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Acta Biochimica et Biophysica Sinica 2009 41(1):54-62; doi:10.1093/abbs/gmn006
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

Inactivation of LARS2, located at the commonly deleted region 3p21.3, by both epigenetic and genetic mechanisms in nasopharyngeal carcinoma

Wen Zhou1, Xiangling Feng1, Hong Li1, Lei Wang1, Bin Zhu1, Weidong Liu1, Ming Zhao1, Kaitai Yao1,2 and Caiping Ren1,*

1 Cancer Research Institute, Xiang-Ya School of Medicine, Central South University, Changsha 410078, China
2 Cancer Institute, Southern Medical University, Guangzhou 510515, China

* Corresponding author: Tel, 86-731-2355066; Fax, 86-731-4360094; E-mail, rencaiping{at}xysm.net


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 
Allelic loss of chromosome 3p, including the 3p21.3 region, is found in 95–100% of primary nasopharyngeal carcinoma (NPC) biopsies, suggesting that this region should harbor some tumor suppressor genes (TSGs) closely related to NPC development. Several TSGs located at 3p21.3, such as RASSF1A, LTF and BLU, have been demonstrated to be involved in NPC development. LARS2 (leucyl-tRNA synthetase 2, mitochondrial) is another gene located in the chromosome 3 common eliminated region-1 (C3CER1) at 3p21.3. In this study, we focussed on the epigenetic and genetic alterations of LARS2 in NPC. The mRNA expression of LARS2 was detected in 36 NPC and 8 chronic nasopharyngitis (NP) tissues by semi-quantitative reverse transcription-polymerase chain reaction (RT–PCR) and real-time RT–PCR. Subsequently, the mutation, allelic loss, and methylation status of LARS2 were analysed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), homozygous deletion (HD) analysis and methylation-specific polymerase chain reaction in primary NPC tissues. No expression or down-regulation of LARS2 was observed in 78% of primary NPC tissues. No mutations, assessed by PCR-SSCP and DNA sequencing, were found in the promoter region and exon 1 of LARS2 in NPC tissues, whereas HD was detected in 28% of NPC specimens at the LARS2 locus. In addition, hypermethylation of LARS2 was found in 64% of NPC samples but only in 12.5% of NP biopsies. Our data indicate that inactivation of LARS2 by both genetic and epigenetic mechanisms may be a common and important event in the carcinogenesis of NPC.

Keywords    nasopharyngeal carcinoma; LARS2; homozygous deletion (HD); mutation; methylation

Received: July 21, 2008; Accepted: August 20, 2008
   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 
Nasopharyngeal carcinoma (NPC) is a malignancy with a high incidence of 25–30 per 100,000 in Southern China and southeast Asia [1–3]. Tumorigenesis of NPC is a multi-step process involving several factors, including Epstein–Barr virus infection and accumulation of epigenetic and genetic alterations [4].

Genetic studies using comparative genomic hybridization have shown that chromosomal abnormalities are involved in NPC, such as losses on chromosomes 3p, 11q, 13q, 14q, 16q, 16p, 1p, and 22q, as well as gains on chromosome 12p, 1q, 3q, 8q, 5p, and 7q [5–8]. Among these chromosomal abnormalities, deletion on chromosome 3p is extremely important because 3p deletion is detected in almost 100% of small-cell lung cancer, renal cell carcinoma, and 95–100% of primary NPC biopsies, and even 75% of pre-cancerous lesions showed loss of heterozygosity (LOH) on 3p [9], implicating that 3p deletion is an early and critical molecular event in the carcinogenesis of NPC and 3p should contain some important tumor suppressor genes (TSGs) closely related to NPC development.

To understand the role of 3p deletion in the development of NPC, we investigated the expression levels of several genes located at 3p21.3, the most frequently rearranged region on 3p. Previous studies in our lab demonstrated that several NPC-related TSGs such as RASSF1A, GNAT1, LTF, and BLU located at 3p21.3 were frequently inactivated by promoter hypermethylation and/or LOH in NPC [10–13]. LARS2 (leucyl-tRNA synthetase 2, mitochondrial), first reported by Kiss et al in 1999, is another gene located in the chromosome 3 common eliminated region-1 (C3CER1) at 3p21.3. It is identified by using the sequence of two overlapping PACs from C3CER1 and encodes the precursor of mitochondrial leucyl-tRNA synthetase which catalyzes the charging of tRNALeu(UUR) with leucine, an essential step in protein synthesis. It performs essential roles in group I intron RNA splicing as well as protein synthesis within the mitochondria and is indirectly required for mitochondrial genome maintenance [14].

