Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma
Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma
Wen Zhou 0
Xiangling Feng 0
Caiping Ren 0
Weidong Liu 0
Wei Huang 0
Lei Wang 0
Bin Zhu 0
Jia Shi 0
Jie Liu 0
Chang Zhang 0
Yanyu Liu 0
Kaitai Yao 0
0 Cancer Research Institute, Xiang-Ya School of Medicine, Key Laboratory for Carcinogenesis of Chinese Ministry of Health, Key Laboratory for Carcinogenesis & Cancer Invasion of Chinese Ministry of Education, Central South University , Xiangya Road 110, 410078, Changsha, Hunan , P. R. China
Background: Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet. Methods: Immunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi). Results: The positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities. Conclusions: Our study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.
Nasopharyngeal carcinoma; BCAT1; c-Myc; Proliferation; Migration; Invasion; Gene amplification; Gene regulation
Nasopharyngeal carcinoma (NPC) is a squamous cell
carcinoma that develops from the epithelium of the
nasopharynx with a high incidence in Southeast Asia
and southern China and causes a serious healthcare
problem in these regions . More than 95% of NPC in
southern China is undifferentiated carcinoma with a
high incidence of early metastasis which is the main
cause of death in NPC patients. Currently, radiation
therapy is the first choice for NPC treatment. Although
the radiotherapy equipments and techniques have been
improved tremendously, the five-year survival rate of
NPC patients has not radically changed yet and remains
around 50-60%. Therefore, it is of great importance to
comprehensively explore the new approaches for NPC
The molecular mechanisms of nasopharyngeal
carcinogenesis have not been elucidated clearly yet.
Previous studies have shown that proto-oncogenes (e.g.
HRAS, NRAS2 , cyclin D1 , MDM2 , EVI1 ,
EGFR ) and tumor suppressor genes (TSGs) (e.g. p53
, p16 , RASSF1A , DLC-1 , LTF [11,12],
DLEC1 , TSLC1 ) are aberrantly expressed in
NPC. However, none of them has been confirmed as an
NPC-specific oncogene or TSG. Using comparative
genomic hybridization (CGH) data from 170 primary NPC
cases, we have developed a tree model indicating the
pathogenetic mechanisms of NPC . According to the
tree model, +12p11-12 may represent an early event in
the carcinogenesis of NPC . We further identified
that BCAT1, KCNJ8, PTX1 and KRAS2, four genes
located at 12p11-12, were significantly up-regulated in
NPC tissues compared to the normal controls .
BCAT1 (branched chain aminotransferase 1 gene, also
known as ECA39) is also significantly up-regulated in
Burkitts lymphoma and breast cancer . Therefore,
we selected BCAT1 as a target gene for further study to
explore its relationship with NPC development. In our
previous work, we found that BCAT1 mRNA expression
was over expressed in NPC tissues, and BCAT1
knockdown in 5-8F NPC cell line inhibited cell cycle
progression and cell proliferation.
In this report, we further investigated the expression
of BCAT1 protein in tissues at various stages including
normal epithelia, mild or moderate hyperplasia, severe
atypical hyperplasia and NPC. We also explored how
BCAT1 is up-regulated and its functional roles in NPC
proliferation, migration and invasion.
protein in different stages of precancerous and
cancerous lesions in nasopharyngeal biopsies. Cytoplastic
immunostaining signals of BCAT1 could be detected at
different stages, but the positive rates differed greatly,
which were 23.6% (17/72), 75.0% (18/24), 88.9% (8/9)
and 88.8% (71/80) in normal epithelia, low-to-moderate
grade atypical hyperplasia tissues, high-grade atypical
hyperplasia tissues and NPC tissues, respectively (Figure 1,
Table 1, P < 0.05), indicating that up-regulation of BCAT1
is an early event in NPC pathogenesis.
No mutation of BCAT1 was found in NPC tissues
Since gene mutation and DNA amplification are two
major causes for oncogene up-regulation, we first
performed DNA sequencing of the full-length of 11
exons in BCAT1. Only one polymorphism (G/T) was
detected at +78 in the non-coding region of first exon
(Figure 2A), which was further confirmed in the single
nucleotide polymorphism (SNP) database.
Frequent amplification of BCAT1 was detected in NPC
Three microsatellites (D12S1435, D12S1617 and RH44650)
located within BCAT1 gene were selected for analysis of
BCAT1 amplification. Real-time PCR was employed to
detect DNA samples from 28 NPC tissues and their matched
peripheral blood specimens. The amplification ratios of
D12S1435, D12S1617 and RH44650 were 14% (4/28), 25%
(7/28) and 17% (5/28), respectively (Figure 2B). The total
amplification ratio was 42.4% (12/28).
The transcription factor c-Myc regulated BCAT1
By searching NNPP and TESS, a c-Myc recognition site
(CACGTG) was discovered in the 5 regulatory region of
BCAT1 gene, suggesting that expression of BCAT1 may
be regulated by the transcription factor c-Myc. ChIP
experiment using anti-c-Myc antibody was carried out to
co-precipitate DNA sequences binding to c-Myc. The
specific primers at 233 to -41 bp of BCAT1 were
designed. As shown in Figure 3A, a 193 bp fragment of
BCAT1 sequence was amplified, indicating that c-Myc
transcription factor can directly bind to the specific
promoter region of BCAT1 gene.
