ULF基因沉默对依托泊苷引起的非小细胞肺癌H1299细胞凋亡的影响  

Effect of ULF gene silence on etoposide induced H1299 cell apoptosis

作　　者：刘伟[1] 徐鑫[2] 曹晓慈[3] 袁菲[4] 方会英 

机构地区：[1]河北中医学院护理学院,河北石家庄050000 [2]河北省人民医院急诊科,河北石家庄050000 [3]河北省人民医院肿瘤二科,河北石家庄050000 [4]山东枣庄职业学院口腔系,山东枣庄277000

出　　处：《中国生化药物杂志》2014年第4期59-62,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的探讨RNA干扰靶向抑制ULF的表达对化疗药物依托泊苷(etoposide)引起的非小细胞肺癌H1299细胞凋亡的影响。方法设计3条针对ULF基因的小干扰RNA(small interfering RNA,siRNA)片段及1条阴性对照siRNA,分别在慢病毒载体介导下感染H1299细胞。用real-time PCR及Western印迹法筛选出1条抑制效率最高的ULF序列,对有效干扰组和对照干扰组细胞进行etoposide处理,干扰对照细胞或沉默ULF的H1299细胞,用c-PARP做标记物检测H1299细胞的凋亡程度,MTT法检测H1299细胞增殖情况,流式细胞仪检测细胞周期分布。结果经感染ULF-shRNA-1序列的H1299细胞中ULFmRNA及蛋白表达降低最为明显,抑制率达80%,选择该条siRNA作为有效干扰组进行后续实验。Western blot结果显示etoposide处理后,有效干扰组H1299细胞凋亡水平显著增加,细胞增殖抑制率显著高于对照干扰组,并与etoposide剂量呈正相关,2组比较差异均有统计学意义(P<0.05)。流武结果显示有效干扰组G0/G1期细胞较对照组增加,S期细胞减少,差异有统计学意义(P<0.05)。结论 etoposide处理后通过干扰ULF蛋白表达可诱导H1299细胞的凋亡,抑制细胞增殖,使细胞出现S期阻滞,推测ULF基因可能成为癌症基因治疗的一个新靶点。Objective To explore the effects of knocking down ULF gene on the apoptosis of non-small-cell carcinoma H1299 cell after treatment with etoposide.Methods Three ULF small interfering RNA(siRNA)sequences and one negative control siRNA sequence were designed and synthesized, and then individually transfected into H1299 cell via lentivirus.The interference efficiency of ULF-siRNA were screened by real-time PCR and Western blotting.Then the most target siRNA was used for apoptosis assay after treatment with etoposide,MTT assay for H1299 cell proliferation,flow cytometry for cell cycle distribution. Results The expression of ULF gene and its protein ULF were down-regulated in H1299 cell when transfected with ULF-siRNA,and ULF-siRNA-1 was the most effective one,which had the highest inhibition rate(80%)of ULF expression.Compared with negative control group,ULF-siRNA group showed an obvious apoptosis after treatment with etoposide,and the inhibition rate of was higher than control group,which was positively correlated with etoposide dose,the difference was statistically significant(P<0.05 ).Flow cytometry showed that compared with the control group,G0/G1 cell cycle in ULF-siRNA group was increased,and S phase cells was decreased,the differences were all statistically significant(P<0.05).Conclusion Down-regulation ULF protein expression through treatment with etoposide can induce apoptosis of non-small-cell carcinoma H1299 cells,and inhibit cell proliferation,which lead to cell cycle arrest.ULF gene may become the new target of gene therapy for cancer.

关 键 词：ULF H1299细胞 肺癌 依托泊苷 

分 类 号：R318.04[医药卫生—生物医学工程]