肝癌相关基因的分子克隆和可变剪切分析  

Molecular cloning and alternative splicing analysis of hepatoma associated gene HTA

作　　者：刘艳红[1] 赵艳洁[1] 房淑娟[1] 李跃辉[1] 李官成[1] 

机构地区：[1]中南大学肿瘤研究所,卫生部癌变原理重点实验室,教育部癌变与侵袭原理重点实验室,长沙410078

出　　处：《中南大学学报（医学版）》2013年第9期869-875,共7页Journal of Central South University ：Medical Science

基　　金：国家重点基础研究发展计划("973"计划)(2010CB833605);国家自然科学基金(81201903);湖南省自然科学基金(2010SK3132)~~

摘　　要：目的:获取肝癌相关基因(hepatomaassociatedgene,HTA)的mRNA分子全长序列并对其可变剪接进行分析,检测其转录本在各肝癌细胞系中的表达,为进一步研究该基因在肝癌发生、发展中的作用奠定基础。方法:用3'RACE和5'RACE方法扩增HTA基因的全长序列并对其序列信息和可变剪接进行扩增和测序分析,用Northern印迹检测该基因转录本在不同肝癌和正常肝细胞系中的表达。结果:HTA基因全长序列为1414bp,经分析该序列包含3个外显子,2个内含子,其中2号内含子在可变剪接中被作为外显子表达。Northern印迹显示HTA基因1.7 kb转录本和1.4 kb转录本均表达于恶性肝癌细胞系,而不表达于正常细胞系。结论:成功获得了HTA基因的全长序列并对其可变剪接进行了分析,2个大小不同的转录本均在肝癌细胞系中特异性表达,作为一个肝癌相关基因值得进一步深入研究。Objective：To obtain the full length cDNA sequences of hepatoma associated gene HTA, analyze its alternative splicing, detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines, and to establish a base for further study of HTA gene function in hepatocellular carcinoma （HCC） occurence and development. Methods：The full length cDNA of HTA gene was cloned by rapid amplification of cDNA 3＇ ends （3＇-RACE）, rapid ampliifcation of cDNA 5＇ ends （5＇-RACE） and DNA sequencing. The gene structure and alternative splicing were analysed. Northern blot assay was performed to detect the expression pattern of 2 HTA gene transcripts in different hepatic cell lines. Results：The full length of HTA gene was 1414 bp, composed of 3 exons and 2 introns, and the second intron could be retained in mRNA. Northern blot assay showed that 2 transcripts of HTA mRNA（1.4 kb and 1.7 kb） could express in the HCC cell lines HepG2 and QGY-7703, but not in the non-malignant cell line L-02 and HUVEC. The expression level of 1.4 kb transcript was much higher than 1.7 kb one. Conclusion：This study successfully has obtained the full length cDNA of HTA gene, and analysed the gene sequence and alternative splicing, 2 transcripts of HTA mRNA specifically expressed in HCC cell lines. As a hepatoma associated gene, HTA deserves further investigation.

关 键 词：肝癌相关基因 全长克隆 可变剪接 

分 类 号：R735.7[医药卫生—肿瘤]