Subsequently, we analyzed the regulation of BCAT1 by
c-Myc through knocking down c-Myc expression in
NPC cells. When c-Myc shRNA vectors were transfected
into 5-8F and 6-10B NPC cells, the mRNA expression of
c-Myc decreased by 80% and 70% in 5-8F-Si-c-Myc and
6-10B-Si-c-Myc cells, respectively, as measured by
semiquantitative RT-PCR. As expected, the expression of
BCAT1 was also inhibited by 85% and 72% in
5-8F-Si-cMyc and 6-10B-Si-c-Myc cells, respectively.
Meanwhile, the expression of KRAS and MCAM, two c-Myc
Figure 1 Detection of BCAT1 protein in different pathological stages of NPC. (A) Normal pseudo-stratified ciliated epithelium. (B) Low-to
-moderate grade atypical hyperplasia tissue. (C) High-grade atypical hyperplasia tissue. (D) NPC tissue. The results demonstrated that the
expression level of BCAT1 protein had increased significantly since early pathological stages of NPC.
non-target genes, was unaffected by c-Myc knockdown
(Figure 3B), further supporting that c-Myc can regulate
To further test whether c-Myc regulates BCAT1
expression, we performed luciferase assay. The COS7 cells
with absent expression of c-Myc were co-transfected
with pGL3-233/-41 recombinant and c-Myc expression
vector. The results showed that the luciferase activity of
reporter system in co-transfected cells was markedly
higher than that in parental COS7 cells and COS7 cells
transfected with pGL3-233/-41-M mutant in which the
c-Myc binding site was mutated (Figure 3C). We also
conducted the luciferase assay in 5-8F-Si-c-Myc cells.
Similarly, once c-Myc was knocked down in 5-8F cells,
luciferase activity of pGL3-233/-41 recombinant
dramatically decreased (Figure 3C), but that of
pGL3-233/-41M mutant had no significant change. Together, these
results indicate that c-Myc directly binds to promoter of
BCAT1 and transactivates its expression.
Expression of c-Myc and BCAT1 was detected in NPC
The mRNA expression of c-Myc and BCAT1 was
detected by RT-PCR in 6 chronic nasopharyngitis (CN)
samples and 28 NPC samples. The results showed that
c-Myc and BCAT1 mRNA expression were low or
undetectable in 6 CN tissues, while over expression of
c-Myc and BCAT1 was found in 67.9% (19/28) and 64.3%
Table 1 Statistical analysis for BCAT1 expression in different stages of NPC
Low-to-moderate grade atypical hyperplasia tissues
High-grade atypical hyperplasia tissues
P* value was calculated by comparing the positive rate of BCAT1 in low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues
and NPC tissues with that in normal epithelia, respectively.
Figure 2 Exon mutation and amplification of BCAT1. (A) BLAST analysis result of BCAT1 exon 1. The red box indicates SNP site (+78G/T) by
DNA sequencing. (B) The amplification status of three BCAT1 microsatellite loci in NPC samples, showing that the amplification ratios for
D12S1435, D12S1617 and RH44650 were 14% (4/28), 25% (7/28) and 17% (5/28), respectively, and the total ratio was 42.4% (12/28).
(18/28) of NPC tissues, respectively. In addition, c-Myc
and BCAT1 exhibited the same mRNA expression
patterns in 74% of NPC tissues, as they were lowly
expressed in 21% (6/28) and co-upregulated in 53%
(15/28) of NPC tissues (Figure 3D, Table 2).
The protein expression of c-Myc and BCAT1 was also
examined by IHC in 22 NPC samples. The c-Myc or
BCAT1 protein was positively stained in 73% (16/22)
and 68% (15/22) of NPC tissues, respectively. Among
them, c-Myc and BCAT1 were simultaneously and
positively stained in 59% (13/22), whereas lowly or negatively
in 18% (4/22) of NPC tissues (Figure 3E, Table 2).
The results showed a positive correlation of c-Myc
expression and BCAT1 expression in NPC tissues (Table 2,
P = 0.019 for RT-PCR; P = 0.032 for IHC).
Silencing BCAT1 inhibited colony formation, migration
and invasion of NPC cells
The cell growth of 5-8F NPC cells stably transfected
with BCAT1-shRNA (5-8F-shBCAT1) and empty vector
(5-8F-vector) was observed using clonogenesis assay.
The colony formation ratios of 5-8F-shBCAT1 and
58F-vector cells were 10.7% 0.5% and 52.1% 3.5%,
respectively (Figure 4A), demonstrating that BCAT1 is
critical for maintenance of NPC cell growth.
Using the migration assay, cell mobility was analyzed
in 5-8F-shBCAT1 and 5-8F-vector cells. 5 104 cells
were inoculated on the filter membrane and cultivated
for 18 hrs. Figure 4B showed that as measured by the
numbers of cells migrating through the filter membrane,
5-8F-shBCAT1 cells (141.67 17.9) demonstrated a
noticeable decrease in the mobility compared to 5-8F-vector
cells (180.8 7.35).
