反义端粒酶寡核苷酸诱导膀胱癌细胞凋亡及对凋亡基因表达的影响

Antisense oligonucleotides of telomerase in the cells apoptosis and expression of apoptotic gene in bladder cancer

目的 探讨端粒酶反义核酸对膀胱癌细胞凋亡及凋亡基因表达的影响。方法 以端粒酶 RNA模板区为靶点 ,不同剂量的序列为TAGGGTTAGACAA的硫代磷酸修饰的反义寡核苷酸 (PS- ASON)作为实验组 (A、B、C) ,以硫代磷酸修饰 1 3个核苷酸的随机引物作为对照组 (D) ,仅加入培养液作为空白对照组 (E) ,与人膀胱癌细胞株 BIU87细胞共同孵育 ,光镜、电镜观察细胞形态 ,DNA凝胶电泳检测 ,流式细胞仪测定和分析细胞凋亡。流式免疫荧光技术检测 FAS、ICE的蛋白表达。结果 与随机引物及空白对照组比较 ,ASON实验组细胞在第 2、8天及 8d后出现较明显的凋亡峰和 DNA梯状电泳带 ,以及退化、凋亡形态学变化 ,凋亡率明显增高 (P<0 .0 0 1 ) ,并显示剂量的依赖性 (P<0 .0 1 )。 FAS、ICE蛋白表达的阳性率 ,实验组为 (89.42± 7.9) %、(89.86± 1 1 .4) % ,对照组为 (56.75± 6.36) %、(50 .4± 4.91 ) % ,与对照组比较 ,实验组 FAS及 ICE表达的阳性率均明显升高 (P<0 .0 1 )。结论 以端粒酶 RNA模板区为靶点的反义寡核苷酸引起膀胱癌 BIU87细胞退化、凋亡 ,对膀胱癌的治疗有潜在的意义 ;FAS、ICE的蛋白表达上调 ,共同参与了端粒酶 ASON诱导的膀胱癌 BIU87细胞的凋亡。

Objective To investigate the effects of the antisense nucleotides of telomerase on apoptosis and expression of apoptotic gene of the cell BIU87 derived from bladder cancer. Methods The phosphorothioate antisense oligonucleotides (PS-ASON) of different dose with sequence TTAGGGTTAGACAA targeted to telomerase RNA template as A, B, C experiment group and PS-random primer with 13 nucleotides as control group (D) and blank control group (E) were incubated with BIU87 cell line. The apoptotic rate was assayed by flow cytometer and cell apoptosis was detected by DNA ladder electrophoresis. The cell form was observed with light and electron microscope, and the expressions of Fas, IL-1β converting enzyme (ICE) protein in the BIU87 cell line were detected by flow cytoimmunofluorescence technique. Results Compared with the D group and E group, the experiment group presented transparent apoptotic cusp at flow cytometry and revealed ladder electrophsis zone on the 2nd and 8th and after 8 days, occurred degeneration and apoptosis under the microscope. The efficacy depended on the dose of ASON (P< 0.01) and it was of sequence-selective specificity. The positive rate of Fas, ICE protein was (56.75±6.36)%, (50.4±4.91)% in the control group, (89.42±7.9)%, (89.86±11.4)% in the experiment group. The expressions of two proteins in the experiment group increased significantly compared with the control group (P<0.01). Conclusions The ASON targeted to telomerase RNA template could induce cells degeneration and apoptosis, involving upregulation of FAS, ICE protein expression. These might have potential significance in tumor therapy.