5-杂氮-2′脱氧胞苷对肝癌细胞中TIMP-3基因的作用

摘要

目的：观察多株肝癌细胞中TIMP-3 CpG岛的甲基化状况，观察甲基化药物5-杂氮-2′脱氧胞苷对肝癌细胞株中TIMP-3基因表达及其CpG岛甲基化程度的影响。方法运用MSP定性检测8株肝癌细胞中TIMP-3 CpG岛甲基化状况。使用去甲基化药物5-杂氮-2′脱氧胞苷对甲基化阳性的细胞株进行干预，观察干预组与对照组TIMP-3基因表达差异，并使用焦磷酸测序技术定量检测CpG岛甲基化差异。结果在8株肝癌细胞系中C3A、Hep-3B、HepG23株检测到TIMP-3 CpG岛甲基化阳性。经去甲基化药物干预后，在这3株肝癌细胞中均发现TIMP-3 mRNA的表达较对照组有明显上调（P<0.05）， CpG岛的甲基化率下降（P<0.05）。结论甲基转移酶抑制剂能显著降低肝癌细胞株TIMP-3 CpG岛甲基化程度及上调TIMP-3 mRNA的表达。%Objective The aim of this study is to investigate the CpG island methylation status of TIMP-3 in HCC cell lines, to treate the HCC cells that exhibit positive methylation with DNA methyltransferase in-hibitor (5-Aza-CdR ), and to observe the influence of 5-Aza-CdR on the expression of TIMP-3 mRNA and its CpG island methylation degree. Methods TIMP-3 CpG methylation status of HCC cells were examined using MSP assay. Methylation-positive HCC cells were treated with DNA methyltransferase inhibitor (5-Aza-CdR) (intervention group), and the difference of TIMP-3 mRNA expression was observed between interven-tion group and control group. Pyrosequencing method was applied to quantitatively examine the CpG island methylation percentage. Results The methylation of TIMP-3 CpG islands in C3A, HepG-2, and Hep-3B was positive. The results showed that TIMP-3 mRNA expression was increased (P<0.05) and the methylation level was decreased (P<0.05) in the intervention group compared to control group. Conclusion 5-Aza-CdR could remarkably reduce the level of the CpG island methylation and increase the expression of TIMP-3 mR-NA in HCC cells.