In this study, we examined the expression level, LOH, mutation, and methylation status of LARS2 by reverse transcription-polymerase chain reaction (RT-PCR), homozygous deletion (HD) analysis, polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), and methylation-specific polymerase (MSP) chain reaction in primary NPC tissues in order to investigate the genetic and epigenetic alterations and the possible role of LARS2 in NPC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Statistical analysis
 Results
 Discussion
 Funding
 References
 
Tissue and blood samples
Thirty-six poorly differentiated NPC biopsies of primary tumors were obtained from NPC patients with consent before treatment at the Hunan Cancer Hospital (Changsha, China). In addition, eight chronic nasopharyngitis (NP) tissues were also obtained from patients without NPC at the Hunan Cancer Hospital. Among these 36 NPC biopsies, 25 biopsies as well as their matched peripheral blood samples were utilized for HD, mutation, and methylation analyses of LARS2. All the specimens were reviewed by an otorhinolaryngologic pathologist. Fresh NPC or NP tissues were snap-frozen in liquid nitrogen and stored until required.

Detection of the mRNA expression level of LARS2 by semi-quantitative RT–PCR
Total RNA was extracted by TRIzol reagent (Gibco BRL, Grand Island, USA) and 2 µg of total RNA was subjected to cDNA synthesis using Superscript First-Strand Synthesis Kit (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Primers for RT–PCR were designed to span at least two exons to avoid contamination by PCR products amplified from genomic DNA (gDNA). The primer sequences are listed in Table 1. PCR reactions were performed in a thermocycler under the following conditions: 95°C for 5 min, followed by 32 cycles of 94°C for 40 s, 58°C for 40 s, 72°C for 40 s, and then extension at 72°C for 10 min. At the same time, GAPDH was amplified as an endogenous control. The PCR products were electrophoresed on 1.5% agarose gel, visualized by ethidium bromide (EB) staining.


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  		Table 1 Summary of primer sequences, annealing temperature and PCR product size

 
The intensity of each band was measured by Image Master VDS (Pharmacia Biotech, Piscataway, USA) and analyzed by VDS software version 2.0 for band quantification. The expression levels of LARS2 in NPC and NP tissues were investigated after they were normalized by transforming them into two groups of ratios of the band intensity of LARS2 over that of GAPDH of the same sample. Each RT–PCR reaction was carried out in triplicate.

Detection of the mRNA expression level of LARS2 by real-time RT–PCR
The cDNA generated was used for real-time RT–PCR amplification with SYBR Green I PCR Kit (TaKaRa, Shiga, Japan) as recommended by the manufacturer. The reaction was carried out in a real-time PCR instrument (MX3000P, Stratagene, La Jolla, USA). The primers used for real-time RT–PCR were same as those used for RT–PCR. β-actin was amplified as an endogenous control. PCR conditions were 95°C for 90 s, followed by 40 cycles of 94°C for 40 s, 58°C for 40 s, and 72°C for 40 s, and a final extension at 72°C for 5 min. A series of diluted cDNA samples were used as templates to generate the standard curves, and melting curve analysis (100 cycles of 45–95°C for 10 s) was also performed to verify the presence of a single amplicon.

DNA extraction
gDNA from NPC biopsies as well as NP tissues was extracted using an improved method of extracting high-molecular-weight DNA with phenol/chloroform as described elsewhere [15], with a little modification. Briefly, tissues were ground in liquid nitrogen, lyzed in 500 µl of Tris/EDTA/SDS at 55°C for 30 min and incubated with proteinase K (2 mg/ml) at 37°C overnight, followed by phenol/chloroform extraction and stored at –20°C. gDNA from peripheral blood lymphocytes was extracted using Universal Genomic DNA Extraction Kit (TaKaRa) according to the manufacturer's instructions.

Allelic loss analysis
To examine the allelic loss in the LARS2 locus, we selected two microsatellite markers flanking the LARS2 gene. Primers for amplification of microsatellite markers RH25266 and SHGC-12886 are available through the genome database on the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/) (Table 1). The microsatellite markers were amplified from 50 to 100 ng gDNA extracted from 25 NPC tissues and their matched blood samples which were used as controls. Reaction was initiated at 95°C for 5 min, 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final elongation at 72°C for 5 min. After amplification, 6–8 µl of the reaction mixture was mixed with 8 µl of loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol), heat denatured, chilled on ice, then electrophoresed on a 6% polyacrylamide gel containing 8 M urea. The DNA bands were visualized by silver staining. HD was scored if one of the alleles showed at least a 50% reduction in intensity in tumor DNA compared to that in the matched blood DNA.

