Download MiR-196a binding-site SNP regulates RAP1A

Carcinogenesis vol.33 no.11 pp.2147–2154, 2012
doi:10.1093/carcin/bgs259
Advance Access publication August 2, 2012
MiR-196a binding-site SNP regulates RAP1A expression contributing to esophageal
squamous cell carcinoma risk and metastasis
Kai Wang1,†, Juan Li1,†, Hong Guo1, Xueqing Xu1,
Gang Xiong2, Xingying Guan1, Botao Liu1, Junxia Li1,
Xuedan Chen1, Kang Yang2 and Yun Bai1,*
1
Department of Medical Genetics, Third Military Medical University,
Chongqing 400038, China and 2Department of Thoracic and Cardiac Surgery,
Southwest Hospital, Third Military Medical University, Chongqing 400038,
China
*
To whom correspondence should be addressed. Tel: +86 23 68752258;
Fax: +86 23 68752224;
Email: [email protected]
Correspondence may also be addressed to Kang Yang.
Tel/Fax: +86 23 68754183;
Email: [email protected]
Polymorphisms in 3′ untranslated region (UTR) of cancer-related
genes might affect regulation by microRNA (miRNA) and contribute to carcinogenesis. In this study, we screened several single nucleotide polymorphisms (SNPs) in 3′UTR of cancer-related
genes and investigated their effects on the risk of esophageal squamous cell carcinoma (ESCC). First, we used SNaPshot assay to
genotype seven 3′UTR SNPs in 537 ESCC cases and 608 normal
controls in a Chinese Han population and found that SNP rs6573
in 3′UTR of RAS-related proteins (RAP1A) was significantly
associated with ESCC risk [P = 0.02, odds ratio (OR) = 0.43;
95% confidence interval (CI): 0.21–0.91] and pathologic stage
(P = 0.03, OR = 1.89; 95% CI: 1.06–3.36). A putative binding
site for miRNA-196a (miR-196a) exists in the 3′UTR of RAP1A,
and the genetic variant, rs6573 A→C, is present in this binding
region. We confirmed that miR-196a regulated the expression of
RAP1A by luciferase reporter assay and that the regulation was
affected by the RAP1A genotype. SNP rs6573 A to C change interfere in the interaction of miR-196a binding to RAP1A 3′UTR,
resulting in higher constitutive expression of RAP1A. Moreover,
we observed that RAP1A was overexpressed in the majority of
ESCC tissues and correlated with RAP1A genotype and lymph
node metastasis. In vitro study indicated RAP1A might function
as a promoter for esophageal cancer cell migration and invasion
through matrix metalloproteinase 2. Our study highlights RAP1A
and SNP rs6573 functioning as potential personal diagnostic and
prognosis markers for ESCC.
Introduction
Esophageal cancer is one of the most aggressive cancers worldwide
and the incidence rate is significantly increasing in recent years.
Overall survival of this cancer is <10% and the 5 years survival rate
is 5–20% after surgery (1). The major reason for this poor prognosis
is that most patients have already had distant metastasis at the time
of diagnosis. After complete surgical removal of primary tumor, the
5 years survival rate is 50–80% for stage I disease, 10–40% for stage
II disease and 10–15% for stage III disease. Patients with distant
metastatic recurrence (stage IV) who are treated with palliative
chemotherapy have a median survival of <1 year (2). Therefore, more
efforts are needed to identify the mechanism underlying esophageal
cancer metastasis.
