下调53BP1基因表达对喉部鳞癌细胞放射增敏作用的研究

Downregulation of 53BP1 by Short Hairpin RNA Enhances Radiosensitivity in Laryngeal Carcinoma Cells

中文关键词:  P53结合蛋白1基因（53BP1） 周期阻滞 放射敏感性 RNA干扰

英文关键词:53BP1 Arrest of cell cycl Radiosensitivity RNA interference

中文摘要:

      目的 利用RNA干扰技术下调喉部鳞癌Hep-2细胞株P53结合蛋白1基因（53BP1）的表达，观察其对肿瘤细胞周期及放疗敏感性的影响。方法 构建53BP1短发夹RNA（shRNA）重组质粒，然后转染Hep-2细胞株，构建稳定低表达53BP1的Hep-2细胞系，通过Western blot检测转染空载质粒的Hep-2细胞（NC）、野生型Hep-2细胞（未转染，Hep-2组）和稳定转染下调53BP1表达的Hep-2细胞组（P6组）细胞53BP1蛋白的表达水平，MTT法检测转染对细胞正常情况下增殖的影响，通过流式细胞术检测经放射线照射后的细胞周期分布，并采用克隆形成实验检测下调53BP1表达的Hep-2细胞系对放射的敏感性变化。结果 成功构建了稳定低表达53BP1的Hep-2细胞株P6，Western blot显示P6组Hep-2相较野生型Hep-2细胞中53BP1蛋白水平下降（P<0.05)。MTT结果示下调53BP1蛋白表达并未影响细胞正常的生长。P6组下调53BP1后，经不同剂量放射后G2/M期周期阻滞较NC组和野生型Hep-2细胞减弱（P<0.05），周期阻滞比例随放射剂量增加而增加。在0、2、6、10 Gy剂量的照射后，P6组放射敏感参数（D0、N、Dq、SF2）均低于野生型Hep-2组和对照组（P<0.05)。结论 RNA干扰可下调53BP1表达的Hep-2细胞，减少G2/M周期阻滞，从而增强对放射的敏感性。

英文摘要:

      Objective To determine the effect of downregulation of 53BP1 expression on cell growth and radiosensitivity in laryngeal carcinoma Hep-2 cells. Methods HEP-2 cell lines were established with a stable knockdown of 53BP1 with short hairpin RNA (shRNA). The level of expressed protein of negative control group（NC），wild type Hep-2 and downregulation of 53BP1 （P6）group was determined by Western blotting. Proliferation on normal conditions was detected by MTT. Radiosensitivity and growth of cells were detected by flow cytometry. Results A stable 53BP1 downregulated cell line P6 was established. Similar cell growth was observed between the 53BP1 downregulated cells and the control cells. The downregulation of 53BP1 reduced the number of clonogenic cells exposed to radiation. Compared with wild type Hep-2 group and NC group,Western blot results showed a decrease in 53BP1 protein level in P6 group (P<0.05)，reducing the ratio of arrest of G2/M with radiation dose increased (P<0.05). MTT results revealed the lower 53BP1 protein level did not affect the cell proliferation. After exposure to 0, 2, 6, 10 Gy ionizing radiation (IR), P6 cells had lower, radiosensitivity parameters (D0, SF2, Dq, N) than controls and wild type Hep-2 (P<0.05). Conclusion RNA interference could effectively down-regulate the expression of 53BP1 and reduce arrest of G2/M phase, thus enhancing the radiosensitivity of Hep-2 cell line.