Moreover, we also used real-time RT–PCR analysis to more accurately calculate the HD frequency of LARS2 in NPC. Samples and microsatellite markers were the same as those mentioned above. β-actin was amplified as an endogenous control. PCR conditions were 95°C for 90 s, followed by 40 cycles of 94°C for 40 s, 58°C for 40 s, and 72°C for 40 s, and a final extension at 72°C for 5 min. The initial relative copy number DNA was given by 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct = {Delta}Cttumor – {Delta}Ctnormal and each {Delta}Ct = {Delta}Cttarget – {Delta}Ctreference. Alleles were considered as homozygously deleted if the highest value of calculated range was below 0.5 and hemizygously deleted if this value was below 1.0 [16].

Detection of LARS2 gene mutations using PCR-SSCP and DNA sequencing analysis
DNA samples from 25 primary NPC tissues as well as their matched peripheral blood samples were subjected to PCR-SSCP analysis for screening mutations in the promoter region and exon 1 of LARS2 gene. We designed two pairs of primers located at –305 to –50 bp and exon 1 of LARS2, respectively. The primer sequences are listed in Table 1. PCR amplification was carried out in 20 µl reaction volume containing 50 ng of gDNA template, 1 x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 µM primer mixture (forward and reverse primers), and 1 U of Taq polymerase. For PCR reactions, after initial denaturation at 95°C for 5 min, 32 cycles, each consisting of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 30 s, were performed, followed by a final elongation at 72°C for 10 min. Then the PCR products were denatured in loading dye at 99°C for 8 min and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel. The results were visualized after the gel was stained with 0.2% AgNO3. PCR products of primary NPC tissues showing distinct PCR-SSCP patterns from those of their matched peripheral blood samples were purified by the PCR product purification kit (TaKaRa) and then sequenced on the 377 ABI PRISM DNA sequencer (Shanghai Invitrogen Company, Shanghai, China).

Methylation analysis by MSP
gDNA from primary NPCs and NP tissues was treated with bisulfite, similar to our previous methods [17,18]. gDNA (10 µg) was denatured with 0.3 M NaOH, mixed with 333 µl of freshly prepared solution (10 mM hydroquinone and 3 M sodium bisulfite), covered with paraffin oil, and then deaminated in the dark for 4 h at 55°C. Bisulfite-treated DNA was purified with purification columns (TaKaRa), desulfonated with 0.3 M NaOH at room temperature for 10 min, neutralized with ammonium acetate, precipitated by ethanol, and resuspended in 20 µl of Tris–EDTA buffer. In this method, the sodium bisulfite reaction converts unmethylated cytosine in DNA to uracil while leaving the methylcytosine unchanged, so that methylated and unmethylated alleles can be distinguished by MSP. The MSP primers of LARS2 were designed using the MethPrimer-Design software (http://www.urogene.org/methprimer). Detailed information concerning MSP primers for LARS2 is shown in Table 1. Bisulfite-modified gDNA (~100 ng) was amplified by MSP in a total volume of 20 µl containing 1x PCR buffer, 0.25 mM dNTPs, 0.2 µM specific primer mixture (forward and reverse primers) and 0.5 U Hotstart (HS) Taq polymerase (TaKaRa). The MSP parameters were: 95°C for 5 min, followed by 32 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 30 s and then an extension at 72°C for 10 min. The PCR products were electrophoresed on a 1.5% EB-stained agarose gel. The unbisulfited DNA was used as negative control and MSP primers were tested for not amplifying any unbisulfited DNA.


   Statistical analysis
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 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
Results
 Discussion
 Funding
 References
 
Statistical analysis was performed using the Wilcoxon rank sum test, {chi}2 test, and Student's t-test, when appropriate. In all analyses, SPSS 10.0 statistical software (SPSS, Chicago, USA) was used and the statistical significance level was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
Discussion
 Funding
 References
 