The invasion capability associated with BCAT1
expression was examined with matrigel-coated transwell
chambers. The 5-8F-shBCAT1 and 5-8F-vector cells were
inoculated on matrigel-coated membrane and cultivated
for 48 hrs. The numbers of cells migrating through the
membrane were 105 33 and 168 29.35, respectively
(Figure 4C). Clearly, down-regulation of BCAT1
remarkably impairs NPC cell invasion.
CGH-array is a newly developed technique for detecting
genetic lesions in cancer and other diseases .
Numerous genetic abnormalities have been identified in
multiple chromosomal regions in NPC tissues and cell
lines . Frequent gains on 1q, 3q, 8q, 11q, 12p and
12q, and losses on 3p, 9p, 11q, 14q and 16q, have been
found. Moreover, several minimal regions of gains
including 3q27.3-28, 8q21-24 and 11q13.1-13.3 have been
identified and several minimal deleted regions have been
mapped to 3p14.1-22, 11q13.3-24, 13q14.3-22,
14q24.332.1 and 16q22-23 . We have analyzed 170 comparative
genomic hybridization (CGH) samples and constructed
a tree model to predict NPC tumorigenesis. We are
particularly interested in the gain of 12p11-12
(+12p11-12) since +12p11-12 is a region frequently
amplified and may be an early event in the
development of NPC .
BCAT1 is located at 12p12.1, and codes for the
cytosolic form of branched-chain amino acid transaminase
which catalyzes the reversible transamination of
branched-chain alpha-keto acids to branched-chain
L-amino acids essential for cell growth. BCAT1 has been
reported to be highly conserved in evolution and
disruption of its yeast homolog affects cell growth [20,21].
Several groups have confirmed that BCAT1 is involved in
cell proliferation, cell cycle progression, differentiation
and apoptosis, and plays an important role in several
malignancies, especially in the progression of nonseminomas
[22-24]. The mouse homologue of BCAT1 has been
shown to be amplified and overexpressed in a
teratocarcinoma cell line . Retroviral transduction of BCAT1
into fetal rat brain cells with SV40 large T-antigen induced
tumor formation with characteristic features of
Figure 3 The regulation of BCAT1 by c-Myc. (A) ChIP confirmed that transcription factor c-Myc can specifically bind to the regulatory region of
BCAT1. Lane 1 and 2 represent gDNA untreated or treated with ultrasonication, respectively. (B) Detection of the mRNA level of BCAT1 in 5-8F
cells and 6-10B cells transfected with pRNAT-U6.1/Si-c-Myc vector or blank vector. BCAT1 mRNA level was reduced when the endogenous
expression of c-Myc was blocked both in 5-8F cells and 6-10B cells, while the expression of KRAS or MCAM, two non-target genes of c-Myc, was
stable despite the change of c-Mycs level in these cells. (C) Luciferase reporter assay demonstrated the influence of c-Myc on BCAT1 promoter
activity. The results showed that the luciferase activity was positively correlated to the expression level of c-Myc. Here, we used 5-8F-vector cells
instead of 5-8F cells as control. (D) The co-expression of BCAT1 and c-Myc was detected in NPC tissues by RT-PCR. Lanes 13 represent BCAT1 and
c-Myc expression in CN tissues. Lanes 410 represent BCAT1 and c-Myc expression in NPC tissues. GAPDH was used as an internal control. (E) IHC
analysis of the same batch of NPC biopsies demonstrated that BCAT1 and c-Myc were co-expressed in most NPC tissues.
Previously, RT-PCR results have presented that BCAT1
is significantly up-regulated in NPC tissues and silencing
its expression blocks NPC cell proliferation and the G1/
S transition, indicating that high expression of BCAT1
may play an important role in NPC cell survival .
Here, we further performed IHC analysis of different
stages of NPC and found that BCAT1 protein level
increased in the low-to-moderate grade atypical
hyperplasia tissues as well as high-grade atypical hyperplasia
tissues, in situ and invasive carcinomas, suggesting that
Table 2 Correlation analysis between c-Myc and BCAT1
expression in the same batch of NPC tissues
P = 0.019
P = 0.032
U up-expression, L low expression.
BCAT1 overexpression may be an important early event
in NPC occurrence and maintain throughout NPC
progression. There are several factors that can account for
the abnormalities of gene expression, such as gene
mutation, DNA amplification, transcriptional regulation and
epigenetic changes, alone or synergistically. Gene
mutation and amplification are two common causes for
genetic activation of oncogenes. It is well known that Ras
mutation is closely related to various malignancies such
as breast cancer and lung cancer , and TRK
mutation is also found to be associated with neuroblastoma
. HER-2/neu amplification is frequently detected in
node-negative breast carcinoma tissues and it is a good
example for oncogene activation by gene amplification
. We first analyzed whether BCAT1 has mutation by
sequencing 11 exons of BCAT1 in 20 cases of NPC. Only
one SNP site in exon1 was detected, suggesting that
gene mutation of BCAT1 is a rare incident in NPC. By
using real-time PCR, we also analyzed three
microsatellite loci including D12S1435, D12S1617 and RH44650 to
examine whether BCAT1 is amplified in NPC. Our results
demonstrated that 42.4% (12/28) of NPC tissues manifested
amplification, revealing that BCAT1 overexpression may be
due to its amplification in a portion of NPC tissues. Genes
such as CDH13 , p16 and p27  have been reported
to be involved in the early development of NPC. BCAT1
over-expression, together with abnormal expression of
CDH13, p16, p27 and others, may result in transition from
normal epithelia to hyperplastic epithelia.
