反义锁核酸封闭c-myc第二外显子翻译起始区对肝癌细胞凋亡的影响

Effect of antisense locked nucleic acid blocking translation initiation region of c-myc exon 2 on apoptosis of hepatocellular carcinoma cells

目的:探讨针对c-myc第二外显子翻译起始区的反义锁核酸对肝癌细胞活力和凋亡的影响。方法:设计合成能特异性封闭c-myc基因第二外显子翻译起始区的反义寡核苷酸、硫代寡核苷酸和锁核酸,分别以阳离子脂质体介导转染HepG2细胞,采用RT-PCR检测细胞内c-Myc的mRNA表达变化;Western blot检测细胞内cMyc的蛋白表达变化;流式细胞技术检测细胞凋亡情况;MTT法检测反义锁核酸的细胞毒性作用。结果:转染第5天后,反义锁核酸组c-Myc的mRNA相对表达量为0.335±0.016,明显低于对照组(P<0.05);c-Myc蛋白相对表达量为0.448±0.037,也明显低于对照组(P<0.01);凋亡细胞比例为32%±6%,显著高于对照组(P<0.05)。结论:针对c-myc第二外显子翻译起始区的反义锁核酸能有效促进肝癌细胞的凋亡。

AIM： To observe the effect of antisense locked nucleic acid（anti-LNA） blocking the translation initiation region of c-myc exon 2 on the viability and apoptosis of hepatocellular carcinoma cells. METHODS： The antiLNA that was complementary to the translation initiation region of c-myc exon 2 was designed,synthesized,and introduced into the HepG2 cells by cationic liposome-mediated transfection. The mRNA and protein levels of c-Myc in the cells were determined by RT-PCR and Western blot. The change of cell apoptosis was analyzed by flow cytometry,and the toxicity of anti-LNA to the cells was detected by MTT assay. RESULTS： Five days after transfection,the mRNA level of c-Myc in anti-LNA group was 0. 335 ± 0. 016,and the protein level was 0. 448 ± 0. 037,significantly lower than those in control group（ both P 0. 05）. The ratio of apoptotic cells in anti-LNA group was 32% ± 6%,which was higher than that in control group（ P 0. 05）. CONCLUSION： Antisense locked nucleic acid targeting at the translation initiation region of cmyc exon 2 shows strong inhibitory effects on the apoptosis of hepatocellular carcinoma cells.