The Mechanism Of USF1 Down-regulating FHL2 Gene Expression In In Colorectal Cancer

BackgroundFHL2(4-1/2 LIM protein 2),also known as DRAL(down-regulated in rhabdomyosarcoma LIM protein),was initially cloned by its abundant expression in the human heart.It is the second member of a small family of proteins with 4-1/2 LIM domains.The acronym LIM is derived from the names of 3 transcription factors, Lin-11,Isl-1,and Mec-3,in which such a domain was first identified.FHL2 expression is abundant in the heart,but FHL2 is also observed in several other organs,such as brain,liver,lung,cortex et al.FHL2 is not expressed in spleen, thymus,blood leukocytes,skin,skeletal muscle or the stromal smooth muscle cells of the prostate.It exerts different functions in different types of cells.We first showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.We firstly showed that FHL2 expression is elevated in gastrointestinal cancer cell lines and cancer tissues.The human FHL2 gene maps to 2q12-q14 and consists of seven exons,of which the first three are non-coding.Computer analysis of the promoter reveals a plethora of putative transcription factor binding sites p53,Nkx2.5,SRF,MEF-2,E-box et al. Although the actual contribution of particular transcription factors has not been scrutinized,some evidence exists that p53,Nkx2.5,SRF,MEF-2 may be involved in the transcription of the FHL2 gene.Yet there is no evidence that E-box regulates the FHL2 gene expression.We speculate that USF1 bind to E-box motif of FHL2 gene promoter by computer analysis of the promoter. USF is a group of basic helix-loop-helix(bHLH) transcription factors encoded by two distinct genes whose products correspond to 43 kDa USF1 and 44 kDa USF2. USF in the expression of several tissue-specific or developmentally regulated genes, including human growth 2,hormone,mouse metallothionein I,rat gamma,fibrinogen and Xenopus TFâ…¢A Cox-2,Osteopontin,IL-10,PAI-1,BRCA2,surfactant protein A,calcyclin,MHC I gene,OPN,BMP-4,TGF-β₂,CyclinB1,TGF-βⅡreceptor et al.Objective1.To investigate the role of USF 1 in human colorectal cancer.2.To indentify the role of USF1 in the regulation of FHL2 gene Transcription.Methods1.RT-PCR and immunoblotting methods were performed to detect USF 1 expression in two gastric cancer and six colorectal cancer cell lines.2.USF1 expression was assessed by Immunohistochemistry in human colorectal cancer tissues.3.USF1 binding to the FHL2 promoter were indentified by EMSA and ChIP assay in the SW480 human colorectal cancer cell line in vivo and in vitro.4.The activity of FHL2 gene promoter and the mutant FHL2 gene promoter were investigated by dual luciferase reporter assay.5.Genetic manipulation was performed to up-regulate of knock down expression of USF1,and followed by RT-PCR,western blot and dual luciferase repoter assay to research the effect of USF1 genes on FHL2.Result1.All of the gastric cancer cells and six colon cancer cells expressed differernt level of USF1.2.The role of USF1 in human colorectal cancer progression was assessed by immunohistochemistry.USF1 immunoactivity was lower in normal colorectal mucosa.USF1 expression increased in adenoma with atypical hyperplasia.An even stronger expression of USF1 was found in all of colorectal cancer samples.In colorectal cancer on different Dukes'stage,USF1 expression was no difference.These data demonstrated that upregulation of USF1 protein occurs in most colorectal cancer tissues,implying that USF1 may play a role in colorectal cancer progression.3.EMSA showed that USF1 recognized the E-box motif located within -817/-812 bp of the FHL2 promoter.ChIP revealed in vivo USF1 binded to the FHL2 promoter in the SW480 human colorectal cancer cell line.4.The E-Box mutation increased the FHL2 promoter activity.5.Overexpression of USF1 down-regulated the expression of FHL2 and the activity of FHL2 gene promoter.Transfection of USF1-siRNA enhanced the expressions of FHL2 and the activity of FHL2 gene promoter.Conclusion1.USF1 expression had no correlaion with colorectal cancer progression.2.USF1 protein interacts with FHL2 promoter in the SW480 human colorectal cancer.3.USF1 and FHL2 expressions had negative correlation with colon cancer cells.4.USF1 mediated the down-regulation of FHL2 transcription.