人肝细胞癌相关基因LAPTM4β启动子结构及SP1结合活性分析

Structural and SP1-binding Analysis of Promoter of LAPTM4β, A Novel Gene Associated with Hepatocellular Carcinoma

研究在溶酶体相关4次跨膜蛋白(lysosomeassociatedproteintransmembrane4beta,LAPTM4β)基因启动子区起重要调控作用的转录因子,为进一步揭示LAPTM4β在肝癌高表达的机制提供理论基础.采用报告基因重组体转染肝癌相关细胞系HepG2细胞和荧光素酶活性测定的方法,确定LAPTM4β的核心启动子区,然后应用凝胶阻滞电泳的方法验证在LAPTM4β核心启动子区起调控作用的转录因子及其特异性.LAPTM4β基因6种不同长度的启动子报告基因重组体,转染肝癌细胞系HepG2.荧光素酶活性比较发现,位于转录起始点上游558bp的一段DNA(PF3)的启动活性最强,表明在这段序列中存在着重要的转录因子结合位点.通过检索Object-orientedTranscriptionFactorsDatabase数据库发现,此段启动子序列中含有多种潜在的转录因子结合位点,其中包括1个SP1结合位点.应用凝胶阻滞电泳(electrophoreticmobilityshiftassay)的方法,证明人肝细胞癌细胞系HepG2及SMMC7721细胞核提取物均能与包含SP1结合位点的寡核苷酸探针结合,并且这种结合可以被相应50倍或者100倍非标记探针竞争抑制.以上结果表明,位于转录起始点上游558bp的一段DNA(PF3)是LAPTM4β基因的核心启动子;而核心启动子序列中的转录因子SP1结合位点具有与肝癌相关细胞系的细胞核蛋白结合的活性.

To investigate the important transcription factors that function in the lysosome-associated protein transmembrane 4 beta （LAPTM4β） promoter and make a basic research for the upregulation of LAPTM4β in hepatocellular carcinoma, hepatocellular carcinomar derived cells HepG2 cells were transfeeted with promoter plasmids of LAPTM4β and luciferase reporter assays were applied to identify the core promoter of LAPTM4β; electrophoretic mobility shift assay （EMSA） was applied to identify the important transcription factor that regulate the LAPTM4β promoter. HepG2 cells were transfected with six different lengths of promoter plasmids of LAPTM4β. Luciferase reporter assays showed that the fragment（PF3）558 bp upstream of the origin site of transcription had the highest promotive activity. As indicated by the Object-oriented Transcription Factors Database, there were multiple putative transcription factor binding sites in this region （PF3）. Among of these transcription binding sites, there was a SP1 binding site. As indicated by EMSA, the oligonucleotides that contain the SP1 binding sites can bind to the nuclear extract of HepG2 and SMMC7721 cells. The binding could be competed by the 50 or 100 molar excess unlabeled oligonucleotides competitor. It was concluded that the fragrnent（PF3）558 bp upstream of the origin site of transcription was the core promoter of LAPTM4β. The oligonucleotides that contain the SP1 binding sites in the core promoter can bind to the nuclear extracts of hepatocellular related cells.