人滋养层细胞表面抗原2基因表达对舌鳞状细胞癌细胞增殖、凋亡的影响及机制研究

Effect of human trophoblast cell-surface antigen 2 gene expression by RNA interference on proliferation and apoptosis of tongue squamous cell carcinoma and its mechanism

目的探讨抑制中人滋养层细胞表面抗原2（human trophoblast cell-surface antigen 2，Trop2）基因表达对舌鳞状细胞癌（tongue squamous cell carcinoma，TSCC）细胞增殖、凋亡的影响及机制，以期为预防和治疗TSCC提供依据。方法选取2014年2月至2016年5月于郑州大学第一附属医院口腔颌面外科行TSCC根治手术的患者46例，实时荧光定量PCR和蛋白质印迹法检测TSCC组织及相应癌旁组织中Trop2 mRNA及蛋白表达。分别用阴性对照小干扰RNA（阴性对照小干扰RNA组）、Trop2小干扰RNA（Trop2小干扰RNA组）转染CAL-27细胞，另设空白对照组，转染48 h后行蛋白质印迹法检测Trop2、Ki-67增殖指数、细胞周期蛋白D1、裂解胱天蛋白酶3、Notch1蛋白和Hes1蛋白表达，细胞计数法检测细胞增殖，流式细胞仪检测细胞周期及细胞凋亡率。结果TSCC中Trop2基因mRNA（5.72±1.13）及蛋白（0.77±0.06）表达均显著高于癌旁组织（分别为0.92±0.15、0.11±0.01）（P〈0.05）；转染Trop2小干扰RNA后CAL-27细胞中Trop2的蛋白表达（0.08±0.01）显著低于空白对照组（0.46±0.05），Trop2小干扰RNA组细胞存活率[（64.28±4.12）%]、S期细胞[（14.54±1.02）%]、Ki-67蛋白（0.12±0.01）、细胞周期蛋白D1（0.04±0.01）表达均显著低于空白对照组[分别为（100.00±1.02）%、（27.33±1.11）%、0.24±0.02及0.20±0.02]和阴性空白对照小干扰RNA组[分别为（96.55±2.43）%、（26.67±1.23）%、0.26±0.03及0.21±0.02]，细胞凋亡率（23.55±1.45）%、G0/G1期细胞[（72.32±0.94）%]及裂解胱天蛋白酶3（0.16±0.02）、Notch1蛋白（0.62±0.06）、Hes1蛋白（0.50±0.05）表达均显著高于空白对照组和阴性对照小干扰RNA组（P均〈0.05）。结论抑制TSCC细胞中Trop2基因表达可降低癌细胞增殖，阻滞细胞于G1期并促进细胞凋亡，与上调Notch1信号通路相关。

ObjectiveTo investigate the effect and its mechanism of human trophoblast cell-surface antigen 2 （Trop2） gene expression was inhibited in squamous cell carcinoma of tongue on the proliferation and apoptosis of cancer cells.MethodsA total of 46 patients from February 2014 to May 2016 received radical treatment of tongue cancer from oral and maxillofacial surgery of the First Affiliated Hospital of Zhengzhou University were enrolled in this study. Real time PCR and Western blotting were used to detect mRNA and protein expression of Trop2 in tongue squamous cell carcinoma and corresponding adjacent tissues; NC-siRNA and Trop2-siRNA were transfected into human tongue squamous cell carcinoma CAL-27 cells, a blank control group （control） was set, the expression of Trop2, Ki-67, cyclin D1, cleaved caspase3, Notch1, Hes1 protein after transfected for 48 h were detected by Western bloting; cell proliferation was detected by cell counting kit-8; cell cycle and apoptosis rate were detected by flow cytometry.ResultsThe mRNA （5.72±1.13） and protein expression （0.77±0.06） of Trop2 gene in tongue squamous cell carcinoma were significantly higher than those in adjacent tissues （0.92±0.15, 0.11±0.01, P〈0.05）; Trop2 protein expression after transfeced Trop2-siRNA （0.08±0.01） in CAL-27 cells decreased significantly （0.46±0.05）; the survival rate （64.28±4.12）%, S cells （14.54±1.02）% and Ki-67 （0.12±0.01）, cyclin D1 （0.04±0.01） protein expression in Trop2-siRNA group were significantly lower than those in control group [（100.00±1.02）%, （27.33±1.11）%, （0.24±0.02）, （0.20±0.02）] and NC-siRNA group [（96.55±2.43）%, （26.67±1.23）%, （0.26±0.03）, （0.21±0.024）], the apoptosis rate （23.55±1.45）%, G0/G1 cells （72.32±0.94）% and the expression of cleaved caspase 3 （0.16±0.02）, Notch1 （0.62±0.06） and Hes1 （0.50±0.05） protein were significantly higher than control group and NC-siRNA group （P〈0.05）.ConclusionsThe expression of Trop2 gene was inhibited in tongue squamous carcinoma cells can reduce the proliferation of cancer cells, block the cells in phase G1, and promote the apoptosis of cells. The mechanism is related to the up regulation of Notch1 signaling pathway.