The molecular mechanisms leading to metastasis of esophageal squamous cell carcinoma (ESCC) have not been fully elucidated, although
several molecules are known to be involved. The expression of mucin
1 and matrix metalloproteinase 13 (MMP13) are strongly correlated
to lymph node metastasis (3). MicroRNAs (miRNAs) are non-proteincoding RNA molecules that can function as tumor suppressors and/or
oncogenes (4). They play key roles in regulating the translation and degradation of messenger RNA (mRNA) genes by sequence complementarity (5–7). It is estimated that about 30% of human genes are regulated
by miRNAs (8). MiRNA-375 inhibits tumor growth and metastasis in
ESCC through repressing insulin-like growth factor 1(9). MiRNA92a promotes lymph node metastasis of human ESCC via decreasing
E-cadherin (10). Single nucleotide polymorphisms (SNPs) located in
miRNA binding sites are probably to affect the expression of miRNA target genes and may contribute to the susceptibility of humans to common
diseases (11), including cancers (12–16). Therefore, we hypothesized
that SNPs in potential miRNA binding sites might affect the susceptibility and progression of ESCC. For this purpose, seven miRNA-binding
site SNPs, namely rs2239680 in baculoviral IAP repeat containing 5,
rs1476215 in fibroblast growth factor 2, rs6573 in RAS-related proteins (RAP1A), rs473698 in transforming growth factor, rs1057035 in
ribonuclease type III, rs1049931 in collagen, type IV, α 2 and rs3757
in DiGeorge syndrome critical region gene 8 (DGCR8), were chosen
by bioinformational tools. An association study between SNPs in the
potential miRNA binding sites and ESCC was performed in the Chinese
Han population. We found that SNP rs6573 in the 3′ untranslated region
(UTR) of the RAP1A gene was significantly associated with ESCC risk
[P < 0.05, odds ratio (OR) = 0.43; 95% confidence interval (CI): 0.21–
0.91) and pathologic stage (P = 0.03, OR = 1.89; 95% CI: 1.06–3.36).
RAP1A is a member of the Ras oncogene family of small G proteins, which play important roles in a variety of cellular processes,
including proliferation, adhesion and cancer progression (17). Recent
studies have indicated that abnormal RAP1A activation can contribute to malignancy via distinct biological effects in different cell types
(18). RAP1A activation promotes epidermal growth factor receptordependent pancreatic carcinoma cell metastasis (19). Activation of
RAP1A promotes prostate cancer metastasis (20). Prior findings suggested that abnormal RAP1A activation can contribute to malignancy
via distinct signal pathways, but the regulation of RAP1A mRNA levels involved in carcinogenesis remains undefined.
In this study, we used a case-control study to demonstrate that the
SNP rs6573 is an important genetic variant for esophageal squamous
cell cancer risk. We also validated that SNP rs6573 was related to
RAP1A expression through affecting miR-196a binding to RAP1A
3′UTR. Finally, we provided evidence that RAP1A gene was an
important regulator of esophageal cancer cell invasion and migration
through MMP2. Our results will provide a new insight into ESCC
tumorigenesis and metastasis by resolving the genetic background.
Materials and methods
Abbreviations: CI, confidence interval; ESCC, esophageal squamous
cell carcinoma; GAP1, GTPase-activating protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; miRNA,
microRNA;NC, negative control; OR, odds ratio; RAP1A, RAS-related proteins; RT-PCR, reverse transcription–polymerase chain reaction; siRNA, small
interfering RNA;SNP, single nucleotide polymorphism; UTR, untranslated
region
†
These authors contributed equally to this work.
Study population and tissue samples
All people were genetically unrelated Han Chinese from Chongqing city of
southwest China. The ESCC patients were histopathologically diagnosed and
confirmed at the Southwest Hospital, the Third Military Medical University.
The exclusion criteria included metastasized cancer and previous radiotherapy
or chemotherapy. The controls were healthy individuals who participated in a
physical examination in Chongqing, who had no history of cancer and who were
frequency matched to cases based on age, sex and residential area. Informed
consent was obtained from the subjects, and the study was performed with the
© The Author 2012. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
2147
K.Wang et al.
approval of the ethical committee of Third Military Medical University. The
interviewers collected the demographic data and environmental exposure history using a questionnaire. An individual who smoked >100 cigarettes in his or
her lifetime was defined an ever smoker. Former smokers were those who had
quit smoking at least 1 year before diagnosis (for cases) or enrollment in this
study (for controls). Recent quitters were those who had quit within 1 year of
diagnosis (for cases) or enrollment in this study (for controls). After the interview, about 5 ml of venous blood was collected from each participant. Tumors
and adjacent non-tumor tissues were collected from patients who underwent
surgery at the Thoracic and Cardiac Surgery of Southwest Hospital.