BCAT1 was first identified from a c-Myc-induced
tumor and has been proven to be directly regulated by
c-Myc through its binding to the specific DNA
sequence, CACGTG [17,25]. C-Myc is an oncogene and
transcription factor involved in the tumorigenesis of
multiple cancers, such as Burkitts lymphoma and breast
cancer . Both BCAT1 and c-Myc were found to be
Figure 4 Detection of the colony formation ability, migration and invasion capacities of NPC cells. The colony formation ability (A),
migration (B) and invasion capacities (C) of 5-8F cells decreased when the expression of BCAT1 was blocked.
overexpressed in NPC . We thus used IHC, RNAi,
ChIP and Luciferase reporter system to investigate
whether BCAT1 is directly regulated by c-Myc in NPC.
59% of NPC tissues were double positive for c-Myc and
BCAT1. Silencing the endogenous expression of c-Myc
by RNAi also decreased BCAT1 mRNA level in
5-8F-Sic-Myc and 6-10B-Si-c-Myc cells. Using luciferase assay,
we found transcription factor c-Myc up-regulated
BCAT1 expression. Furthermore, we confirmed that
c-Myc can directly bind to the BCAT1 promoter. Our
results revealed that c-Myc, together with BCAT1
amplification, up-regulates BCAT1 expression and leads
to BCAT1 activation in NPC tissues.
One of the major clinical features of NPC is early
metastasis. Several genes have been found to be associated
with the metastasis of NPC, for example, LMP1,
LMP2A, p16, nm-23, CD44v6, TSLC1, NGX6, MMP9
and LTF [6,9,32-35]. However, they cannot fully
elucidate the mechanisms underlying NPC metastasis. In this
study, we indicated increased expression of BCAT1 in
the premalignant and NPC tissues. By performing colony
formation, migration and invasion assays, we showed
that colony formation, cell mobility and invasion abilities
of 5-8F cells were reduced in response to knockdown of
BCAT1 expression. Consistent with our data, high
BCAT1 expression is associated with a high incidence of
metastasis resulting in an adverse disease-free survival in
colorectal adenocarcinomas . Both mRNA and
protein levels of BCAT1 are higher in medulloblastoma
patients with metastasis compared with those without
metastasis (P < 0.01) . Taken together, BCAT1 may be
a favorable biomarker to indicate NPC early metastasis.
In summary, for the first time, we demonstrate that
expression of BCAT1, which locates in the frequently
amplified 12p12 region, increases at early pathological stage
of NPC. Gene amplification is an important cause for
overexpression of BCAT1 in NPC, while c-Myc also
plays a critical role in regulation of BCAT1 expression.
We also confirm that high expression of BCAT1 is
associated with the mobility of NPC cells, indicating that it
may be a promising target for NPC diagnosis and
5-8F, 6-10B and COS7 cells were cultured in RPMI1640
(Gibco BRL, Bethesda, MD) media with 10% fetal bovine
serum (FBS) at 37C in an atmosphere containing 5%
CO2. The NPC cell lines 5-8F and 6-10B were derived
from the same NPC cell line SUNE-1. Although sharing
almost the same genetic background, the two NPC cell
lines have different metastatic capability, for 5-8F cell
line had high metastasis potential, while 6-10B cell line
was non-metastatic .
Patients and tissues
Six chronic nasopharyngitis (CN) biopsies and 28
primary poorly-differentiated NPC biopsies were obtained
from CN and NPC patients with consent before
treatment at Hunan Tumor Hospital (Changsha, Hunan,
China), Xiangya Hospital of Central South University
(CSU), the Second Xiangya Hospital of CSU and the
Third Xiangya Hospital of CSU (Changsha, Hunan,
China) in 2006 and 2007.
A total of 120 paraffin-embedded specimens, including
7 normal nasopharyngeal epithelia samples, 24 mild or
moderate atypical hyperplasia samples, 9 severe atypical
hyperplasia samples and 80 NPC samples, were supplied
by Hunan Tumor Hospital and the Second Xiangya
Hospital of CSU. All the specimens were stained with
haematoxylin and eosin (HE) for histological
examination and reviewed by an otorhinolaryngologic
pathologist. The present study was approved ethically by Cancer
Research Institute review board of CSU. All patients
provided informed written consent.
Immunohistochemistry (IHC) staining
Investigating the expression of BCAT1 in different stages
of precancerous lesions can help us evaluate the
significance of this gene in NPC pathogenesis. Therefore, we
used IHC method to analyze the expression of BCAT1
protein in the normal nasopharyngeal epithelia including
pseudo-stratified ciliated epithelia and stratified
epithelia, low-to-moderate grade atypical hyperplasia tissues,
high-grade atypical hyperplasia tissues and NPC tissues,
according to the protocol described in our previously
published paper . Meanwhile, the co-expression of
BCAT1 and c-Myc in NPC tissues was also detected by
IHC. Incubation with anti-BCAT1 (BD, Franklin Lakes,
NJ) or anti-c-Myc (Calbiochem, Darmstadt, Germany)
was carried out overnight at 4C. Semi-quantitative
assessment of BCAT1 and c-Myc immunostaining was
performed by consensus and comprised both intensity
of staining (0, 1, 2, or 3) and extent of staining (0, 0%;
1, <10%; 2, 10-50%; 3, >50%). The scores for the
intensity of staining and extent of staining were multiplied
to give a weighted BCAT1 or c-Myc score for each case
(maximum possible, 9). The cases with at least
moderate staining intensity (2 or 3) in a minimum of 10% of
tumor cells were regarded as BCAT1 or c-Myc positive
(++ or +++, total weighted score of > 4 out of 9), while
the cases with weighted score of 0 () or 13 (+) were
regarded as BCAT1 or c-Myc negative. BCAT1
immunostaining in normal or hyperplastic nasopharyngeal
epithelia was similarly assessed. All of the biopsy
samples were detected under the exactly same condition.
