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Acta Biochim Biophys Sin 2009, 41: 54–62
doi: 10.1093/abbs/gmn006.

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 Yao^1,2, 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:

Caiping Ren, M.D, Tel: 86-731-2355066; Fax: 86-731-4360094; E-mail: rencaiping@xysm.net

Running title: Epigenetic and genetic alterations of LARS2 in NPC

Abstract

Allelic loss of chromosome 3p, including 3p21.3 region, is found in 95% to100% of primary nasopharyngeal carcinoma (NPC) biopsies, suggesting that this region should harbor some tumor suppressor genes (TSGs) closely related to NPC development. Several tumor suppressor genes (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 the current study, we focused 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 RT-PCR and real-time RT-PCR. Subsequently, the mutation, allelic loss and methylation status of LARS2 were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), homozygous deletion (HD) analysis and methylation-specific polymerase chain reaction (MSP) in primary NPC tissues. Absent expression or down-regulation of LARS2 were 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, while 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

Nasopharyngeal carcinoma (NPC) is a malignancy with a high incidence of 25-30 per 100000 in Southern China and South-East Asia [1-3]. The 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 (CGH) 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 precancerous 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 2 overlapping PACs from C3CER1 and encodes the precursor of mitochondrial leucyl-tRNA synthetase which catalyzes the charging of tRNA^Leu(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), HD analysis, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and methylation-specific polymerase chain reaction (MSP) in primary NPC tissues in order to investigate the genetic and epigenetic alterations and the possible role of LARS2 in NPC.

Materials and Methods

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, 8 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 mg 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 were listed in Table 1. PCR reactions were performed in a thermocycler under the following conditions: 95 ºC for 5 min, followed by 32 cycles through 94 ºC for 40 s, 58 ºC for 40 s, 72 ºC for 40 s and then extension at 72 ºC 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.

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 was as same as those for RT-PCR. b-actin was amplified as an endogenous control. PCR conditions were 95 ºC for 90 s, followed by 40 cycles of 94 ºC for 40 s, 58 ºC for 40 s, and 72 ºC for 40 s and a final extension at 72 ºC 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 ºC 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 ml of Tris/EDTA/SDS at 55 ºC for 30 min and incubated with proteinase K (2 mg/ml) at 37 ºC overnight, followed by phenol/chloroform extraction and stored at -20 ºC. 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 50-100 ng gDNA extracted from 25 NPC tissues and their matched blood samples which were used as controls. Reaction was initiated at 95 ºC for 5 min, 30 cycles of 94 ºC for 30 s, 58 ºC for 30 s, and 72 ºC for 30 s, followed by a final elongation at 72 ºC for 5 min. After amplification, 6-8 ml of the reaction mixture was mixed with 8 ml 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. b-actin was amplified as an endogenous control. PCR conditions were 95 ºC for 90 s, followed by 40 cycles of 94 ºC for 40 s, 58 ºC for 40 s, and 72 ºC for 40 s and a final extension at 72 ºC for 5 min. The initial relative copy number DNA was given by 2^-DDCt, where DDCt=DCt_tumor-DCt_normal and each DCt=DCt_target-DCt_reference. Alleles were considered as homozygously deleted if the highest value of calculated range was below 0.5 and hemozygously 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 bp~-50 bp, and exon 1 of LARS2, respectively. The primer sequences were listed in Table 1. PCR amplification was carried out in 20 ml reaction volume containing 50 ng of gDNA template, 1´PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, 0.25 mM primer mixture (forward and reverse primers) and 1 unit of Taq polymerase. For PCR reactions, after initial denaturation at 95 ºC for 5 min, 32 cycles, each consisting of denaturation at 94 ºC for 30 s, annealing at 60 ºC for 30 s and elongation at 72 ºC for 30 s, were performed, followed by a final elongation at 72 ºC for 10 min. Then the PCR products were denatured in loading dye at 99 ºC for 8 min and separated by electrophoresis on an 8% non-denaturing polyacrylamide gel. The results were visualized after 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 mg) was denatured with 0.3 M NaOH and mixed with 333 ml of freshly prepared solution (10 mM hydroquinone and 3 M sodium bisulfite), covered with paraffin oil then deaminated in the dark for 4 h at 55 ºC. 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 ml 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 was shown in Table 1. Bisulfite-modified gDNA (~100 ng) was amplified by MSP in a total volume of 20 ml containing 1´PCR buffer, 0.25 mM dNTPs, 0.2 mM specific primer mixture (forward and reverse primers) and 0.5 unit Hotstart (HS) Taq polymerase (TaKaRa). The MSP parameters were 95 ºC for 5 min, followed by 32 cycles of 95 ºC for 30 s, 56 ºC for 30 s, 72°C for 30 s and then extension at 72 ºC 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

Statistical analysis was performed using Wilcoxon rank sum test, c² test and student 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

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 8 NP tissues. The results showed that all the 8 NP tissues expressed stable LARS2 mRNA level, while 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, Tables 2, 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), while LARS2 down-regulation was significantly correlated with lymph node metastasis (P =0.01) (Table 4).

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 LARS2 in NPC.

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, Fig. 4(B)]. Statistical analysis indicated that there was a significant difference in methylation frequency of LARS2 between NP and primary NPC tissues (c² test, P=0.017) (Table 5). Meanwhile, we also found hypermethylation of LARS2 showed a significant correlation with lymph node metastasis (Fisher’s exact test, P=0.010) (Table 5).

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 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 was 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 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 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 HD frequency was 28% in NPC specimens at the LARS2 locus, in accordance with previously reported high deletion frequency of 3p21.3 in NPC, suggesting that allelic loss may be one of mechanisms leading to inactivation of LARS2 in NPC. Nevertheless, more conspicuous downregulation of LARS2 detected in NPC strongly suggested that downregulation 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 our current 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 have 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 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 LARS2 in NPC (for example, in samples 6, 14, 17, 22) (Table 2), also supporting our point of view. However, in two cases (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. 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 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 mitochondrial DNA alterations including point mutations and microsatellite instability (MSI) 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.

Acknowledgements:

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

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