Esophageal cancer cell lines and plasmids
Human esophageal cancer cell lines EC109 and TE-1 were purchased from
Cell Bank of Chinese Academy of Sciences, Shanghai, China. KYSE150 were
obtained from Cancer Institute and Hospital, Chinese Academy of Medical
Sciences, Beijing, China. The GTPase-activating protein 1 (RAP1GAP1)
plasmid was kindly provided by Dr P.J.Stork (Vollum Institute, Oregon
Health and Science University, Portland, OR) and the enhanced green fluorescent protein (EGFP)-RAP1A plasmid was kindly provided by Dr M.R.Philips
(New York University, New York, NY). The plasmids pcDNA3.1 and pEGFPC1 were purchased from Invitrogen (Carlsbad, CA). The pMIR-REPORT
luciferase miRNA expression reporter vector was purchased from ABI (Foster
City, CA).
SNP selection
First, we used database and literature searches to screen cancer-related genes.
A 3′UTR dataset and a miRNA target dataset of human genes were obtained
from the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgTables).
The miRNA target dataset, developed by Krek et al., contains the human
genes. We used target-scan (http://www.targetscan.org/), miRBase (http://
www.mirbase.org/), miRSNP (http://compbio.uthsc.edu/miRSNP/) and PicTar
(http://pictar.mdc-berlin.de/) to choose the SNPs in the miRNA targeting sites.
We only choose SNPs with a minor allele frequency >5% in the Chinese population based on the HapMap CHB database. The selected SNPs are listed in
Supplementary Table 1, available at Carcinogenesis Online.
Genotyping
We used the SNaPshot assay to genotype the SNPs. The SNaPshot PCR was
run in a 10 µl volume containing 3 µl PCR product mix, 5 µl SNaPshot multiplex kit (ABI), 1 µl primer and 1 µl H2O. The PCR protocol entailed 25 cycles
at 96°C for 10 s, 50°C for 5 s and 60°C for 30 s. These samples and Liz120
(ABI) were separated by capillary electrophoresis using Genetic Analyzer 3130
instrument (ABI). The data were analyzed using the Genemapper software
version 4.0 (ABI). PCR and SNaPshot primers are shown in Supplementary
Table 2, available at Carcinogenesis Online. Genotyping of select SNPs in
esophageal cell lines was performed by direct sequencing.
Luciferase reporter assay
The 3′UTR of the RAP1A gene containing different alleles was amplified and
cloned into the SacI/HindIII site of the pMIR-REPORT Firefly luciferase
reporter vector (Ambion, Grand Island, NY) using standard DNA techniques.
The accuracy of the two plasmid DNA constructs, shown as pMIR-A (for
A allele in 3′UTR of RAP1A) and pMIR-C (for C allele in 3′UTR of RAP1A),
was further identified by sequencing.
For luciferase activity analysis, 293FT and EC109 cells were co-transfected
with 100 ng of luciferase reporter constructs 5 ng of the β-gal control plasmid
and 10 pmol of miRNAs with 1 µl lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, NY). The miRNA mimics that were transfected
into the cells were purchased from GenePharma (Shanghai, China), including the
miR-196a mimics and its negative control (NC). After incubation for 48 h, we carried out the luciferase assay using the luciferase reporter assay system (Promega,
Madison, WI) according to the manufacturer’s protocol. Measurements of luminescence and absorbance of β-gal were performed on a luminometer (Glomax
20/20; Promega) and enzyme-linked immunosorbent assay (Bio-rad, Hercules,
CA) individually. Three independent experiments were performed in triplicate.
RT-PCR
Total RNA was isolated using the RNAiso Plus Kit (TaKaRa, Otsu, Shiga
Japan). Then, 100 ng RNA from each sample was reverse-transcribed into complementary DNA and subjected to conventional PCR (TaKaRa). The PCR primers sequences were as follows: forward 5′-TGTCTCACTGCACCTTCA-3′
and reverse 5′-GACTTCCCAACGCCTCCT-3′. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used for normalization. The analysis was performed in triplicate.