Detection of exon mutation of BCAT1 in NPC tissues
The primers for all the 11 exons of BCAT1 were
designed by Primer 5 software and synthesized by
Invitrogen (Shanghai, China). PCR was carried out using
the genomic DNA from NPC tissues and the matched
blood samples as templates. Then the PCR products
were sequenced after being purified. The primer
sequences are listed in Table 3.
Real-time quantitative PCR (qPCR) and reverse
transcription PCR (RT-PCR)
The qPCR was performed on a Bio-Rad iQ5 system
(Hercules, CA) with SYBR Green I PCR kit (TaKaRa,
Dalian, China) to quantitatively analyze BCAT1
amplification in NPC tissues. Three microsatellite loci located
in the BCAT1 gene were selected for this analysis. Gene
amplification was set as 2-C >2.0 compared with
control. Semi-quantitative RT-PCR was performed to detect
the mRNA expression levels of BCAT1 and c-Myc in CN
tissues, NPC tissues and cell lines. Total RNA was
extracted with TRIzol reagent (Invitrogen, Carlsbad, CA).
cDNA was synthesized from DNase I-digested RNA (2 g)
using oligo(dT) as the primer with a commercially available
reverse transcription system (Promega, Madison, WI)
according to the manufacturers protocol.
GlyceraldehydeTable 3 Primers for amplifying 11 exons of BCAT1
3-phosphate dehydrogenase (GAPDH) was amplified as an
internal control. Due to small size of NPC biopsies, the
NPC samples for RT-PCR and IHC were not the same
batch. The primer sequences for qPCR or RT-PCR are
listed in Table 4.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed with EZ-ChIP kit (Millipore,
Darmstadt, Germany). Two specific primers (5-TGGCA
TAGCACTGAAAGG-3 and 5-CTGACTGGCAGTTG
GTTG-3) were used to amplify a 193 bp fragment
containing the predicted c-Myc binding site in the BCAT1
regulatory region. As a negative control, GAPDH was also
amplified with the corresponding primers (5-CGACC
ACTTTGTCAAGCTCA-3 and 5-AGGGGTCTACATG
Plasmids and recombinants
The plasmids including pGL3-control, pGL3-promoter
and pRL-TK used for luciferase reporter gene expression
analysis were purchased from Promega Ltd. Vector for
knocking down c-Myc expression
(pRNAT-U6.1/Si-cMyc) and c-Myc expression vector (pCMV-HA/c-Myc)
were both presented by Dr. Huaying Liu from our
institute. For cloning pRNAT-U6.1/Si-c-Myc, a vector
expressing shRNA, an oligonucleotide encoding a
stemloop structure targeting c-Myc with the targeting
sequence AGACTCTGACACTGTCCA, was designed and
then subcloned into the pRNAT-U6.1 vector (Genscript,
Piscataway, NJ) under the control of the U6 promoter.
Table 4 Summary for primer sequences and product sizes
F: 5'- CTCCGCGTCTACAAAGCTCC-3'
R: 5'- ACCACTCGACTCCACAGTCT-3'
Primers for microsatellite loci of BCAT1
pGL3-233/-41 vector was constructed by amplifying a
193 bp fragment comprising 233 ~ 41 bp upstream of
BCAT1 transcription start site (TSS) which contained
the predicted c-Myc binding site (CACGTG) and
inserting it into pGL3-promoter vector. Meanwhile,
pGL3-233/-41-M vector with a mutated c-Myc binding
site (CGCGTT) in the BCAT1 regulatory region was also
constructed. Key regions in all constructs were verified
by DNA sequencing.
Knockdown of c-Myc in NPC cell lines
5-8F-Si-c-Myc and 6-10B-Si-c-Myc cell lines with
suppressed endogenous c-Myc expression were established
by introducing pRNAT-U6.1/Si-c-Myc vector into 5-8F
cells and 6-10B cells, respectively. For comparison,
58F-vector and 6-10B-vector cell lines were also yielded
by transfecting pRNAT-U6.1 blank vector into 5-8F
cells and 6-10B cells. Stable transfection was performed
using Lipofectamine 2000 reagent (Invitrogen)
following the manufacturers instructions. G418 (500 g/ml,
Millipore) was used to select the stable clones. The mRNA
expression levels of c-Myc, BCAT1, KRAS and MCAM in
NPC cell lines were detected by RT-PCR as previously
described. Since the optimal PCR amplification parameters
of them were not identical, we examined their expressions
in different tubes. GAPDH was used as endogenous
reference gene for normalizing variance in the quality of RNA
and the amount of input cDNA. The same volume of PCR
products was used to be analyzed by electrophoresis on
the same agarose gel. The intensity of each band was
measured by Image Master VDS (Pharmacia Biotech,
Piscataway, NJ) and was analyzed by Bandleader software
version 3.0. The expression levels of c-Myc, BCAT1, KRAS
and MCAM in NPC cells were evaluated after they were
normalized by transforming them into the ratio of the
band intensity of each gene over that of GAPDH of the
same samples. Each sample was repeated in triplicate.
