LHScDNA的部分克隆及其表达对肝癌细胞行为的影响

Partial cloning of LHS cDNA and effect of its expression on behavior of hepatocellular carcinoma cells

目的 :克隆在肝癌组织中低表达的差异显示片段L2的基因cDNA序列 ,并初步探讨其在肝癌发生、发展过程中的作用。方法 :将L2在GenBank中拼接后 ,分析其序列结构 ,并构建正义真核表达质粒 ,转染BEL 740 2肝癌细胞后 ,对转染细胞增殖、粘附、迁移和侵袭等指标进行测定。结果 :克隆得到一条 10 0 5bp的cDNA ,其 5′端 712bp与人Liprinβ1cDNA 5′端调控序列和外显子 1,2 ,3序列同源 (10 0 % ) ,而 3′端 2 93bp与Liprinβ1基因内含子 4同源 (10 0 % )。其间有一个 5 10bp开放阅读框架 ,起始密码位置与Liprinβ1基因相同 ,推断的蛋白产物除C端 13个氨基酸残基外 ,其余均与Liprinβ1同源。此cDNA暂命名为LHS (Liprinβ1 homologoussequence)。LHScDNA正义转染BEL 740 2肝癌细胞系 ,对于细胞的增殖活性和侵袭能力无明显影响 ,但可降低肝癌细胞在层粘连蛋白上的粘附和迁移能力。结论

Objective: To clone cDNA sequence of differential expressed fragment L2 which preferentially down regulated in hepatocellular carcinoma (HCC), and to preliminarily study its biological function in hepatocarcinogenesis. Methods: Homologous sequences were spliced with L2, and the structure of the spliced gene was analyzed. Sense eukaryotic expression plasmid of the candidate cDNA was constructed, and the proliferation, adhesion, migration and invasion of the transfected BEL 7402 HCC cells were analyzed. Results: A 1 005 bp cDNA was cloned. 5′ end 712 bp sequence of the cDNA was homologous (100%) to 5′ non encoding sequence and exon 1, 2, 3 sequence of Liprin β1 gene, while 3′ end 293 bp sequence of the cDNA was homologous to intron 4 of Liprin β1 gene. The cDNA was temporarily termed LHS (Liprinβ1 homologous sequence). There was an open reading frame of 510 bp in LHS cDNA sequence, and the position of initiation codon was the same as that of Liprinβ1. Except for the last 13 amino acid residues, LHS was homologous to Liprinβ1 at protein level. BEL 7402 cells transfected with sense sequence of LHS cDNA showed lower ability for adhesion and migrating, while no effect on proliferation and invasion was observed. Conclusion: The down regulation of LHS expression may play an important role in HCC metastasis.