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目的:探讨印迹基因父系表达基因10(paternally expressed gene10,PEG10)在肝癌组织中高表达的分子机制,及其对肝癌细胞生长的影响。方法:采用半定量RT-PCR法检测30例肝癌患者术后肝癌和癌旁组织中以及15株肝癌细胞株中PEG10基因的表达水平;甲基化特异PCR(methylation specific PCR,MSP)技术检测17例PEG10基因高表达的肝癌和癌旁组织中PEG10基因启动子区域CpG岛甲基化状态的差异,及其与PEG10基因表达水平的关系。采用小干扰RNA(small interfering RNA,siRNA)技术沉默肝癌QGY-7703细胞中PEG10基因的表达,并观察对细胞克隆形成能力的影响。结果:与癌旁组织相比,PEG10基因在67%(20/30)的肝癌组织中表达水平明显提高,在15株肝癌细胞株中均有表达。PEG10基因CpG岛2在29.4%(5/17)肝癌组织中显示有肝癌特异性低甲基化,同时采用亚硫酸氢盐测序验证了PEG10基因CpG岛2在肝癌病例中总体低甲基化的状态。与对照组相比,短发夹状RNA(short hairpin RNA,shRNA)干扰PEG10表达后,肝癌细胞株QGY-7703的克隆数目明显减少(P<0.05)。结论:印迹相关基因PEG10在肝癌中高表达,可能与其启动子区域CpG岛2的低甲基化密切相关,沉默PEG10表达可以抑制肝癌细胞的生长,提示PEG10基因可能是一个新的肝癌治疗的药物靶点。
OBJECTIVE: To investigate the molecular mechanism of overexpression of paternally expressed gene 10 (10%) in hepatocellular carcinoma (HCC) and its effect on the growth of hepatoma cells. Methods: The expression of PEG10 gene was detected by semi-quantitative RT-PCR in 30 cases of hepatocellular carcinoma (HCC) and adjacent non-cancerous tissues as well as 15 hepatocellular carcinoma cell lines. Methylation-specific PCR (MSP) The differences of methylation status of CpG island in the promoter region of PEG10 gene in hepatocellular carcinoma and adjacent non-cancerous tissues with high expression of PEG10 gene and its relationship with the expression of PEG10 gene. Small interfering RNA (siRNA) was used to silence the expression of PEG10 gene in QGY-7703 hepatocellular carcinoma cell line and the effect on cell clonality was observed. Results: Compared with the adjacent tissues, the expression level of PEG10 gene in 67% (20/30) HCC tissues was significantly increased, and it was found in all the 15 HCC cell lines. CpG island 2 of PEG10 gene showed hepatocarcinoma-specific hypomethylation in 29.4% (5/17) of HCC tissues, and bisulfite sequencing verified the overall hypomethylation of CpG island 2 of PEG10 gene in HCC cases status. Compared with the control group, the short hairpin RNA (shRNA) interfered with the expression of PEG10, and the number of cloned QGY-7703 cells was significantly decreased (P <0.05). Conclusion: The high expression of imprinted gene PEG10 in hepatocellular carcinoma may be closely related to the hypomethylation of CpG island 2 in its promoter region. Silencing PEG10 expression can inhibit the growth of hepatoma cells, suggesting that PEG10 gene may be a new drug target for hepatocellular carcinoma point.