人肝细胞癌差异表达基因TCTP的克隆和分析

Cloning and analysis for translationally controlled tumour protein (TCTP) gene differentially expressed in human hepatocellular carcinoma

目的 筛选和鉴定在人肝细胞癌中差异表达的基因。方法 采用抑制性消减杂交技术建立人肝细胞癌cDNA消减文库,通过DNA测序分析、Northern印迹、cDNA末端快速扩增和RT-PCR等方法对其中一个克隆基因加以分析。结果 从所建cDNA消减文库中分离到一个插入子长度为479bp的克隆,该片段含有3’端Poly(A)结构,Northern杂交证明其表达水平在癌组织和肝癌细胞株均显著升高,同时经5’末端快速扩增获得一个长度为865bp的全长cDNA序列,具有一个编码172个氨基酸的完整开放阅读框,与GenBank数据库作同源性分析显示其为已报道的TCTP基因。RT-PCR分析表明TCTP基因在癌组织样本中的扩增水平高于癌旁非癌组织样本。结论 TCTP基因的差异表达可能在肝癌发生机制中扮演重要角色。

Objective To screen and characterize a differentially expressed gene in human hepatocellular carcinoma (HCC) .Methods Suppression subtractive hybridization was used to construct a subtracted cDNA library of HCC, then the analysis of a cloned gene from the library was performed by means of DNA sequencing analysis, Northern blot, rapid amplification of cDNA end (RACE) and RT-PCR.Results It has been found that a clone with an insert of 479bp cDNA fragment containing poly (A) in 3' end was isolated from the subtracted library of HCC. Northern hybridization identified that the expression level of this target cDNA was increased significantly in HCC tissue and SMMC-7721 hepatoma cells. In the meantime, the full-length cDNA was explored by RACE of 5' end, and a sequence of 865bp was obtained, which has an entire open reading frame encoding 172 amino acids. By homology analysis compared with GenBank database, this gene was shown as a reported translationally controlled tumour protein (TCTP) . RT-PCR analysis of TCTP displayed that the amplification of HCC specimens was higher than that of adjacent non-HCC specimens. Conclusion Differential expression of TCTP might play an important role in hepatocarcinogenesis.