人CLDN10a启动子的克隆及活性分析

Cloning and Activity Analysis of Human CLDN10a Promoter

CLDN基因家族编码构成细胞间紧密连接的主要跨膜蛋白Claudins,在维持细胞极性、信号转导和恶性肿瘤复发转移中发挥重要作用。CLDN10基因属于CLDN家族,有包括CLDN10a在内的多个转录本。本研究利用多种生物信息学方法分析CLDN10a的5′侧翼序列的特征和转录因子结合位点,采用基因合成、PCR扩增和分子克隆构建5′缺失的不同长度的CLDN10a启动子荧光素酶报告基因载体,瞬时转染HEK-293T细胞,通过双荧光素酶报告基因系统检测不同长度片段的启动子活性。生物信息学分析结果显示,CLDN10a的上游转录调控区序列(-2000~+460 bp)含有TATA box、GC box和CAAT box,存在1个CpG岛,有4个启动子位置,有1个RNA聚合酶Ⅱ核心启动子;MatInspector和Jaspar分析发现,有75种可能的转录因子结合位点(评分≥0.99)。PCR和测序结果显示,成功构建了6个不同长度的CLDN10a启动子的双荧光素酶报告基因质粒。荧光素酶活性检测表明,-1890~-1100 bp、-1000~-890 bp、-890~-150 bp和-150~+460 bp是人CLDN10a启动子的主要活性区域,-150~+460 bp包含CLDN10a的核心启动子区域。本实验为进一步研究CLDN10基因的表达及调控机制提供了依据。

The CLDN gene family encodes Claudins,the main transmembrane protein that constitutes tight junctions between cells and plays an important role in maintaining cell polarity,signal transduction,and recurrence and metastasis of malignant tumors.CLDN10 gene belongs to the CLDN family and has multiple transcripts including CLDN10 a.In this study,a variety of bioinformatics methods were used to analyze the characteristics of 5′flanking sequence of CLDN10 a and transcription factor binding sites.Gene synthesis,PCR amplification and molecular cloning were used to construct CLDN10 a promoter luciferase reports of different lengths with 5′deletions.The gene vector was transiently transfected into HEK-293 T cells,and the promoter activity of different length fragments was detected by the dual luciferase reporter gene system.The results of bioinformatics analysis showed that the upstream transcription regulatory region sequence(-2000~+460 bp)of CLDN10 a contained TATA box,GC box,CAAT box,1 CpG island,4 promoter positions,and 1 RNA polymeraseⅡcore promoter.And 75 transcription factor binding sites were predicted by MatInspector and Jaspar(Score≥0.99).PCR and sequencing results showed that 6 dual luciferase reporter gene plasmids with diffe-rent lengths of CLDN10 a promoter were successfully constructed.The luciferase activity test showed that-1890~-1100 bp,-1000~-890 bp,-890~-150 bp and-150~+460 bp were main active regions of human CLDN10 a promoter,while-150~+460 bp contained the core promoter region of CLDN10 a.This study provides experimental basis for further investigation of CLDN10 gene expression and regulation mechanism.