The primer sequences for RT-PCR are listed in Table 4.
Luciferase activity assay
COS7 cells with no endogenous c-Myc expression,
58F-Si-c-Myc cells with inhibited c-Myc expression,
58F-vector cells with endogenous c-Myc expression,
were plated in 24-well plates at a density of 1 104 cells/
well. After 24 hrs, pGL3-233/-41 (or pGL3-233/-41-M),
pCMV-HA/c-Myc and pRL-TK vectors were introduced
into COS7 cells at a ratio of 10:10:1, and pGL3-233/-41
(or pGL3-233/-41-M) and pRL-TK vectors were
cotransfected into 5-8F-Si-c-Myc and 5-8F-vector cells at a
ratio of 10:1 by Fugene 6 transfection reagent (Roche,
Switzerland). Another 36 hrs later, cells were washed
twice, suspended in 500 l reporter lysis buffer (Promega),
and then the firefly luciferase activity was measured using
the dual luciferase reporter assay system and a GloMax
20/20 luminometer (Promega) according to the
manufacturers protocol. The Renilla luciferase vector pRL-TK
(Promega) was co-transfected to standardize transfection
efficiency in each experiment. As a positive control, the
pGL3-control vector was also co-transfected into COS7
cells with pCMV-HA/c-Myc and pRL-TK vectors at a
ratio of 10:10:1 or co-transfected into 5-8-Si-c-Myc and
58F-vector cells with pRL-TK vector at a ratio of 10:1.
Colony formation assay
Colony formation assay was conducted as described in
our published paper  with minor modification. 5-8F,
5-8F-shBCAT1 and 5-8F-vector cells were seeded in
sixwell plates at the density of 700 cells per well. 5-8F-shBCAT1
and 5-8F-vector cells were established in our previous work
. After incubation for 8 days at 37C in a 5% CO2
incubator, the cells were fixed with methanol and stained with
crystal violet. Colonies containing at least 50 cells were
counted under inverse microscope (Nikon, Japan). Colony
formation ratio was also calculated.
Cell migration and invasion assays
The in vitro migration and invasion abilities were
compared between 5-8F-shBCAT1 and 5-8F-vector cells by
using transwell chambers and matrigel-coated invasion
chambers (Corning, Tewksbury, MA). For invasion
assay, 8 m pore transwell inserts coated with matrigel
in cold serum-free media were seeded with 5 104 cells
per well and incubated for 48 hrs. Non-invasive cells on
the upper surface of the filter were removed by wiping
with a cotton swab, and cells that migrated through the
membrane and stuck to the lower surface of the
membrane were fixed with 10% paraformaldehyde and
stained with 0.1% hexamethylpararosaniline for 30 mins.
For quantification, the cells were counted in five
predetermined fields under a microscope. Data were
expressed as the average number of cells migrating
through the filters. The procedures of migration assay
were similar to those described in matrigel invasion
assay except there was no matrigel, the incubation time
was 18 hrs and the fixative was methanol.
Some bioinformatics tools such as Neural Network
Promoter Prediction (NNPP) (http://www.fruitfly.org/seq_tools/
promoter.html) and Transcription Element Search Software
(TESS) (http://www.cbil.upenn.edu/tess) were used to
predict the possible regulatory relationship and interaction
mode between c-Myc and BCAT1. SNPs database (http://
www.ncbi.nlm.nih.gov/snp/) was utilized to discriminate
between mutation and SNP of BCAT1.
Statistical analysis was performed using Wilcoxon rank
sum test, chi-square test, and student t-test. In all
analyses, SPSS 13.0 statistical software was used and the
statistical significance level was set at P < 0.05.
CGH: Comparative genomic hybridization; ChIP: Chromatin
immunoprecipitation; CN: Chronic nasopharyngitis; CSU: Central South
University; HE: Haematoxylin and eosin; IHC: Immunohistochemistry;
NNPP: Neural network promoter prediction; NPC: Nasopharyngeal carcinoma;
qPCR: Real-time quantitative PCR; RNAi: RNA interference; RT-PCR: Reverse
transcription PCR; SNP: Single nucleotide polymorphism; TESS: transcription
element search software; TSG: Tumor suppressor gene; TSS: Transcription
RC designed the general study, wrote the protocols, revised the manuscript
and provided the funding. ZW and FX performed most of the experiments.
JX, HW, Liu Z, Li Z, ZL and WL contributed to administrative, technical or
material support (such as clinical samples collection). ZB, SJ, LJ and LY were
in charge of the literature searches, analyses and partial experiments. WL
undertook the statistical analysis. ZW and HW drafted the manuscript. YK
provided critiques of the manuscript. All authors read and approved the final
This work was supported by National Basic Research Program of China
(2010CB833605), Program for New Century Excellent Talents in University
(NCET-10-0790), Specialized Research Fund for the Doctoral Program of
Higher Education (SRFDP) (20110162120037), National Natural Science
Foundation of China (30801322, 81272972), Foundation of Hunan Provincial
Science and Technology Department (2010FJ3005), Incubation Program for
National Natural Science Funds for Distinguished Young Scholar of Central
South University (2010QYZD006), Open-End Fund for the Valuable and
Precision Instruments of Central South University.
