RNA干扰靶向沉默诱骗受体3基因增加人胰腺癌细胞的放射敏感性

siRNA targeting decoy receptor 3 gene increases the radiosensitivity of human pancreatic cancer cells

目的:探讨RNA干扰沉默诱骗受体3(decoy receptor 3,DcR3)基因对胰腺癌细胞放射敏感性的影响及其相关机制。方法:构建带有DcR3 siRNA序列的稳定表达质粒,通过脂质体转染至胰腺癌AsPC-1细胞株,设对照组、siRNA(-)阴性对照组和DcR3 siRNA组,应用Western blotting检测AsPC-1细胞中DcR3表达的变化,平板克隆形成实验检测转染DcR3 siRNA后AsPC-1细胞放射敏感性的变化,流式细胞术检测细胞凋亡,RT-PCR和Western blotting检测Caspase-8、Caspase-3和PARP-1表达的变化。结果:DcR3 siRNA组细胞中DcR3蛋白表达水平较对照或siRNA(-)组明显降低(均P<0.01);DcR3 siRNA组的克隆形成率明显低于对照或siRNA(-)组,其存活分数(survival fraction,SF)降低、α/β比值升高(均P<0.01);放射后DcR3 siRNA组肿瘤细胞凋亡率明显高于对照或siRNA(-)组(均P<0.01);转染DcR3 siRNA后可以明显上调Caspase-8、Caspase-3的表达和下调PARP-1的表达。结论:RNA干扰沉默DcR3基因通过激活凋亡因子Caspase-8和Caspase-3促进细胞凋亡,从而增加胰腺癌细胞对放射的敏感性。

Objective： To investigate the effect of siRNA targeting decoy receptor 3 gene on the radiosensitivity of pancreatic cancer cells and its related mechanism. Methods： Plasmid stably expressing DcR3 siRNA sequence was constructed and transfected into pancreatic cancer AsPC-1 cell line by liposome; control group, siRNA（-） negative control group and DcR3 siRNA group were set up. Then DcR3 expression in AsPC-1 cells was detected by Western blotting. Effect of DcR3 siRNA transfection on radiation sensitivity of AsPC-1 cells was detected by plate clone formation assay. Cell apoptosis was analyzed by Flow cytometry; the expressions of Caspase-8, Caspase-3 and PARP-1were detected by Western blotting and RT-PCR after transfecting with DcR3 siRNA. Results： The expression of DcR3 protein in DcR3 siRNA group was significantly lower than that in control or siRNA（-） group（P〈0.01）. The colony formation rate of DcR3 siRNA group was significantly lower than that in control group and siRNA（-） group（P〈0.01）, and the survival fraction（SF） value of DcR3 siRNA group was decreased and the ratio of α/β was increased（P〈0.01）. The cell apoptosis rate of DcR3 siRNA group was significantly higher than that of control group or siRNA（-） group（P〈0.01）. DcR3 siRNA could significantly up-regulate the expression of Caspase-8 and Caspase-3 and down-regulate the expression of PARP-1. Conclusion： siRNA silencing DcR3 gene can increase the radiosensitivity of pancreatic cancer cells by activating the apoptosis factors Caspase-8 and Caspase-3, promoting cell apoptosis.