靶向沉默人基因MDC1表达对食管癌细胞放射增敏作用的实验研究

Short hairpin RNA-mediated MDC1 gene silencing enhances the radiosensitivity of esophageal squamous cell carcinoma cell line ECA109

目的利用RNA干扰技术抑制食管癌细胞ECA109细胞MDC1基因表达,观察其对细胞周期和放射敏感性的影响。方法构建MDC1基因的干扰质粒pMDC1-shRNA,与慢病毒包装质粒混合物共同转染293T细胞,收集病毒液,感染ECA109细胞,采用Real-TimePCR和westernblotting测定MDC1在mRNA水平和蛋白水平的表达。采用流式细胞术和克隆形成实验,检测RNA干扰对ECA109细胞放射敏感性的影响。结果成功构建pMDC1-shRNA质粒,转染ECA109细胞,获得稳定转染细胞株ECA109/MDC1,基因MDC1在mRNA水平和蛋白水平的表达均明显降低;5Gy射线照射后12h、24h、48h,ECA109/MDC1的G2/M期比例明显低于阴性对照组和空白对照组;ECA109、ECA109/NEGATIVE、ECA109/MDC1细胞的D0值分别为3.06Gy、2.90Gy、1.88Gy;SF2值分别为0.91、0.89、0.84;Dq值分别为1.59、1.47、1.20,显示ECA109/MDC1的D0值、SF2值、Dq值均明显降低。结论 RNAi技术可以有效地抑制食管癌细胞ECA109中基因MDC1的表达,从而增强ECA109细胞对放射线的敏感性。

Objective To explore the effect of MDC1 gene silencing by RNA interference on the radiosensitivity of human esophageal carcinoma cell line ECA109. Methods The vectors containing short hairpin RNA （shRNA） targeting MDC1 gene （pMDC1-shRNA） were cotransfected with pPACKH1-lentivector packaging system into 293T cells to package the lentivirus particles. Forty-eight hours after the transfection with specific or control lentiviral vectors, the stable integrants were selected using copGFP reporter gene; real-time RT-PCR and Western blotting were used to detect the expression levels of MDC1 mRNA and protein in the transfected ECA109 cells, respectively. The cell cycle distribution was measured with flow cytometry at 12, 24 and 48 h after a 5 Gy irradiation, and the radiosensitivity of esophageal carcinoma cell was evaluated by clone formation array. Results Sequence analysis confirmed correct insertion of MDC1-shRNA construct into pSIH1-H1-copGFP. The percentage of G2/M phase ECA109/ MDC1 cells was lower than that of ECA109 and ECA109/negative cells. The value of D0, SF2 and Dq of ECA109/ MDC1 cells were 1.88 Gy, 0.84 and 1.20, respectively, lower than those of ECA109 cells （3.06 Gy, 0.91 and 1.59） and those of ECA109/negative cells （2.90 Gy, 0.89 and 1.47）. Conclusion RNA interference can inhibit MDC1 gene expression and enhance the radiosensitivity of ECA109 cells in vitro.