1. Yao KT : The application and prospect of nasopharyngeal carcinoma etiology . China Cancer 1997 , 6 ( 7 ): 3 - 4 .
2. Yung WC , Sham JS , Choy DT , Ng MH : ras mutations are uncommon in nasopharyngeal carcinoma . Eur J Cancer B Oral Oncol 1995 , 31B : 399 - 400 .
3. Lin HS , Berry GJ , Sun Z , Fee WE Jr: Cyclin D1 and p16 expression in recurrent nasopharyngeal carcinoma . World J Surg Oncol 2006 , 4 : 62 .
4. Wu HC , Lu TY , Lee JJ , Hwang JK , Lin YJ , Wang CK , Lin CT : MDM2 expression in EBV-infected nasopharyngeal carcinoma cells . Lab Invest 2004 , 84 : 1547 - 1556 .
5. Guo X , Lui WO , Qian CN , Chen JD , Gray SG , Rhodes D , Haab B , Stanbridge E , Wang H , Hong MH , et al: Identifying cancer-related genes in nasopharyngeal carcinoma cell lines using DNA and mRNA expression profiling analyses . Int J Oncol 2002 , 21 : 1197 - 1204 .
6. Leong JL , Loh KS , Putti TC , Goh BC , Tan LK : Epidermal growth factor receptor in undifferentiated carcinoma of the nasopharynx . Laryngoscope 2004 , 114 : 153 - 157 .
7. Porter MJ , Field JK , Lee JC , Leung SF , Lo D , Van Hasselt CA : Detection of the tumour suppressor gene p53 in nasopharyngeal carcinoma in Hong Kong Chinese . Anticancer Res 1994 , 14 : 1357 - 1360 .
8. Baba Y , Tsukuda M , Mochimatsu I , Furukawa S , Kagata H , Satake K , Koshika S , Nakatani Y , Hara M , Kato Y , Nagashima Y : Reduced expression of p16 and p27 proteins in nasopharyngeal carcinoma . Cancer Detect Prev 2001 , 25 : 414 - 419 .
9. Zhou L , Jiang W , Ren C , Yin Z , Feng X , Liu W , Tao Q , Yao K : Frequent hypermethylation of RASSF1A and TSLC1, and high viral load of EpsteinBarr Virus DNA in nasopharyngeal carcinoma and matched tumoradjacent tissues . Neoplasia 2005 , 7 : 809 - 815 .
10. Peng D , Ren CP , Yi HM , Zhou L , Yang XY , Li H , Yao KT : Genetic and epigenetic alterations of DLC-1, a candidate tumor suppressor gene, in nasopharyngeal carcinoma . Acta Biochim Biophys Sin (Shanghai) 2006 , 38 : 349 - 355 .
11. Yi HM , Li H , Peng D , Zhang HJ , Wang L , Zhao M , Yao KT , Ren CP : Genetic and epigenetic alterations of LTF at 3p21.3 in nasopharyngeal carcinoma . Oncol Res 2006 , 16 : 261 - 272 .
12. Zhang H , Feng X , Liu W , Jiang X , Shan W , Huang C , Yi H , Zhu B , Zhou W , Wang L , et al: Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines . J Cell Biochem 2011 , 112 : 1832 - 1843 .
13. Kwong J , Chow LS , Wong AY , Hung WK , Chung GT , To KF , Chan FL , Daigo Y , Nakamura Y , Huang DP , Lo KW : Epigenetic inactivation of the deleted in lung and esophageal cancer 1 gene in nasopharyngeal carcinoma . Genes Chromosomes Cancer 2007 , 46 : 171 - 180 .
14. Lung HL , Cheng Y , Kumaran MK , Liu ET , Murakami Y , Chan CY , Yau WL , Ko JM , Stanbridge EJ , Lung ML : Fine mapping of the 11q22-23 tumor suppressive region and involvement of TSLC1 in nasopharyngeal carcinoma . Int J Cancer 2004 , 112 : 628 - 635 .
15. Huang Z , Desper R , Schaffer AA , Yin Z , Li X , Yao K : Construction of tree models for pathogenesis of nasopharyngeal carcinoma . Genes Chromosomes Cancer 2004 , 40 : 307 - 315 .
16. Zhou W , Feng X , Li H , Wang L , Zhu B , Zhang H , Yao K , Ren C : Functional evidence for a nasopharyngeal carcinoma-related gene BCAT1 located at 12p12 . Oncol Res 2007 , 16 : 405 - 413 .
17. Ben-Yosef T , Yanuka O , Halle D , Benvenisty N : Involvement of Myc targets in c-myc and N-myc induced human tumors . Oncogene 1998 , 17 : 165 - 171 .
18. Pinkel D , Albertson DG : Array comparative genomic hybridization and its applications in cancer . Nat Genet 2005 , 37 : S11 - S17 .
