Identification And Characterization Of The Promoter Of Cancer-related Gene LOXL2

Lysyl oxidase like 2,LOXL2,as a member of the lysyl oxidase(LOX)family,has been shown to function similarly to LOX in the extracellular matrix(ECM)by promoting crosslinking of collagen and elastin.LOXL2 is also engaged to transcription regulation,cell signaling transduction and cell adhesion regulation.It has been reported that LOXL2 is highly expressed in several types of tumors and promotes cell proliferation and migration in various cancer cells.However,the regulatory mechanism of LOXL2 expression remains largely unknown.To further investigate its transcriptional regulatory mechanism,LOXL2 promoter region has been cloned and identified in the present study.1?Gene organization and chromatin state of LOXL2 gene locusAnalysis of the structure and chromatin status of the LOXL2 gene by the UCSC genome database(http://genome.ucsc.edu/)has revealed that the LOXL2 gene is located on chromosome 8p21 and consists of 14 exons and13 introns.An active promoter is contained near the first exon.2?Cloning of LOXL2 gene promoter regionWe then constructed five different LOXL2 gene promoter luciferasereporter constructs covering 1.7 kb upstream of LOXL2 gene transcription initiation site,such as P1773(-1651 / + 122),P1207(-1085 / + 122),P636(-514 / + 122),P401(-279 / + 122),P185(-63 / + 122).3?Identification of the LOXL2 promoter regionSeries luciferase reporter assay in 293 T cells and HCT116 cells demonstrated that all the five constructs showed notable promoter activity,and LOXL2 core promoter was located in a region of 185 bp near the transcription initiation site.Transcriptional factor binding analysis indicated that,LOXL2 promoter lacked classical TATA box,but contained putative binding sites for classic transcriptional factors such as Sp1 and NF-?B.4?Regulation of Sp1 on LOXL2 gene expressionIn 293 T cells and HCT116 cells,transiently co-transformed the Sp1 overexpression plasmid and the LOXL2 gene promoter plasmid,and tested by Dual-Luciferase? Reporter Assay,quantitative RT-PCR,and Western Blot experiments.LOXL2 promoter activity was significantly enhanced by ectopic overexpression of Sp1 as well as its endogenous expression in cells.In contrast,mithramycin A(a selective Sp1 inhibitor)treatment repressed LOXL2 promoter as well as its endogenous transcription.Site directed mutagenesis assay further confirmed that the Sp1 binding sites were essential for proximal prompter activity of LOXL2 gene.Chromatin immunoprecipitation(ChIP)assay revealed that Sp1 bound LOXL2 promoter in vivo.5?Clinical significance of Sp1 and LOXL2 expression in colorectal cancerFinally,to further understand whether LOXL2 and Sp1 are related to clinical significance in colorectal cancer,we examined their expression levels and clinical outcomes.Of note,the expression of Sp1 and LOXL2 are positively correlated,and the higher expression of LOXL2 is associated with poor prognosis in colorectal cancer,strongly suggesting the implication of Sp1-mediated LOXL2 transactivation in the pathogenesis of colorectal cancer.In conclusion,our study for the first time experimentally identified the promoter region of the LOXL2 gene and demonstrated that Sp1 transcription factor can directly regulate the transcriptional activity of LOXL2,which not only lays a solid foundation for further elucidating the mechanism of expression and regulation of LOXL2 gene,but also helps further analyze the biological function of LOXL2 in cell proliferation and carcinogenesis.