Western blotting
Western blotting was performed using antibodies directed against RAP1
(1:500; Abcam, Cambridge, UK), MMP2 (1:1000; Epitomics, Burlingame,
2148
CA). GAPDH was used for normalization. Quantity One software was used to
compare the intensity of bands on the western blot.
RNA interference
To silence RAP1A expression, we transfected small interfering RNA
(siRNA) targeting RAP1A, using lipofectamine 2000 (Invitrogen, NY).
RAP1A siRNAs were synthesized by GenePharma. The siRNA sequence was
5′-GCAAGACAGTGGTGTAACT-3′ (RAP1A siRNA). A non-related, scrambled siRNA was used as a control.
Cell proliferation assay
The proliferation of EC109 cells was determined using Cell-Light EdU DNA
Cell Proliferation Kit (RiboBio Co. Ltd, Guangzhou, Guangdong, China)
according to manufacturer’s instructions. Briefly, 5 × 103 cells/well were
seeded in a 96 well plate, grown at 37°C for 24 h. Subsequently, cells were
transfected with RAP1A GAP, EGFP-RAP1A, siRNA or their controls in the
presence of 10% fetal bovine serum for 48 h. And then, EdU (50 µM) was
added to the wells. After fixation in 4% paraformaldehyde for 30 min and a
series of rinses in phosphate-buffered saline, the cells were then incubated with
the EdU reaction cocktail for 30 min, followed by rinses in phosphate-buffered
saline with 0.2% Triton X-100. Finally, after 5 min of incubation with Hoechst
33342, the cells were observed under a fluorescent microscope (Olympus,
Tokyo, Japan).
Cell migration and invasion assays
For the invasion assay, 24 well Millicell (8 µm Polyethylene Terephthalate,
PET; Millipore, Bedford, MA) was coated with a 50 µl ECMgel (Sigma, St
Louis, MO). For migration assays, the ECMgel was not needed. After transfection with RAP1A siRNA, EGFP-RAP1, RAP1GAP or their NCs, cells were
incubated in serum-free medium for starvation and then cells were starved and
1 × 105 or 5 × 104 cells in 200 µl Dulbecco’s modified Eagle’s medium were
placed into the upper chamber of the Milliwell for invasion and migration assay,
respectively. Dulbecco’s modified Eagle’s medium containing 20% fetal bovine
serum was added to the bottom well. After incubation for 48 h at 37°C in a CO2
incubator, cells in the top well were removed with cotton swabs. Membranes
were then fixed in 4% paraformaldehyde and then stained with gentian violet.
Finally, the cells on the membranes were counted using a phase-contrast microscope. Six randomly selected fields were counted for each membrane.
Statistical analysis
Differences between cases and controls were evaluated by the Student’s t-test
for continuous variables and the χ2 test for categorical variables. The association between SNPs and ESCC risk was estimated by the OR and 95% CI
using the general genetic model. The potential gene–environment interaction
was evaluated by logistic regression analysis and tested by comparing changes
in deviance (−2log likelihood) between the models of main effects with or
without the interaction term. The χ2 test for Hardy–Weinberg equilibrium was
applied to the SNPs among controls. Comparisons between groups were analyzed by the t test (two-sided). All statistical analyses were performed using
Statistical Package for Social Sciences software (SPSS version 13.0, Chicago,
IL). P-values <0.05 were considered statistically significant.
Results
Association study between SNPs and ESCC
To identify candidate genes that contribute to ESCC, we performed an
association study between seven SNPs located in the potential miRNA
binding site of cancer-related genes and ESCC. The characteristics of
patients and controls are shown in Supplementary Table 3, available at
Carcinogenesis Online. There was no difference between patients and
controls in regard to age, sex and drinking status (P > 0.05). However,
about 66.5% of cases were smokers, which is much higher than the
controls (P < 0.01). In the clinical stage, most patients were divided
into stage II (71.8%) and stage III (24.6%). In the association study,
we found that there were no significant differences between these
SNPs except rs6573 in the RAP1A 3′UTR. Table I shows the allele
frequencies and genotype distributions of these SNPs in the cases and
controls. Multivariate logistic regression analysis of the SNP rs6573
showed that subjects carrying the CC genotype had an increased risk
for ESCC than carrying CA (OR = 0.31; 95% CI: 0.23–0.41) or AA
genotype (OR = 0.43; 95% CI: 0.21–0.91), indicating that C allele is
a risk factor in ESCC.
