人肝癌组织中γ-谷氨酰转移酶异常表达及其基因甲基化程度的研究

Abnormal expression and methylation status of glutamyl transferase (GGT) genes in human hepatoma tissue

目的探讨γ谷氨酰转移酶（ＧＧＴ）基因在人类肝癌发生过程中的表达及改变机制。方法以病理组织学、生物化学和分子生物学方法，将自身配对的肝癌、癌旁组织和远癌组织中总ＲＮＡ纯化，对ＧＧＴ的表达与改变进行了分析；并设计两套引物，以逆转录巢式ＰＣＲ法扩增ＧＧＴ５′非编码区（ＮＣ）基因和限制性内切酶消化分析了不同肝癌组织中ＧＧＴ基因的甲基化程度。结果在病理组织学证实的２４例肝癌、癌旁和远癌组织中：①从肝癌灶至远癌的不同组织中，总ＲＮＡ浓度呈逐步升高趋势，三种不同组织间的差异均有显著性（Ｐ＜０．０５）；②癌组织中总ＧＧＴ比活性（Ｕ／ｇ）较癌周和远癌组织明显升高（Ｐ＜０．０５）；③以自行设计建立的逆转录巢式ＰＣＲ法，成功地扩增ＧＧＴ的５′ＮＣ基因片段，特异性强且区带清晰，ＰＣＲ产物经酶切分析证明，ＧＧＴ基因５′ＮＣＭ３位点（ＣＣＧＧ），其内位Ｃ在癌灶、癌周和远癌组织中的低甲基化频率分别为７０．８３％，５８．３３％和５４．１７％，非常明显地高于同组中已甲基化程度。结论肝癌组织中ＧＧＴ基因甲基化程度较低，且与肝癌ＧＧＴ的异常表达关系密切，对肝细胞癌变可能起促进作用。

Objective To explore the mechanisms of expression and alteration of glutamyl transferase (GGT) genes during the development of human hepatic carcinoma. Methods The total RNA concentration and the expressions and changs of different molecular forms of GGT in the selfcontroled liver cancerous, paracancerous and distal cancerous tissues were investigated by pathologic examination, biochemical and molecular biological assays. Moreover, the part GGT genes of the 5NC region were amplified with two sets of primers by using a nest RTPCR assay, and methylation status of GGT genes were also studied by a restriction endonuclease RTPCR technique. Results Of the 24 tissue specimens confirmed by pathological examination: An increasing tendency of total RNA concentrations was found from cancer to distal cancerous tissues, and there was a marked difference (P<0.05) between those in liver cancers and paracancerous /or distal cancerous tissues; The specific activities (U/g) of total GGT was significantly higher (P<0.05) in hepatoma tissues than in those of paracancerous/or distal cancerous tissues; Analysed by using a restriction endonuclease RTPCR assay, the inner C of M3CCGGsite of GGT 5NC genes in their PCR products showed universal hypomethylation. The frequency of hypomethylated M3 site was 70.83% in hepatomas, 58.33% in paracancerous and 54.17% in distal cancerous tissues and was significantly higher than that of the M3 site methylated cases in the same group. Conclusions The hypomethylation status of GGT genes in hepatomas is strongly correlated with the abnormal expression of hepatoma GGT and hypomethylated GGT gene might promote the liver canceration during hepatocarcinogenesis.