5-氮杂胞苷抑制肝癌细胞恶性表型和逆转甲基化状态的作用及机制

Effects of 5-azacytidine on the growth inhibition of human hepatocellular carcinoma cells and reversion of p16 hympermethylation

目的:探讨DNA异常甲基化与肝细胞肝癌间的相关性及5-氮杂胞苷(5-aza-CR)抑制肝癌细胞恶性表型和逆转甲基化状态的作用及其机制.方法:用5-aza-CR处理肝癌细胞株HuH-7和裸鼠移植瘤模型,然后用相差显微镜观察药物处理前后细胞形态变化,MTT法观察细胞生长速度变化,流式细胞仪检测细胞周期、细胞凋亡率,甲基化特异性PCR(MSP)检测p16基因5'CpG岛甲基化状态,RT-PCR法检测p16 mRNA的表达情况.结果:5-aza-CR对肿瘤细胞HuH-7和裸鼠移植瘤细胞均有明显的抑制作用;实验组HuH-7细胞周期G0/G1期延长41.1%±3.2%,S期缩短39.0%±1.4%,G2期缩短2.2%±0.7%,凋亡细胞增加30.0%±4.5%;裸鼠移植瘤细胞周期G0/G1期延长27.4%±3.1%,S期缩短25.8%±2.1%,G2期缩短1.6%±1.8%,凋亡细胞增加2.9%±0.6%;对照组HuH-7与裸鼠细胞仅甲基化引物扩增出特异PCR条带,实验组HuH-7细胞仅非甲基化引物扩增出特异PCR条带,而实验组裸鼠细胞甲基化和非甲基化引物均扩增出特异PCR条带;实验组肿瘤细胞HuH-7和裸鼠移植瘤细胞的p16 mRNA均有表达,而对照组则无表达.结论:5-aza-CR在体外和体内均有抑制肝癌细胞生长、阻滞细胞周期G1/S和促进p16 mRNA表达的作用;5-aza-CR可抑制肝癌细胞恶性表型和逆转p16甲基化状态.

AIM： To investigate correlation between DNA methylation alteration and hepatocellular carcinoma as well as to explore mechanisms of 5-aza-CR inhibiting hepatoma cell lines and reversed methylation. METHODS： Human hepatoma cell lines, HUH-7, and the murine xenograft model were treated with 5-aza-CR. Cell morphology changes were determined under phase contrast microscopy, cell growth speed was measured using MTT assay,cell cycle distribution and apoptosis rate were estimated using flow cytometry, methylation status of p16 was determined using methylation-specific PCR and mRNA expression of p16 was determined using RT-PCR. RESULTS： After treatment with 5-aza-CR, significant inhibiting effects were detected both in hepatoma cell lines HUH-7 and the murine xenograft model cells. In the treatment group, G1 of HUH-7 increased by 41.1% ＋ 3.2%, S and G2 ＋ M decreased by 39.0% ＋ 1.4% and 2.2% ＋ 0.7%, respectively, and apoptosis rate increased by 30.0% ＋ 4.5%. In the murine xenograft model group, G1 increased by 27.4% ＋ 3.1%, S and G2＋ M decreased by 25.8% ＋ 2.1% and 1.6% ＋ 1.8%, respectively, and apoptosis rate increased by 2.9% ＋ 0.6%. Only methylating PCR product appeared before treatment with drugs, Conversely while only demethylating PCR amplification product was detected after drug treatment. For the murine xenograft model group, methylated PCR product was detected in the control group, however, methylated and demethylated PCR amplification products were observed in the experimental group. Both cell and xenografted nude mice presented the expression of p16 mRNA in experimental group. No expression of p16 mRNA was detected in the control group. CONCLUSION： 5-aza-CR inhibits tumor cell growth, decreases cell cycle and increases mRNA expression of p16 in hepatoma cell lines both in vitro and in vivo. 5-aza-CR inhibits the malignant phenotypes of human hepatocellular carcinoma cells and reverses hympermethylation of p16.