title: Promoter hypermethylation-mediated inactivation of LRRC4 in gliomas.
abstract: BACKGROUND
  Leucine-rich repeat C4 protein (LRRC4) is a new member of the leucine-rich repeat (LRR) superfamily. It is not only a brain-specific gene but also a novel candidate for tumor suppression. LRRC4 inactivation is commonly found in glioma cell lines and primary glioma biopsies. However, little is known about the mechanism controlling LRRC4 expression. In a previous study, we did not find any genetic alteration in LRRC4 in primary glioma, which led us to explore an alternative mechanism underlying this phenomenon.
  
  
  METHODS
  In the present paper, we cloned the LRRC4 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the LRRC4 promoter region in glioma cell lines and primary gliomas was examined by methylation-specific PCR and bisulfite DNA sequencing. In order to demonstrate a functional association between LRRC4 promoter methylation and its gene inactivation, we performed DNA demethylation analysis with two human glioma cell lines using methylation-specific PCR and RT-PCR.
  
  
  RESULTS
  The sequence spanning positions -835 to -293 relative to the translation start site was identified as the LRRC4 promoter; this sequence is a TATA- and CAAT- less, high GC content region. It was found that LRRC4 promoter activity is strongly suppressed after treatment with SssI methylase in vitro. Furthermore, LRRC4 promoter methylation was observed by methylation-specific PCR in two glioma cell lines and all 30 primary glioma specimens, but not in normal brain tissue. Bisulfite DNA sequencing showed that most of the CpG sites were located around the LRRC4 promoter methylated in glioma cells and tissues, but not in normal brain tissue. In addition, the methylase inhibitor 5-Aza-2'-deoxycytidine could induce LRRC4 mRNA expression and LRRC4 promoter partial demethylation in SF126 and SF767 glioma cells.
  
  
  CONCLUSIONS
  Methylation-mediated inactivation of LRRC4 is a frequent and glioma-specific event, and it may be a potential biomarker for diagnosis or prognosis, or serve as a therapeutic target.
authors.affiliation: Cancer Research Institute, Central South University, Changsha 410078, Hunan, PR China. zhangzp74@126.com
authors.forename: Zuping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Minghua
authors.initials: M
authors.lastname: Wu
authors.forename: Bo
authors.initials: B
authors.lastname: Xiang
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.forename: Pan
authors.initials: P
authors.lastname: Chen
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Li
authors.forename: Shourong
authors.initials: S
authors.lastname: Shen
authors.forename: Guiyuan
authors.initials: G
authors.lastname: Li
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018899:CpG Islands; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D005260:Female; D005910:Glioma / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C503331:LRRC4 protein, human; D009419:Nerve Tissue Proteins; D004247:DNA