Promoter Hypermethylation-mediated Inactivation of LRRC4 in Gliomas
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The Third Affiliated Hospital of Xiang Ya School of Medicine, Central South University
,
Changsha 410078, Hunan
,
PR China
1
Department of Parasitology, Central South University
,
Changsha 410078, Hunan
,
PR China
2
Cancer Research Institute, Central South University
,
Changsha 410078, Hunan
,
PR China
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 methylationspecific 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. Conclusion: 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.
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Background
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 promoter
hypermethylation has also been shown to occur [4-6]. Aberrant
promoter methylation of CpG island-associated genes is a
common epigenetic alteration associated with the
inactivation of tumor suppressor and other genes in human
cancers [7-9]. Unmethylated in normal tissues, promoters
of these genes can become methylated de novo in cancer
cells. This change is accompanied by alterations in histone
modification and chromatin conformation, rendering the
promoter transcriptionally inert [10].
Such epigenetic mechanisms have been implicated in the
inactivation of several key regulators of the cell cycle (RB,
p16INK4A, p73), DNA repair (O6MGMT), apoptosis (DAP
kinase), angiogenesis (THBS1), and invasion (TIMP3) in
glioma [11]. Recently, novel hypermethylated genes in
glioma have been identified using a candidate gene
approach or by a genome-wide screening method. The
former revealed that genes such as EMP3, TMS1/ASC,
SLC5A8, hMLH and PTEN are frequently targeted for DNA
methylation-mediated silencing in glioma [4,5,12,13]. A
genome-wide screen using a combined approach of
pharmacologic inhibition of epigenetic modifications and
gene expression microarrays also revealed that several
novel genes are subject to aberrant hypermethylation in
glioma [14,15]. Thus, aberrant methylation events have
become critical to our understanding of the initiation and
progression of human brain malignancies and may serve
as a biomarker for diagnosis, prognosis and susceptibility
to treatment.
Leucine-rich repeat C4 protein (LRRC4) is a new member
of the leucine-rich repeat (LRR) superfamily located at
7q31-32 [16]. It was found to be predominantly expressed
in normal brain tissue and involved in early nervous
system development and differentiation [17], but the
expression of LRRC4 was absent in several malignant glioma cell
lines [18]. Similarly, it was absent or significantly
downregulated in 87.5% of primary glioma biopsies [16]. More
importantly, LRRC4 had the potential to suppress
tumorigenesis of U251 malignant glioma cells in vivo and cell
proliferation in vitro [19]. Recent studies show that
LRRC4 (...truncated)