In addition, we performed stratification analyses according to TNM
(Tumor Node and Metastasis) stages. The risk of ESCC in relation to
Metastasis biomarker and personalized medicine
Table I. Main effect of SNPs on ESCC
Genotype
Cases, N
RAP1A rs6573
CC
AC
AA
BIRC5 rs2239680
TT
CT
CC
FGF2 rs1476215
AA
AT
TT
TGFA rs473698
GG
GC
CC
DICER rs1057035
AA
AG
GG
COL4A2 rs1049931
TT
CT
CC
DGCR8 rs3757
GG
AG
AA
Controls, N
OR (95% CI)a
Pa
444
82
11
368
219
21
1.00 (reference)
0.31 (0.23–0.41)
0.43 (0.21–0.91)
P < 0.01
P = 0.02
325
184
14
381
204
14
1.00 (reference)
1.06 (0.82–1.35)
1.17 (0.55–2.49)
P = 0.7
P = 0.7
467
51
16
531
39
25
1.00 (reference)
1.49 (0.96–2.30)
0.73 (0.38–1.38)
P = 0.08
P = 0.33
304
195
31
290
232
45
1.00 (reference)
0.80 (0.62–1.03)
0.66 (0.40–1.07)
P = 0.08
P = 0.09
207
323
3
222
373
6
1.00 (reference)
0.93 (0.73–1.18)
0.54 (0.13–2.17)
P = 0.55
P = 0.30
78
306
148
83
323
184
1.00 (reference)
1.01 (0.71–1.42)
0.86 (0.59–1.25)
P = 0.96
P = 0.42
75
449
10
97
484
12
1.00 (reference)
1.20 (0.86–1.66)
1.08 (0.44–2.63)
P = 0.27
P = 0.87
BIRC5, baculoviral IAP repeat containing 5; COL4A2, collagen, type IV, α 2; DGCR8, DiGeorge syndrome critical region gene 8; DICER, ribonuclease type III;
FGF2, fibroblast growth factor 2; TGFA, transforming growth factor.
a
Adjusted for age, sex and pack-years of smoking. Significant p-values (<0.05) are in bold.
this SNP was examined with stratification by clinical stages (Table II).
An association between RAP1A genotypes and ESCC clinical status
was detected (P = 0.03, OR = 1.89; 95% CI: 1.06–3.36).
SNP rs6573 affects miR-196a binding to the 3′UTR of RAP1A
SNP rs6573 was significantly associated with ESCC development
and clinical status. MiRNA regulates gene expression by binding
to the 3′UTR of its target gene. In silico analysis showed that SNP
rs6573 lay within a putative binding site of RAP1A for miR-196a.
In addition, the A allele matches the predicted seed region of miR196a, whereas the C allele represents a C:U mismatch base pairing
(Figure 1A). To determine whether miR-196a could regulate the
expression of RAP1A, we performed a luciferase reporter assay. First,
we constructed two different reporter vectors containing RAP1A
3′UTR with the AA or CC genotype, named pMIR-A and pMIR-C,
respectively (Figure 1B). Then, we transfected the two reporter plasmids into 293FT or EC109 cell lines along with a miR-196a mimics
or its NC. We found that luciferase activity significantly decreased
in the presence of the pMIR-A plasmids but not with the pMIR-C
plasmids in both the 293FT and EC109 cell lines (Figure 1C). These
data indicated that miR-196a could suppress luciferase activity with
the A allele, but not with the C allele. Thus, SNP rs6573 may affect
miR-196a binding to the RAP1A 3′UTR.
Table II. Association of rs6573 genotypes and ESCC stage in cases
Genotype
I/II (n = 396),
n (%)
III/IV (n = 141),
n (%)
CC
AC/AA
319 (80.5)
77 (19.5)
125(88.7)
16(11.3)
a
Adjusted for age, sex and smoking.
