EVI1表达与食管癌细胞放疗敏感性的关系及机制

Study on the relationship between EVI1 expression and radiosensitivity of esophageal cancer cells and its mechanism

目的探讨异位病毒整合位点1(EVI1)对食管癌细胞放疗敏感性的影响及作用机制。方法选择在我院接受放射治疗的食管癌患者组织标本67例,qRT-PCR检测EVI1在食管癌组织中的表达水平,Spearman等级相关分析EVI1的表达与放疗敏感性的相关性。常规培养ECA109细胞,采用分次放疗递增法诱导建立放射抵抗型细胞株ECA109(ECA109R),MTS检测ECA109和ECA109R细胞48 h的放射剂量IC50值,western blotting检测ECA109和ECA109R细胞中的EVI1表达。ECA109R细胞分为对照组(si-NC组)和干扰EVI1表达组(si-EVI1组)并进行siRNA转染,采用western blotting检测各组细胞中EVI1的表达,MTS检测各组细胞48 h的放射剂量IC50值。si-NC组和si-EVI1组细胞经辐射后,平板克隆检测各组细胞平板克隆形成率,流式细胞仪检测各组细胞的凋亡率,western blotting检测各组细胞中凋亡相关蛋白活化caspase 3、Bcl-2和Bax蛋白的表达。结果EVI1在放疗抵抗食管癌组织中的表达高于其在放疗敏感食管癌组织中的表达(P<0.05),EVI1的表达与食管癌患者分化程度和临床分期相关(P<0.05),EVI1的表达与放疗敏感性呈负相关(P<0.05)。与ECA109细胞相比,ECA109R细胞48 h的放射剂量IC50值和EVI1的表达增加(P<0.05)。siRNA转染ECA109R细胞后,与si-NC组相比,si-EVI1组细胞中EVI1的表达降低和放射剂量IC50值降低(P<0.05)。与辐射si-NC组相比,辐射si-EVI1组细胞平板克隆形成率降低,细胞凋亡率增加(P<0.05)。与辐射si-NC组相比,辐射si-EVI1组细胞中活化caspase 3和Bax蛋白的表达均增加,Bcl-2蛋白表达降低(P<0.05)。结论EVI1可能通过调控凋亡相关蛋白增加食管癌细胞放疗敏感性,是治疗食管癌的潜在靶标。

Objective To investigate the effect and mechanism of ectopic viral integration site 1(EVI1)on the sensitivity of esophageal cancer cells to radiotherapy.Methods 67 tissue samples of esophageal cancer patients received radiotherapy in our hospital were selected.The expression of EVI1 in esophageal cancer tissues was detected by qRT-PCR.Spearman rank correlation analysis was used to analyze the correlation between EVI1 expression and radiotherapy sensitivity.The relationship between EVI1 expression level and clinical pathological parameters of patients were analyzed by statistical analysis.ECA109 cells were routinely cultured,and the radiation-resistant cell line ECA109(ECA109R)was induced by incremental radiotherapy.MTS was used to detect the 48-hour radiation dose of ECA109 and ECA109R cells for 48 hours.Western blotting was used to detect the expression of EVI1 in ECA109 and ECA109R cells.ECA109R cells were divided into a control group(si-NC group)and an interfering EVI1 expression group(si-EVI1 group)and transfected with siRNA.Western blotting was used to detect the expression of EVI1 in each group of cells,and MTS detected the 48-hour radiation dose of each group cells for 48 hours.After the si-NC group and si-EVI1 group cells were irradiated,the plate clones were used to detect the plate clone formation rate of each group cells,the flow cytometry was used to detect the apoptosis rate of each group cells,and the Western blotting was used to detect the expression of apoptosis-related protein activated caspase 3,Bcl-2 and Bax proteins.Results The expression of EVI1 in esophageal cancer tissues in the radiotherapy-resistant group was higher than that in radiotherapy-sensitive esophageal cancer tissues(P<0.05).The expression of EVI1 was related to the differentiation and clinical stage of esophageal cancer patients(P<0.05),EVI1 expression was negatively correlated with radiotherapy sensitivity(P<0.05).Compared with ECA109 cells,ECA109R cells had an increased IC50 value and EVI1 expression at 48 h(P<0.05).After siRNA transfection into ECA109R cells,compared with si-NC group,the expression of EVI1 in si-EVI1 group cells decreased and the radiation dose IC50 value decreased(P<0.05).Compared with the radiation si-NC group,the cell plate clone formation rate of the radiation si-EVI1 group decreased,and the apoptosis rate increased(P<0.05).Compared with the irradiated si-NC group,the expression of activated caspase 3 and Bax protein in the irradiated si-EVI1 group were increased,and the expression of Bcl-2 proteine was decreased(P<0.05).Conclusion EVI1 may increase the radiosensitivity of esophageal cancer cells by regulating apoptosis-related proteins and is a potential target for the treatment of esophageal cancer.