TBX15基因启动子甲基化在肝细胞癌中的表达及其功能研究

目的  探讨TBX15基因在肝细胞癌中的表达及其启动子甲基化对细胞生物学行为的影响。方法 分别采用亚硫酸氢盐测序法（bisulfite sequencing PCR，BSP）和实时荧光定量PCR（qPCR）检测3种肝癌细胞系（HepG2、MHCC97H、SNU449）中TBX15基因启动子甲基化状态和表达水平，再将空白质粒、空载质粒pc3.1和TBX15过表达质粒转染肝癌ＳＮＵ499细胞，通过CCK-8实验和流式细胞术检测各组肝癌细胞的增殖和凋亡情况。 结果 3种肝癌细胞系中，TBX15基因在肝癌 SNU449细胞中的启动子甲基化程度最高，甲基化率为89.5%，而TBX15 mRNA表达水平最低。TBX15过表达质粒组培养48 h后，肝癌SNU449细胞的增殖能力高于空载质粒组，差异有统计学意义（0.549±0.080 vs 0.457±0.506，P=0.015）；TBX15过表达质粒组肝癌SNU449细胞的总凋亡比例高于空白质粒组，差异有统计学意义［（5.12±1.42）% vs （2.16±0.41）%，P=0.014］。 结论 启动子区异常甲基化可能是肝癌细胞TBX15基因失活的主要原因，且与肝癌恶性生物学行为密切相关。TBX15可能是预测肝癌发生发展的指标。

Research of TBX15 promoter methylation on its expression and functional in hepatocellular carcinoma

Objective To investigate the promoter methylation and expression level of TBX15 gene in hepatoma cells，and explore the effect of promoter methylation on its mRNA expression and cell tumor behavior. Methods Bisulfite sequencing PCR （BSP） and quantitative real-time PCR（qPCR） were used to detect the promoter methylation and expression level of TBX15 in three hepatocellular carcinoma cell lines（HepG2，MHCC97H，SNU449）. Then the blank plasmid，empty plasmid pc3.1 and TBX15 overexpression plasmid were transfected into the hepatoma cell SNU449，and the proliferation and apoptosis in three different groups of hepatoma cells were detected by CCK-8 assay and flow cytometry. Results Among the three hepatocellular carcinoma cell lines，the TBX15 gene had the highest promoter methylation rate（89.5%） and the lowest mRNA expression level in SNU449 hepatoma cell. The overexpression plasmid group proliferating for hepatoma cells after culturing 48 h was higher than that of the empty plasmid group. The difference was statistically significant（0.549±0.080 vs 0.457±0.506，P=0.015）. The results of total apoptosis counting showed that the overexpression plasmid group had a higher effect on the apoptosis ability of hepatoma cells than the blank plasmid group. The difference was statistically significant［（5.12±1.42）% vs （2.16±0.41）%，P=0.014］. Conclusions Abnormal methylation in the promoter region may be the main reason for the inactivation of TBX15 gene，and it was closely related to the malignant biological behavior of hepatocellular carcinoma. It was a predictor for the progression of hepatocellular carcinoma.