Pa
ORa (95% CI)
P = 0.03
Reference
1.89
(1.06–3.36)
SNP rs6573 affects inhibition of RAP1A expression induced by
miR-196a
MiR-196a can inhibit a reporter gene driven by the RAP1A 3′UTR and
that the regulation was affected by the RAP1A genotype. To determine
if miR-196a can regulate the expression of RAP1A in esophageal cancer cells, reverse transcription–polymerase chain reaction (RT-PCR)
and western blot analysis were used to detect RAP1A expression after
transfecting the miR-196a mimics or NC into esophageal cancer cells
or normal cells with different genotypes. First, we genotyped cell
lines by direct sequencing. We found that EC109 and KYSE150 cells
are CC homozygote, whereas TE-1 and 293FT are AC heterozygote
(Figure 2A). Then, miR-196a mimics or NC were transfected into
these cells. After 48 h of transfection, we measured RAP1A mRNA
and protein levels. In TE-1 and 293FT cells, we found that miR-196a
significantly reduced the expression of RAP1A compared with the NC
(Figure 2B). However, in EC109 and KYSE150 cells, there was no
obvious reduction in RAP1A expression after miR-196a overexpression (Figure 2C). Taken together, these data showed that miR-196a
could regulate the mRNA and protein expression of RAP1A, and SNP
rs6573 might affect this regulation.
RAP1A expression in ESCC tissues
Because SNP rs6573 was significantly associated with ESCC in the
Chinese Han population, we next examined RAP1A protein levels in tumor tissues and corresponding adjacent non-tumor tissues
from 35 surgical patients by western blot (Figure 3A). We found
that cancerous tissues exhibited significantly higher RAP1A protein
levels compared with corresponding adjacent normal tissues (23/35,
65.7%, Figure 3B), which led us conclude that RAP1A expression was increased in ESCC. In addition, the result showed that the
expression of RAP1A in CC homozygote patients was higher than
that in AC heterozygote and AA homozygote patients (P = 0.006,
Figure 3C). These data indicate that RAP1A might play an oncogenic role in the development of ESCC and SNP rs6473 related to
2149
K.Wang et al.
Fig. 1. Luciferase reporter gene expression assays with constructs containing pMIR-A and pMIR-C. (A) A schematic shows the potential binding site of
RAP1A 3′UTR. M: SNP rs6573 A/C. (B) Schema of the constructs harboring different alleles of miRNA binding sites. (C) MiR-196a mimics or its NC was
co-transfected with the pMIR-REPORT constructs containing A allele or C allele into EC109 or 293FT cell lines. Data shown are the mean fold increase ± SD
from two independent experiments. **P < 0.01.
RAP1A expression in ESCC tissues, which is consistent to in vitro
experiment. We next analyzed RAP1A expression in patients with
different clinical features and found that expression of RAP1A in
patients with lymph node metastasis was higher than that in patients
without lymph node metastasis (P = 0.003, Figure 3D). This further suggested that RAP1A might involve in the regulation of ESCC
metastasis.
RAP1A promotes esophageal cancer cell migration and invasion
in vitro
Migration and invasion of cancer cells are essential steps of cancer
metastasis. Because we found that SNP rs6573 was associated
with the pathologic stage of ESCC and the expression of RAP1A
correlated to ESCC lymph node metastasis, the impact of RAP1A
on esophageal cancer cell migration and invasion was further
investigated using transwell assay. Three approaches were used
to change RAP1A status in EC109 cells: (i) downregulating
RAP1A expression by siRNA; (ii) inhibiting RAP1A activation by
RAP1GAP and (iii) increasing RAP1A levels through expression
vectors (EGFP-RAP1A). After 48 h of transfection, cells were
performed with transwell migration and invasion assay. Compared
with the controls, RAP1A siRNA decreased RAP1A protein
levels and EGFP-RAP1A transfection increased the expression
of RAP1A (Figure 4A). Also, transfection of RAP1A siRNA
resulted in a reduced number (~22%) of migrated cells and RAP1A
overexpression caused an increase in cell migration. EC109 cells
transfected with RAP1GAP showed a relative lower ability of
2150
migration (Figure 4B). In the invasion assay, transfected cells were
seeded into transwell Millicell precoated with ECMgel, which
imitates the extracellular matrix. Cells migrating through the matrix
were stained. The ratio of cells invading through matrigel was
40% less in siRNA-transfected cells than NC cells. In RAP1GAPtransfected cells, number of invasive cells was reduced by ~21%.