Down-regulation of LARS2 in NPC tissues detected by RT–PCR and real-time RT–PCR
RT–PCR was performed to analyze the expression of LARS2 at transcription level. LARS2 expression was examined in 36 primary NPC tissues as well as in 8 NP tissues. The results showed that all the eight NP tissues expressed stable LARS2 mRNA level, whereas no LARS2 transcript was amplified in 28% (10 of 36) of NPC tissues and down-regulation of LARS2 was detected in 50% (18 of 36) of NPC tissues, indicating that aberrant expression (loss plus down-regulation) was detected in 78% (28 of 36) of NPC tissues. Compared with NP tissues, the mRNA expression level of LARS2 was significantly down-regulated in NPC tissues (P = 0.019) (Fig. 1 and Tables 2 and 3). To more accurately detect the expression level of LARS2 gene, real-time RT–PCR was also performed in 36 primary NPC tissues and 8 NP tissues. According to the real-time RT–PCR results, LARS2 was found to be down-regulated significantly in NPC tissues (P = 0.02) (Table 3), when the overall LARS2 mRNA expression was compared between NPC and NP tissues. To determine whether the expression level of LARS2 in primary NPC tissues was associated with the clinical features of the NPC patients, we compared the expression level of LARS2 with the gender and lymph node metastasis (Table 4). The expression status of LARS2 did not show any significant correlation with the gender (P = 1.00), whereas LARS2 down-regulation was significantly correlated with lymph node metastasis (P =0.01) (Table 4).


Figure 1
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  		Fig. 1 Semi-quantitative RT–PCR analysis for detecting the mRNA expression level of LARS2 in primary NPC tissues    Compared with NP tissues, primary NPC tissues expressed lower levels of LARS2. (A) Stable expression of LARS2 was detected in NP tissue (lane 1) and primary NPC tissue (lane 4), but no LARS2 transcript was detected in primary NPC tissues (lanes 2–3 and 5–7). (B) Stable expression of LARS2 was detected in NP tissue (lane 1), but down-regulation of LARS2 mRNA was detected in primary NPC tissues (lanes 2–9); M, DNA marker; GAPDH gene was used as an internal control.

 


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  		Table 2 Genetic and epigenetic changes of LARS2 in primary NPC tissues

 


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  		Table 3 Statistical analysis of RT–PCR and real-time RT–PCR results of LARS2

 


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  		Table 4 Relationship between LARS2 expression and clinical features of NPC patients

 
No mutations detected in the promoter region and exon 1 of LARS2
We analyzed mutations in the promoter region (–164 to –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 NPC samples tested 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).


Figure 2
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  		Fig. 2 Detection of mutations in promoter region, exon 1 of LARS2 by PCR-SSCP and sequencing    No mutations were detected in the promoter region and exon 1 of LARS2. (A) PCR-SSCP results. N, matched control peripheral blood samples; T, primary NPC tissues. (B) DNA sequencing results for promoter region –164 to –18 bp. (C) DNA sequencing results for exon 1.

 
Allelic deletion of LARS2 gene in NPC
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 the mechanisms involving in the inactivation of LARS2 in NPC.


Figure 3
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  		Fig. 3 Representative results illustrating HD of LARS2 using two microsatellite sites (RH25266 and SHGC-12886)    HD of LARS2 was detected in the primary NPC tissues, compared with the matched control peripheral blood samples. N, matched control peripheral blood samples; T, primary NPC tissues.

 
Hypermethylation of LARS2 gene in NPC
We analyzed the methylation status of 25 CpG islands in a 227-bp promoter region of LARS2 in 25 primary NPC samples and 8 NP tissues by MSP [Fig. 4(A)]. Hypermethylation of LARS2 promoter was only detected in 12.5% (1 of 8) of NP tissues; however, it was found in 64% (16 of 25) of primary NPC tissues [Table 2 and Fig. 4(B)]. Statistical analysis indicated that there was a significant difference in methylation frequency of LARS2 between NP and primary NPC tissues ({chi}2 test, P = 0.017) (Table 5). Meanwhile, we also found that hypermethylation of LARS2 showed a significant correlation with lymph node metastasis (Fisher's exact test, P = 0.010) (Table 5).


Figure 4
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  		Fig. 4 The sequence of LARS2 promoter region and the methylation status of LARS2 CpG islands in NPC tissues analyzed by MSP    (A) Positions of the primers used for MSP analysis along with CpG sites of LARS2 gene. The positions of CpG sites are numbered 1–25. Horizontal arrows indicate the positions of MSP primers. (B) MSP analysis of LARS2 in primary NPC and NP tissues. m, MSP products using methylation-specific primers; u, MSP products using unmethylation-specific primers; N, NP tissues; T, primary NPC tissues.

