集落形成法和Western Blot法研究ELAC2的辐射增敏作用

目的 研究ELAC2基因对乳腺癌细胞MCF-7辐射敏感性的影响。方法 利用脂质体转染法将ELAC2基因转入MCF-7细胞,同时设立未转染MCF-7和转染空载体的MCF-7/pcDNA3作为对照。RT-PCR法检测ELAC2基因的mRNA表达;集落形成法检测ELAC2基因对肿瘤细胞辐射敏感性的影响;Western Blot法检测ELAC2、FEN-1、MGMT、MRE11及Ku-70基因的蛋白表达水平。结果 ELAC2基因的mRNA和蛋白水平在转染细胞中表达明显增高。细胞接受0、0.5、1、2、4、6Gy的X射线照射后,ELAC2基因高表达使MCF-7细胞的剂量-效应曲线左移,细胞辐射敏感性增强。细胞经5Gy的X射线照射,ELAC2基因高表达未使MGMT蛋白和MRE11蛋白表达发生明显变化,而FEN-1蛋白及Ku-70蛋白表达水平显著下降。结论 ELAC2基因高表达使细胞辐射敏感性增高,其涉及的分子机制可能与ELAC2下调FEN-1蛋白及Ku-70蛋白表达水平有关。

Study the Radiosensitive Effect of ELAC2 by Using Colony Formation Assay and Western Blot Assay

Objective To study the effects of ELAC2 gene on the human breast cancer cell MCF-7＇s radiosensitivity.Methods ELAC2 gene was transfected into MCF-7 via Lipofectamine transfection assay,while MCF-7 cells transfected with/without empty vector MCF-7/pcDNA3 were also established as negative control.The mRNA expression of ELAC2 gene was detected by RT-PCR assay.The colony formation assay was performed to test the radiosensitivity.The protein expression of ELAC2,FEN-1,MGMT,MRE11and Ku-70 were detected by western blot assay.Results Overexpressed ELAC2 level was both observed at mRNA and protein level of the cells transfected with pcDNA3.0/ELAC2.After 0,0.5,1,2,4,6Gy X-ray exposure,the radiation dose-response curve shifted to the left when ELAC2 overexpressed,and ELAC2 enhanced the radiosensitivity of MCF-7 cells.After 5Gy X-ray irradiation,no significant difference of MGMT and MRE11 protein expression levels were detected between ELAC2 overexpressed MCF-7 cells and control cells.While FEN-1 protein and Ku-70 protein expression levels were reduced significantly in MCF-7 cells with high level ELAC2.Conclusion ELAC2 gene could radiosensitize MCF-7 cells to X-ray irradiation,probably by the mechanisms of reducing the FEN-1 protein and Ku-70 protein expression.