And EGFP-RAP1A vector overexpression of RAP1A increased
esophageal cell invasion by 40% (Figure 4C). These results
indicated that RAP1A might have a role in promoting esophageal
squamous cancer cell migration and invasion.
RAP1A promotes esophageal cancer cell migration and invasion
through MMP2
Remodeling of the extracellular matrix is essential for tumor cell invasion and metastasis. Degradation of this matrix requires several matrix
metalloproteinases (MMPs). MMP2 as a downstream molecule of
RAP1A plays a critical role in esophageal cancer cell migration and
invasion. We detected the expression of MMP2 after transfecting of
miR-196a mimics, RAP1A siRNA or EGFP-RAP1A into esophageal
cancer cells. MiR-196a significantly reduced the expression of MMP2
compared with the NC (Supplementary Figure 1A, available at
Carcinogenesis Online). RAP1A siRNA and RAPGAP also reduced
MMP2 expression. Otherwise, EGFP-RAP1A increased the expression of MMP2 compared with the control vector (Supplementary
Figure 1B, available at Carcinogenesis Online). These results indicate
that RAP1A promoted esophageal cancer cell invasion and metastasis, most probably via MMP2.
Metastasis biomarker and personalized medicine
Fig. 2. RT-PCR and western blotting were used to detect mRNA and protein expression of RAP1A after transfection of miRNA-196a into cells with different
genotypes. (A) Using sequencing technology to genotype the SNP in EC109, KYSE150 (CC homozygote) and TE-1, 293FT (AC heterozygote). (B) Cells with
AC heterozygote transfected with miRNA-196a mimics or its NC. After 48 h of transfection, the cells were harvested for measurements of mRNA and protein
expression of RAP1A using RT-PCR and western blot analysis, respectively. GAPDH served as a control. (C) Cells with CC homozygote transfected with
miRNA-196a mimics or NC. After 48 h of transfection, cells were harvested for measurement of mRNA and protein expression of RAP1A using RT-PCR and
western blot analysis, respectively. GAPDH served as a control. *P < 0.05.
Discussion
Because ESCC is one of the most aggressive cancers with very
poor prognosis, a clear understanding of the genetic factors for the
development and metastasis of this cancer is very important. In this
study, we screened genetic variations in the 3′UTR of cancer-related
genes by a population-based association study approach and identified
SNP rs6573 located in the binding site of miR-196a for RAP1A was
significantly associated with susceptibility and metastasis of ESCC
in a Chinese Han population. Though there have been many ESCC
association studies, including the genome wide association study
(21,22). SNPs involved in ESCC development and metastasis have
remained unclear. Our research suggested a novel potential marker
for predicting the susceptibility and malignancy of ESCC. To our
knowledge, this is the first study evaluating the relationship between
SNP rs6573 and human disease.
RAP1A has been reported to implicate in a wide range of biological
processes, from cell proliferation, differentiation to cell mobility.
