靶向Slug基因诱导PUMA上调增强AsPC-1细胞对γ射线的敏感性研究

Radiosensitivity of AsPC-1 cell to γ-rays enhanced by up-regulation of PUMA induced by targeted Slug gene

目的 探讨siRNA干扰Slug基因表达对胰腺癌AsPC-1细胞γ射线敏感性的影响.方法 以0、10、50、100感染复数（MOI）的rAAV-EGFP-Slug-siRNA（Slug-siRNA）转染AsPC-1细胞72 h,免疫组织化学法和Western blotting检测转染前后细胞内Slug和PUMA表达.采用4 Gy γ射线细胞进行照射,MTT比色法和Hoechst 33342和PI双染法检测对照组、转染组（MOI 50）、照射组、转染（MOI 50）＋照射组细胞存活率和凋亡率,DNA琼脂糖凝胶电泳检测细胞凋亡,Giemsa染色观察细胞形态.结果 细胞受到MOI为10、50、100作用72 h后,Slug蛋白表达相对值分别为0.831±0.14、0.546±0.12和0.178±0.08,明显低于对照组1.17±0.152,差异有统计学意义（F=4.992,P＜0.05）;而细胞受到MOI为10、50、100作用72 h,PUMA蛋白表达相对值分别为0.325±0.07、0.593±0.11和0.978±0.12,明显高于对照组0.143±0.04,差异有统计学意义（F=4.324,P＜0.05）;转染（MOI 50）＋照射组细胞增殖抑制率为（78.76±9.36）%,明显高于转染组和单独射线照射组[（43.68±6.71）%和（19.25±3.72）%],差异有统计学意义（F=5.056,P＜0.05）.另外,转染（MOI 50）＋照射组细胞的凋亡现象较其他组更加明显.结论 siRNA干扰Slug基因表达能够解除Slug对PUMA的抑制,增强胰腺癌细胞对γ射线的敏感性.

Objective To explore the influence of PUMA on radiosensitivity of pancreatic cancer AsPC-1 cells after Slug gene inhibition by transfected short interferencing RNA（siRNA）. Methods The AsPC-1 cells were infected with MOI 10,50,100 for 72 h, respectively. The expression of Slug and PUMA was analyzed by Western blotting and immunohistochemistry methods. The transfected and control cells were exposed to 4 Gy γ-rays. The cells inhibition rate was examined by MTT, Hoechst 33342 and IP double staining. DNA ladder and Giemsa staning was used to observe apoptosis. Results The relative value of Slug expression was 0.831 ±0.14,0. 546 ±0.12 and 0.178 ±0.08 after AsPC-1 was infected with Slug-siRNA （ MOI 10,50,100） for 72 h, significantly lower than that of control group （ F = 4. 992,P ＜ 0.05 ）.The relative value of PUMA was 0. 325 ±0. 07,0. 593 ±0. 11 and 0. 978 ±0. 12, after AsPC-1 was infected with Slug-siRNA （ MOI 10,50,100） for 72 h, significantly higher than that of control group （ F = 4. 324,P ＜ 0. 05 ）. The cell proliferation rate was （ 78.76 ± 9. 36 ） % in transfection combined with radiosensitivity group, significantly higher than that of transfection group [ （ 43.68 ± 6.71 ） % ] and radiosensitivity group alone [（ 19.25 ± 3.72）% ] （F = 5.056, P ＜ 0.05）. The apoptosis of transfection combined with radiosensitivity group was significantly higher than that of others. Conclusions Slug gene targeting siRNA could inhibit the expression of Slug, and consequently increase the activation of PUMA expression, and so enhance the radiosensitivity to γ-rays.