Promoter Hypermethylation-mediated Inactivation of LRRC4 in Gliomas

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.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.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.Gliomas are the most common malignant tumors in the adult central nervous system and account for 50 to 60% of primary brain tumors. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, and thus usually result in a poor prognosis [1]. Although multiple genetic alterations are involved in the development and progression of malignant gliomas [2,3], epigenetic silencing of wild-type tumor suppressor genes via aberrant promo