19. Tao Q , Chan AT : Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments . Expert Rev Mol Med 2007 , 9 : 1 - 24 .
20. Bledsoe RK , Dawson PA , Hutson SM : Cloning of the rat and human mitochondrial branched chain aminotransferases (BCATm) . Biochim Biophys Acta 1997 , 1339 : 9 - 13 .
21. Eden A , Simchen G , Benvenisty N : Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branchedchain amino acid aminotransferases . J Biol Chem 1996 , 271 : 20242 - 20245 .
22. Schuldiner O , Eden A , Ben-Yosef T , Yanuka O , Simchen G , Benvenisty N : ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast . Proc Natl Acad Sci USA 1996 , 93 : 7143 - 7148 .
23. Eden A , Benvenisty N : Involvement of branched-chain amino acid aminotransferase (Bcat1/Eca39) in apoptosis . FEBS Lett 1999 , 457 : 255 - 261 .
24. Rodriguez S , Jafer O , Goker H , Summersgill B , Zafarana G , Gillis AJ , van Gurp RJ , Oosterhuis JW , Lu YJ , Huddart R , et al: Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2-p12 .1. Oncogene 2003 , 22 : 1880 - 1891 .
25. Ben-Yosef T , Eden A , Benvenisty N : Characterization of murine BCAT genes: Bcat1, a c-Myc target, and its homolog, Bcat2 . Mamm Genome 1998 , 9 : 595 - 597 .
26. Weggen S , Preuss U , Pietsch T , Hilger N , Klawitz I , Scheidtmann KH , Wiestler OD , Bayer TA : Identification of amplified genes from SV40 large T antigen-induced rat PNET cell lines by subtractive cDNA analysis and radiation hybrid mapping . Oncogene 2001 , 20 : 2023 - 2031 .
27. Rodenhuis S , Slebos RJ : The ras oncogenes in human lung cancer . Am Rev Respir Dis 1990 , 142 : S27 - 30 .
28. Brodeur GM , Maris JM , Yamashiro DJ , Hogarty MD , White PS : Biology and genetics of human neuroblastomas . J Pediatr Hematol Oncol 1997 , 19 : 93 - 101 .
29. Press MF , Bernstein L , Thomas PA , Meisner LF , Zhou JY , Ma Y , Hung G , Robinson RA , Harris C , El-Naggar A , et al: HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas . J Clin Oncol 1997 , 15 : 2894 - 2904 .
30. Sun D , Zhang Z , Van do N , Huang G , Ernberg I , Hu L : Aberrant methylation of CDH13 gene in nasopharyngeal carcinoma could serve as a potential diagnostic biomarker . Oral Oncol 2007 , 43 : 82 - 87 .
31. Fan CS , Wong N , Leung SF , To KF , Lo KW , Lee SW , Mok TS , Johnson PJ , Huang DP : Frequent c-myc and Int-2 overrepresentations in nasopharyngeal carcinoma . Hum Pathol 2000 , 31 : 169 - 178 .
32. Kong QL , Hu LJ , Cao JY , Huang YJ , Xu LH , Liang Y , Xiong D , Guan S , Guo BH , Mai HQ , et al: Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma . PLoS Pathog 2010 , 6 : e1000940 .
33. Huang GW , Mo WN , Kuang GQ , Nong HT , Wei MY , Sunagawa M , Kosugi T : Expression of p16, nm23-H1, E-cadherin, and CD44 gene products and their significance in nasopharyngeal carcinoma . Laryngoscope 2001 , 111 : 1465 - 1471 .
34. Lung HL , Cheung AK , Xie D , Cheng Y , Kwong FM , Murakami Y , Guan XY , Sham JS , Chua D , Protopopov AI : TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma . Cancer Res 2006 , 66 : 9385 .
35. Peng SP , Li XL , Wang L , Ou-Yang J , Ma J , Wang LL , Liu HY , Zhou M , Tang YL , Li WS , et al: The role of NGX6 and its deletion mutants in the proliferation, adhesion and migration of nasopharyngeal carcinoma 5-8F cells . Oncology 2006 , 71 : 273 - 281 .
36. Yoshikawa R , Yanagi H , Shen CS , Fujiwara Y , Noda M , Yagyu T , Gega M , Oshima T , Yamamura T , Okamura H , et al: ECA39 is a novel distant metastasis-related biomarker in colorectal cancer . World J Gastroenterol 2006 , 12 : 5884 - 5889 .
37. de Bont JM , Kros JM , Passier MM , Reddingius RE , Sillevis Smitt PA , Luider TM , den Boer ML , Pieters R : Differential expression and prognostic significance of SOX genes in pediatric medulloblastoma and ependymoma identified by microarray analysis . Neuro Oncol 2008 , 10 : 648 - 660 .
38. Song LB , Yan J , Jian SW , Zhang L , Li MZ , Li D , Wang HM : [Molecular mechanisms of tumorgenesis and metastasis in nasopharyngeal carcinoma cell sublines] . Ai Zheng 2002 , 21 : 158 - 162 .
39. Wanggou S , Jiang X , Li Q , Zhang L , Liu D , Li G , Feng X , Liu W , Zhu B , Huang W , et al: HESRG: a novel biomarker for intracranial germinoma and embryonal carcinoma . J Neurooncol 2012 , 106 : 251 - 259 .