半乳糖配体介导C-myc反义锁核酸对肝癌细胞增殖和凋亡的影响

Effects of C-myc locked nucleic acid mediated by galactose ligand on proliferation and apoptosis of hepatoma carcinoma cell

目的探讨针对增殖相关基因(C-myc)第二外显子翻译起始区的反义锁核酸对肝癌细胞增殖和凋亡的影响。方法设计合成能特异性封闭C-myc基因mRNA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,分别以阳离子半乳糖配体介导转染Hep G2细胞,采用RT-PCR法检测细胞内C-myc的mRNA表达变化;Western blot法检测细胞内C-myc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 d后,反义锁核酸组C-myc mRNA相对表达量为(0.335±0.016),明显低于对照组(1.014±0.022),差异有统计学意义(P=0.015);C-myc蛋白相对表达量为(0.448±0.037),也明显低于对照组(1.00±0.00),差异有统计学意义(P=0.008);细胞凋亡比例为(32±6)%,显著高于对照组(0),差异有统计学意义(P=0.032)。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抑制肝癌细胞增殖和促进细胞凋亡。

Objective To observe effect of proliferation-associated gene Myc locked nucleic acid( LNA) on the second exon of translation initiation region on proliferation and apoptosis of hepatoma carcinoma cell. Methods Antisense oligonucleotides,phosphorothioate oligonucleotides and nucleic acids on the second exon of translation initiation region which could specifically block C-myc mRNA were designed and synthesized. And transfection Hep G2 cells were mediated by cationic galactose ligand,mRNA expression in C-myc was detected by RT-PCR,expression of C-myc protein in cells was detected by Western blot method,cell apoptosis was detected by flow cytometry,and cytotoxicity of locked nucleic acid was detected by MTT,respectively. Results 5 days after transfection,relative expression of C-myc mRNA levels in LNA group was( 0.335±0.016),significantly lower than that( 1.014±0.022) of control group,difference was statistically significant( P = 0. 015).Relative expression quantity of C-myc protein of the LNA group was( 0.448±0.037),also significantly lower than that( 1.00±0.00) of the control group,difference was statistically significant( P = 0.008). The ratio of cell apoptosis in the LNA group was( 32±6) %,statistically higher than that( 0) of the control group,difference was statistically significant( P = 0.032).Conclusion Antisense locked nucleic acid that targeting at the translation initiation region of C-myc exon 2 shows strong inhibitory effects on the proliferation and apoptosis of hepatocellular carcinoma cells.