05).The results of double luciferase experiment showed that compared with the pGL3-basic+TFAP4+PRL-TK Vector transfection cells,the luciferase relative activity increased in the pGL3-basic-promoter+TFAP4+PRL-TK Vector transfection cells(P<0.05).Conclusion Low expression of TFAP4 can inhibit the invasion and migration of gastric cancer cells,and the mechanism may be related to the transcriptional regulation of HMGB1 and activation YAP signal by TFAP.