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目的采用RNA干扰技术沉默过氧化物酶Ⅰ(peroxiredoxinⅠ,PrxⅠ)基因,观察乳腺癌MCF-7细胞对X线敏感性变化,探讨其作用机制。方法将PrxⅠshRNA真核表达载体pGPU6-PrxⅠ和阴性对照质粒pGPU6-HK转染入乳腺癌MCF-7细胞中,经G418稳定筛选后RT-PCR和Western blot检测PrxⅠ表达变化。6 MV X线照射6 Gy后48 h,流式细胞术检测细胞的活性氧水平、细胞周期和细胞凋亡;MTT法测定细胞增殖能力;克隆形成实验观察克隆形成率,计算Do、N、Dq、SF2及放射增敏比(sensitive enhancement ratio,SER)等放射生物学参数,Western blot法检测Rad51及γ-H2AX蛋白变化。结果获得2个稳定转染细胞株:pGPU6-HK和pGPU6-PrxⅠ。RT-PCR和Western bolt检测显示pGPU6-PrxⅠ组中PrxⅠmRNA和蛋白表达均明显抑制。6 Gy的X线照射48 h后,pGPU6-PrxⅠ及联合照射(ionizing radiation,IR)组细胞中的活性氧水平、G1期细胞和细胞凋亡明显增加,而细胞增殖能力和S期细胞明显降低,Western blot分析表明Rad51蛋白表达降低,而γ-H2AX蛋白表达升高(P<0.05),以pGPU6-PrxⅠ+IR组变化最显著,其Rad51蛋白表达下降了79.3%,而γ-H2AX蛋白表达升高了3.7倍。pGPU6-PrxⅠ组细胞的Do、N、Dq和SF2值分别为1.354、3.106、1.547和0.504,均低于pG-PU6-HK组,SF2时的SER为1.353。结论下调PrxⅠ基因表达可提高乳腺癌MCF-7细胞的放射敏感性,其作用机制与细胞内活性氧清除能力减弱、DNA双链断裂增加及DNA损伤后修复能力降低有关。PrxⅠ可能是1个理想的肿瘤放射增敏分子靶点。
OBJECTIVE: To silence peroxiredoxin Ⅰ (PrxⅠ) gene by RNA interference and observe the changes of sensitivity to breast cancer MCF-7 cells and to explore its mechanism. Methods The Prx I shRNA eukaryotic expression vector pGPU6-PrxⅠ and the negative control plasmid pGPU6-HK were transfected into breast cancer MCF-7 cells. The expression of Prx Ⅰ was detected by RT-PCR and Western blot. Cell viability, cell cycle and apoptosis were measured by flow cytometry (FCM) after 6 Gy irradiation for 6 Gy. Cell proliferation was measured by MTT assay. Clonal formation rate was determined by clonogenic assay. Calculations of Do, N, Dq , SF2 and sensitive enhancement ratio (SER), and Western blot was used to detect the changes of Rad51 and γ-H2AX protein. Results Two stable transfected cell lines were obtained: pGPU6-HK and pGPU6-PrxⅠ. The results of RT-PCR and Western blot showed that the expression of PrxⅠmRNA and protein in pGPU6-PrxⅠgroup were significantly inhibited. After 48 h of X-ray irradiation at 6 Gy, the level of reactive oxygen species (ROS) in cells of pGPU6-PrxⅠand ionizing radiation (IR) increased significantly in G1 phase and markedly decreased in cell proliferation and S phase , Western blot analysis showed that the expression of Rad51 protein was decreased and the expression of γ-H2AX protein was increased (P <0.05), the expression of Rad51 protein was decreased by 79.3% in pGPU6-PrxⅠ + IR group, while γ-H2AX protein expression Increased by 3.7 times. The values of Do, N, Dq and SF2 in pGPU6-PrxⅠgroup were 1.354, 3.106, 1.547 and 0.504, respectively, lower than those in pG-PU6-HK group. Conclusion The down-regulation of PrxⅠ gene expression can enhance the radiosensitivity of breast cancer MCF-7 cells. The mechanism is related to the decrease of intracellular reactive oxygen species scavenging ability, DNA double-strand breaks and the decrease of repair ability after DNA damage. Prx I may be an ideal tumor radiosensitization molecular target.