RNA干扰MDC1基因对食管癌细胞X线照后细胞周期及相关蛋白表达影响

Effect of RNA interference for MDC1 gene on cell cycle and expression of related proteins in esophageal carcinoma cells after X-ray radiation

目的：利用RNA干扰技术降低食管癌细胞ECA109中MDC1基因表达，观察照射后细胞周期和放射敏感性变化并探讨相关机制。方法针对MDC1mRNA序列设计合成3对有效干扰序列和阴性对照序列，与载体pSIH1？H1？copGFP形成重组质粒，RT？PCR和蛋白印迹法测定MDC1mRNA和蛋白水平表达。克隆形成实验检测细胞放射敏感性，流式细胞术检测细胞周期，蛋白印迹法检测CHK1、CHK2蛋白表达，激光共聚焦显微镜观察细胞核内MDC1斑点数量。单因素方差分析组间差别。结果成功构建 pMDC1？shRNA 质粒并感染 ECA109细胞，获得稳定转染细胞 ECA109M。ECA109M细胞MDC1mRNA、蛋白表达水平低于ECA109N、ECA109细胞（ P＝0．032、P＝0．041）。5 Gy照射后 ECA109M 细胞 G2＋M 期比例低于 ECA109N、ECA109（P＝0．026）。5 Gy 照射后 ECA109、ECA109N、ECA109M细胞中CHK1和CHK2蛋白表达相近（P＝0．345和P＝0．451），ECA109M 细胞CHK2T68蛋白表达低于ECA109、ECA109N细胞（ P＝0．012）。 ECA109细胞D0值为3．06 Gy，SF2值为0．91；ECA109N、ECA109M细胞的D0值分别为2．90、1．88 Gy；SF2值分别为0．89、0．84（ P＝0．021；P＝0．037）。结论 RNA干扰降低MDC1蛋白表达后可以降低细胞周期相关蛋白的表达，解除细胞周期阻滞，增强食管癌细胞ECA109的放射敏感性。

Objective To apply RNA interference technique for reducing the expression of MDC1 gene in esophageal carcinoma cell line ECA109, observe the changes in cell cycle and radiosensitivity after radiation, and discuss related mechanisms. Methods Three pairs of effective interference sequences and negative control sequences were synthesized for MDC1 mRNA sequence, and a recombinant plasmid was constructed with the vector pSIH1？H1？copGFP. RT？PCR and Western blot were used to determine the expression levels of MDC1 mRNA and protein. Colony？forming assay was applied to measure radiosensitivity, flow cytometry to determine cell cycle, Western blot to determine the expression of CHK1 and CHK2 proteins, and laser scanning confocal microscope to observe the number of MDC1 blotches inside the nucleus. One？way analysis of variance was used to analyze the differences between groups. Results The pSIH1？H1？copGFP plasmid was constructed successfully and ECA109 cells were infected to obtain ECA109M cells with stable transfection. The expression levels of MDC1 mRNA and protein in ECA109M cells were lower than those in ECA109N and ECA109 cells （ P= 0. 032 and 0. 041, respectively ） . After 5？Gy radiation, ECA109M cells had a lower proportion of G2＋M cells than ECA109N and ECA109 cells （ P=0. 026） . After 5？Gy radiation, ECA109, ECA109N, and ECA109M cells had similar expression levels of CHK1 and CHK2 proteins （ P= 0. 345 and 0. 451, respectively ） , and ECA109M cells had a lower expression level of CHK2 T68 protein than ECA109 and ECA109N cells （ P=0. 012） . ECA109 cells had a D0 value of 3. 06 Gy and an SF2 value of 0. 91;the D0 values for ECA109N and ECA109M cells were 2. 90 Gy and 1. 88 Gy, respectively, and the SF2 values for them were 0. 89 and 0. 84, respectively （ P=0. 021 and 0. 037, respectively ） . Conclusions RNA interference can reduce the expression levels of MDC1 protein and cell cycle？related proteins, release cell cycle arrest, and enhance radiosensitivity in esophageal carcinoma ECA109 cells.