沉默ECT2对食管癌EC109细胞增殖、细胞周期和裸鼠成瘤能力的影响

Effects of shRNA silencing ECT2 on proliferation and cell cycle of EC109 cells and tumorigenic ability in nude mice

目的:探讨shRNA沉默ECT2对食管癌EC109细胞增殖和裸鼠成瘤能力的影响及其机制。方法:将体外培养的EC109细胞分为对照组(未感染)、shRNA-NC组(感染shRNA-NC慢病毒)和shRNA-ECT2组(感染shRNA-ECT2慢病毒),采用RT-PCR检测各组细胞中ECT2 mRNA的表达,Western blot检测ECT2、细胞周期蛋白1(CyclinD1)和细胞周期依赖蛋白激酶2(CDK2)蛋白的表达,CCK-8和碘化丙啶染色检测细胞的增殖活力和细胞周期。将3组细胞接种于裸鼠皮下,建立食管癌裸鼠移植瘤模型,每组11只,喂养30 d后处死裸鼠,剥离肿瘤组织,测量肿瘤体积并称取瘤体质量,采用Western blot检测肿瘤组织中P21和P53蛋白的表达情况。结果:与对照组和shRNA-NC组相比,shRNA-ECT2组细胞中ECT2 mRNA和ECT2、CyclinD1、CDK2蛋白的表达水平、细胞增殖活力和S期细胞百分比均明显降低,G0/G1期细胞百分比明显升高(P<0.05);与对照组和shRNA-NC组相比,shRNA-ECT2组移植瘤体积和瘤体质量均减小,而P21和P53蛋白的表达水平均明显升高(P<0.05)。结论:shRNA沉默ECT2可抑制食管癌EC109细胞增殖和成瘤能力,其作用机制可能与促进P21和P53蛋白的表达有关。

Aim:To investigate the effects of shRNA silencing ECT2 on the proliferation of EC109 cells and the tumorigenic ability in nude mice and its mechanism.Methods:EC109 cells cultured in vitro were divided into control group(uninfected),shRNA-NC group(infected with empty vector shRNA-NC lentivirus)and shRNA-ECT2 group(infected with shRNA-ECT2 lentivirus).The expression of ECT2 mRNA in EC109 cells of each group was determined by RT-PCR,the expressions of ECT2,CyclinD1 and CDK2 protein were checked by Western blot,and the cell proliferation and cycle changes in each group were measured by CCK-8 assay and flow cytometry.The cells of control group,shRNA-NC group and shRNA-ECT2 group were inoculated subcutaneously in nude mice to establish the xenograft model of esophageal cancer in nude mice.After 30 days of feeding,the nude mice were executed,the tumor tissue was prepared,the volume of the tumors was measured and the tumors were taken out.The expressions of P21 and P53 protein in tumor tissue were tested by Western blot.Results:Compared with the control group and shRNA-NC group,the expression levels of ECT2 mRNA and protein,CyclinD1 protein,CDK2 protein,cell proliferation activity and S phase cell percentage in shRNA-ECT2 group were significantly decreased,and the percentage of G0/G1 phase cells was significantly increased(P<0.05).Compared with control group and shRNA-NC group,the volume and mass of tumors in the shRNA-ECT2 group were significantly reduced,while the expression levels of P21 and P53 protein were significantly increased(P<0.05).Conclusion:shRNA silencing ECT2 can inhibit the proliferation and tumorigenesis of EC109 cells,and its mechanism may be related to the promotion of the expression of P21 and P53 proteins.