慢病毒介导RNF2基因表达对食管癌细胞X线照射后增殖与迁移等影响

Effect of lentivirus-mediated expression of RNF2 gene on the proliferation and migration of esophageal carcinoma cells following X-ray radiotherapy

目的 利用RNA干扰技术抑制人食管癌细胞中RNF2基因表达,观察其X线照射对细胞增殖活性、迁移能力、凋亡和细胞周期的影响.方法 培养人食管癌细胞ECA-109,RT-PCR检测RNF2 mRNA水平表达;MTT法检测ECA-109食管癌细胞细胞增殖;蛋白印迹法检测ECA-109-R细胞中RNF2蛋白表达变化;流式细胞技术检测照射后不同时间细胞周期和凋亡变化.Transwell侵袭小室模型检测转染对食管癌细胞迁移能力的影响.重复测量设计资料方差分析.结果 照射组食管癌细胞中RNF2 在mRNA水平和蛋白表达水平均较未照射组明显增加（P〈0.01）,且存在剂量-效应关系;MTT结果显示食管癌细胞的增殖水平在照射后不同时间点均明显降低（P〈0.01）;ECA-109-R组细胞中BMI1、RNF2蛋白表达水平较ECA-109组和ECA-109-N组细胞明显降低（P〈0.01）,ECA-109组与ECA-109-N组比较差异无统计学意义（P〉0.05）;Transwell侵袭小室模型检测结果显示照射后3.5 h ECA-109-R组穿膜细胞数明显低于ECA-109组和ECA-109-N组（P〈0.01）.6 Gy照射后各实验组处于G2＋M期比例明显高于相应未照射组（P〈0.01）,且ECA-109-R组处于G2＋M期的比例均明显低于照射后的ECA-109组和ECA-109-N组（P〈0.05）.照射后ECA-109-R组细胞凋亡率明显高于ECA-109组和ECA-109-N组（P〈0.01）.结论 采用RNA干扰技术降低食管癌细胞中RNF2的表达,降低细胞增殖和迁移能力,解除照射后G2＋M期阻滞,促进细胞凋亡,增加放射敏感性.

Objective To examine the effect of X-ray radiotherapy on cell proliferation, migration, apoptosis, and cell cycle of human esophageal carcinoma ECA-109 cells following RNA interference （RNAi）-mediated downregulation of RNF2 gene expression.Methods The level of RNF2 mRNA expression in the human esophageal carcinoma cell line ECA-109 was determined using RT-PCR.Cell proliferation of ECA-109 was measured by MTT assay, and the changes in RNF2 protein expression in ECA-109-R cells were determined using Western blot.The changes in cell cycle and cell apoptosis at different time points following radiation were analyzed by flow cytometry, and the effect of transduction on cell migration was examined using Transwell migration assay.Data were subjected to an analysis of variance with repeated measurement design.Results The mean mRNA and protein levels of RNF2 in ECA-109 cells were significantly increased in a dose-dependent manner in the radiation group than in the control group （P〈0.01）.MTT results demonstrated that ECA-109 cell proliferation was significantly decreased at each time point following radiation compared to those without radiation （P〈0.01）.The levels of BMI1 and RNF2 protein expression were significantly reduced in the ECA-109-R group than in the ECA-109 and ECA-109-N groups （P〈0.01）,and no significant difference in protein levels was observed between the ECA-109 group and ECA-109-N group （P〉0.05）.The Transwell migration assay showed that the number of migrating cells following 3.5 h of radiation was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups （P〈0.01）.The percentage of G2/M phase cells in each group was significantly lower following 6 Gy radiation compared to that in the corresponding untreated group （P〈0.01）, and the percentage of G2/M phase cells was significantly lower in the ECA-109-R group than in the ECA-109 and ECA-109-N groups （P〈0.05）.Furthermore,cell apoptosis following radiation was also significantly higher in the ECA-109-R group than in the ECA-109 and ECA-109-N groups （P〈0.01）.Conclusions RNAi-mediated downregulation of RNF2 expression in esophageal carcinoma cells can reduce cell proliferation and cell migration, rescue post-radiation G2/M cell cycle arrest, promote cell apoptosis,and increase radiosensitivity.