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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.
Objective: To investigate the relationship between DNA abnormal methylation and hepatocellular carcinoma (HCC) and the role of 5-aza-CR in inhibiting the malignant phenotype and reversing methylation of HCC.Methods: The hepatocarcinoma cell line HuH-7 and nude mice were treated with 5-aza-CR. The morphological changes of the cells were observed by phase-contrast microscopy. The cell growth rate was observed by MTT assay. The cell cycle was detected by flow cytometry The methylation status of 5’CpG island of p16 gene was detected by methylation-specific PCR (MSP), and the expression of p16 mRNA was detected by RT-PCR.Results: The inhibitory effect of 5-aza-CR on the expression of HuH-7 and nude The number of apoptotic cells in HuH-7 cells was 41.1% ± 3.2% in G0 / G1 phase, 39.0% ± 1.4% in S phase and 2.2% ± 0.7% in G2 phase in experimental group Increased by 30.0% ± 4.5%. The cell cycle of G0 / G1 phase in nude mice was prolonged by 27.4% ± 3.1%, S phase was shortened by 25.8% ± 2.1%, G2 phase was shortened by 1.6% ± 1.8% and apoptotic cells increased by 2.9% ± 0.6 %. Only specific PCR bands were amplified from the HuH-7 and nude mice only in the control group. Only the non-methylated primers amplified specific PCR bands in the HuH-7 cells in the experimental group, while the nude mouse cells A The specific PCR bands were amplified by both genotyping and unmethylated primers.The expression of p16 mRNA in tumor cells HuH-7 and xenografts in nude mice was not detected in the control group.Conclusion: 5-aza- CR inhibited the growth of hepatoma cells in vitro and in vivo, blocked the cell cycle G1 / S and promoted the expression of p16 mRNA; 5-aza-CR inhibited the malignant phenotype of hepatocellular carcinoma cells and reversed the methylation status of p16.