人LOXL2启动子的鉴定与初步分析

Structural and Functional Analysis of the Human LOXL2 Promoter

LOXL2(lysyl oxidase like 2)是赖氨酰氧化酶(LOX)家族的一个重要成员,不仅可促进细胞外基质中胶原蛋白和弹性蛋白的交联,而且在转录调控、细胞信号转导以及细胞粘附等生物学过程中也有重要作用。多篇研究表明,LOXL2在多种肿瘤中高表达,且与多种肿瘤细胞的增殖迁移等生物学行为密切相关。LOXL2的表达调控机制目前仍不清楚。为了进一步研究LOXL2的转录调控机制,本研究克隆鉴定了LOXL2的启动子。首先通过数据库对LOXL2基因结构及潜在启动子区域进行了分析,进而以人的基因组DNA为模板,通过PCR定向克隆策略,构建了5个长度不同并覆盖LOXL2基因转录起始位点附近约1.7 kb的LOXL2基因启动子荧光素酶报告基因重组体。启动子活性分析结果表明,与对照组相比,5个重组体均具有启动子活性(P<0.05),提示LOXL2基因核心启动子定位于转录起始位点附近约185 bp的区域内。转录因子结合位点分析结果表明,LOXL2基因启动子缺乏典型的TATA盒,但含有GC盒以及Sp1、NFk B等潜在的转录因子结合位点。外源转染Sp1表达质粒能显著增强LOXL2基因启动子的活性(P<0.05),提示Sp1能直接激活LOXL2的转录。

Lysyl oxidase like 2(LOXL2）,a member of the lysyl oxidase(LOX） family,functions similarly to LOX in the extracellular matrix(ECM） by promoting crosslinking of collagen and elastin.LOXL2 is also engaged in transcription regulation, cell signaling transduction and cell adhesion regulation. It has been reported that LOXL2 is highly expressed in several types of tumors and closely related to proliferation and migration of cancer cells. However,the regulatory mechanism of LOXL2 expression remains unknown. To further investigate its regulatory role in transcription, the LOXL2 promoter region has been cloned and identified in the present study. Firstly,the genomic structure and potential promoter region of LOXL2 was analyzed by online genomic databases. Then,by using a PCRbased cloning method,we constructed five different LOXL2 gene promoter luciferase reporter constructs covering 1. 7 kb upstream of the LOXL2 gene transcription initiation site. Luciferase reporter assays demonstrated that all five constructs have promoter activity compared with the control group(P〈0. 05）,and the LOXL2 core promoter is located in a region of 185 bp near the transcription initiation site.Transcriptional factor binding analysis indicated that the LOXL2 promoter lacks the classical TATA box,but contains the classical GC box as well as other putative binding sites for classic transcriptional factors such as Sp1 and NF-κB. Furthermore,ectopic overexpression of Sp1 significantly enhanced the LOXL2 promoter activity(P〈0. 05）,suggesting that Sp1 can directly transactivate the transcription of the LOXL2 gene.