 


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  		Table 5 Relationship between LARS2 promoter methylation and clinical features of NPC patients

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
Funding
 References
 
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, and ovarian cancer [19]. LUCA (also referred to 3p21.3C) and AP20 (also referred to 3p21.3T), two most frequently rearranged regions on 3p, were of high-frequency LOH or HD in multiple epithelial malignancies [16,20]. Besides, another region between D3S32 and D3S2354, which was located distal to LUCA, was also reported to be frequently deleted in lung and other cancers [21,22]. Although the first data suggesting that 3p deletions were involved in nasopharyngeal carcinogenesis had been published more than 10 years ago, only recently has significant progress been achieved in identifying the candidate TSGs and demonstrating their functional roles in NPC development. At present, some genes located on 3p have been considered as promising candidate NPC-associated TSGs, such as RASSF1A, BLU, LTF, and DLEC1 [23]. It has been demonstrated that genetic and epigenetic abnormalities of these promising candidate NPC-associated TSGs residing in chromosome region 3p may be important for the development of NPC; however, it is still obscure how many of them exist and which of the numerous candidate TSGs are the key players in NPC pathogenesis.

LARS2 gene is located at the 1-Mb long common eliminated region 1 (CER1) between D3S32 and D3S3582 at 3p21.3 and spans 160 kb consisting of 22 exons encoding a 903-amino acid 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 and 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, whereas all the NP tissues expressed stable LARS2 mRNA level, indicating that 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 LARS2 in NPC. As LOH or HD can be considered as a reliable indicator for the presence of TSGs in some specific regions with high-frequency LOH or HD [26,27], a number of TSGs have been successfully identified by LOH or HD assay combined with other methods, for example, sFRP1 and DFF45 [28,29]. By amplifying RH25266 and SHGC-12886, two microsatellite markers flanking the LARS2 gene, we found that HD frequency was 28% in NPC specimens at the LARS2 locus, in accordance with the previously reported high deletion frequency of 3p21.3 in NPC, suggesting that allelic loss may be one of the mechanisms leading to inactivation of LARS2 in NPC. Nevertheless, more conspicuous down-regulation of LARS2 detected in NPC strongly suggested that down-regulation of LARS2 might not be only due to genomic deletions and epigenetic mechanism remained investigated.

Recently, 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. RASSF1A, p16, LTF, DLC1) are frequently methylated in NPC tissues but are rarely methylated in the corresponding NP tissues. In this study, aberrant DNA methylation of LARS2 gene was found in 64% of primary NPC tissues but only in 12.5% of NP tissues. Statistical analysis showed that the difference in hypermethylation level of LARS2 gene between NPC and NP tissues was of statistical significance (P = 0.017), implying that promoter methylation may be the major mechanism for inactivation of LARS2 in NPC. In addition, we found that there may be a correlation between LARS2 methylation and lymph node metastasis, but further large-scale studies with more NPC samples are necessary and warranted to prove the strength of this contention.

Further parallel analysis demonstrated that hypermethylation of LARS2 promoter alone could contribute to LARS2 down-regulation in NPC (e.g. in samples 7, 13, 20, 23) or eliminate LARS2 expression in NPC combined with HD (e.g. in samples 3, 9, 18), whereas no hypermethylation or HD could keep a normal level of LARS2 in NPC (e.g. in samples 6, 14, 17, 22) (Table 2), also supporting our point of view. However, in two cases with LARS2 down-regulation (samples 2, 25), no mutation/hypermethylation/HD was detected, indicating that other epigenetic mechanism (e.g. histone deacetylation) may play a certain role in transcriptional silencing of LARS2 in NPC.

As we know, TSGs could be divided into various categories: classical, e.g. RB1, p53; haploinsufficient, e.g. p27Kip1, 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 LARS2 in NPC tissues indicated that LARS2 might act as a haploinsufficient tumor suppressor in NPC. Meanwhile, the relationship between mitochondrial DNA (mtDNA) and human malignancies attracts much attention in the understanding of carcinogenesis in recent years [31]. For example, metabolic catastrophe in mitochondria can promote death of tumor cells that have disabled apoptosis, and somatic mtDNA alterations including point mutations and microsatellite instability have been frequently detected in human cancers such as prostate cancer and gastric cancer [32,33]. LARS2 encoding the precursor of mitochondrial leucyl-tRNA synthetase was found to be dramatically down-regulated in NPC in our work, providing us a new insight into further understanding the process of NPC tumorigenesis.

In 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 LARS2 in NPC, but more work is required to be done to elucidate its role in NPC. Upon further study, these unique aberrations will provide insight into mechanisms of NPC carcinogenesis.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
References
 
This work was supported by a grant from the National Natural Science Foundation of China (No. 30801322).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Statistical analysis
 Results
 Discussion
 Funding
 References
 

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