ABBS 2009,41(01): Inactivation of LARS2, located at the commonly deleted region 3p21.3, by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma

No mutations detected in the promoter region
and exon 1 of LARS2

We analyzed mutations in the promoter region
(–164 bp~–18 bp) and exon 1 of LARS2 by PCR-SSCP and subsequent sequencing
analysis in 25 primary NPC tissues and their matched blood samples. All the
tested NPC samples showed the same mobility DNA bands as their matched blood
samples. Further DNA sequencing revealed no mutations available in these two
regions of LARS2 gene (Fig. 2).

Allelic deletion of LARS2 gene in NPC

By using two microsatellite markers, allelic
deletion of LARS2 was examined in 25
primary NPC biopsies and their matched blood samples. The results showed that
HD frequency for RH25266 and SHGC-12886 was 16% (4 of 25) and 12% (3 of 25),
respectively, resulting in a total HD frequency of 28% in NPC, which was
further confirmed by real-time RT-PCR (Fig. 3). These findings demonstrated
that allelic loss may be one of mechanisms involving in the inactivation of LARS

Hypermethylation of
LARS2 gene in NPC

We analyzed the methylation status of 25 CpG
islands in a 227-bp promoter region of LARS

Discussion

Numerous studies have indicated the presence
of TSGs on the short arm of human chromosome 3 involved in the development of
many cancers, e.g., lung cancer, breast cancer, head and neck cancer, ovarian
cancer [19]. LUCA
(also referred to 3pLARS2 gene is located at the 1-Mb
long common eliminated region 1 (CER1) between D3S32 and D3S3582 at 3p21.3 and spans 160-kb consisted of 22
exons encoding a 903-aa protein (Locuslink ID 23395). It is often found to be up-regulated in the brains of patients with
bipolar disorder and schizophrenia and it may represent a novel type 2 diabetes
susceptibility gene [24,25]. However, there is no literature reporting
its role in tumorigenesis. In this study, we detected its expression level,
genetic and epigenetic alterations in NPC tissues. To our knowledge, this is
the first report showing association between down-regulation of an aminoacyl
tRNA synthetase gene and NPC tumorigenesis.

According to the semi-quantitative RT-PCR and
real-time RT-PCR results, aberrant expression (loss plus down-regulation) of LARS2 was detected in 78% (28 of 36) of
NPC tissues, while all the NP tissues expressed stable LARS2 mRNA level, indicating LARS2
might be involved in the pathogenesis of NPC. Meanwhile, we found that LARS2 down-regulation showed a
significant correlation with lymph node metastasis in NPC patients, implying
that the stable expression of LARS2
may prevent lymph node metastasis of tumor cells in NPC patients to a certain
extent.

To assess the possible molecular mechanisms
causing LARS2 inactivation in NPC
tissues, we detected the genetic (mainly gene mutation, allelic loss) and
epigenetic (mainly promoter methylation) alterations of LARS2 gene in NPC tissues.

We screened the promoter region and exon 1 of
LARS2 gene for mutations by SSCP and
sequencing analysis in 25 NPC tissues. However, no mutations were found in the
two regions in all the tested NPC samples, demonstrating that gene mutation
might not be responsible for LARS2
silencing in NPC. Meanwhile, we analyzed the allelic loss status of LARSRecently, it is recognized that
transcriptional silencing by hypermethylation of CpG islands in promoter region
has become a very common mechanism for the inactivation of TSGs [30]. Previous studies
have demonstrated that the CpG islands in the promoter regions of some TSGs
(e.g. RASSFFurther parallel analysis demonstrated that
hypermethylation of LARS2 promoter
alone could contribute to LARS2
downregulation in NPC (for example, in samples 7, 13, 20, 23) or eliminate LARS2 expression in NPC (for example, in
samples 3, 9, 18); while no hypermethylation or HD could keep a normal level of
LARSAs we know, TSGs could be divided into
various categories: classical, e.g. RB1,
p53; haploinsufficient, e.g. p27^Kip1, Beclin 1; cancer specific or multiple, i.e., involved in several
distinct cancers. Unlike the classical TSGs, haploinsufficient TSGs defy the
identification through mutation analysis and may be quite common. The genetic
and epigenetic alterations of LARSIn summary, we have identified that LARS2 was absent or down-regulated in
NPC. Our findings suggested that HD and methylation should play an important
role in inactivation of LARS

Acknowledgements:

This work was supported by a grant from the
National Natural Science Foundation of China (No. 30801322).

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