Numerous reports have implied that abnormal RAP1A activation
contributed to the tumorigenic processes. Expression of RAP1 at
high levels can morphologically transform Swiss 3T3 fibroblasts and
formed tumors when injected into nude mice (23). In our study, we
found that overexpression of RAP1A existed in most ESCC tissues
and expression of RAP1A was higher in patients with lymph node
metastasis than those without. In vitro study indicated RAP1A
might function as a promoter for esophageal cancer cell migration
and invasion. However, we have not detected any role for RAP1A in
regulating cell proliferation of esophageal cancer cells (Supplementary
Figures 2A–C). Thus, we suggest that RAP1A might play an
oncogenic role in the development of ESCC. Abnormal activation
of RAP1A has also been observed in other type of cancer, such as
melanoma (24), cervical cancer (25), papillary thyroid cancer (26),
squamous cell carcinoma (27), thyroid cancer (28), colon cancer (29)
and pancreatic cancer (30). However, the impact of RAP1A on cancer
cell migration and metastasis was not consistent. Inhibition of RAP1A
reduced invasion of pancreatic cancer cells (19) and thyroid cancer
(31). Activation of RAP1A was shown to promote prostate cancer
metastasis (20). Nevertheless, inhibition of RAP1A has been reported
to correlate with increased invasion of squamous cell carcinoma in
an in vitro study (32). Our results showed that RAP1A promoted
esophageal cancer cell migration and invasion. These opposing
findings indicate that activation of RAP1A may play different roles
in different cancers. MMP2 plays an important role in esophageal
cancer metastasis (33). Previous studies have demonstrated that
2151
K.Wang et al.
Fig. 3 Expression of RAP1A protein in cancer tissues and adjacent non-tumor tissues of 35 ESCC patients. (A) Western blotting was used to measure the protein
expression of RAP1A in esophageal cancer patients and adjacent non-tumor tissues. Ca: cancer tissues. N: non-tumor tissues. (B) The relative expression level
of RAP1A was measured by western blotting. C (cancer)/N (non-cancer) relative protein expression ratios between normalized values were calculated. + and –
indicate that the expression is higher or lower, respectively, in the cancer tissue than in the respective adjacent non-cancer tissue. (C) Gray scale of the western
blot bands indicates the expression of RAP1A related to GAPDH in different genotypes. (D) Gray scale of the western blot bands indicates the expression of
RAP1A related to GAPDH in non-lymph node metastasis (N0) and lymph node metastasis (Nx) patient tissues. GAPDH served as a control.
Fig. 4. RAP1A promotes esophageal cancer cell migration and invasion. (A) RAP1A expression was measured by western blot analysis after transfection with
EGFP-RAP1, control vector EGFP and RAP1A siRNA into the EC109 cell line. GAPDH served as a control. (B) Migration assays of RAP1 siRNA-treated,
RAP1GAP-treated and EGFP-RAP1-treated EC109 cells. (C) Invasion assays of RAP1 siRNA-treated, RAP1GAP-treated and EGFP-RAP1-treated EC109 cells.
Experiments were performed in duplicate and at least six fields were counted. *P < 0.05.
2152
Metastasis biomarker and personalized medicine
RAP1GAP promotes invasion through MMP2 and MMP9 secretion,
which is associated with poor survival in squamous cell carcinoma
(34). Our results also demonstrate that RAP1A promotes esophageal
cancer cell migration and invasion, potentially by regulating MMP2
protein level. Therefore, we first showed the role of RAP1A during
the carcinogenesis of esophageal squamous cell cancer that RAP1A
probably function as a promoter of metastasis of ESCC.
MiRNAs regulate gene expression by mRNA degradation or
by reducing the translation of target mRNAs. Our study has added
RAP1A mRNA as one more target of miR-196a, which has also been
implicated in targeting HOX gene family (35). Further study provided
confirmation of its role in regulation of HOX gene expression (36).
The elevated expression of HOX genes in leukemogenesis is very well
confirmed and this oncogene had myeloid differentiation capacities
(37). In this study, we found that miR-196a could inhibit the expression of RAP1A. Although RAP1A was related to increase MMP2
levels and promote invasive ability of ESCC cells. This suggests miR196a might be prone to suppress the development of ESCC, which
was similar to the role of miR-196a during leukemogenesis. However,
there were also some studies showing that increase of miR-196a levels was the character of breast cancers (38), esophageal adenocarcinoma (39) and pancreatic adenocarcinoma (40). As a single miRNA
can target many genes, the functions of any miRNA as oncogenic or
tumor suppressive might be cancer type-specific and dependent on
the target genes whose expressions it may be regulating. In previous
study, Maru et al. found that miR-196a is a potential marker of progression during Barrett’s metaplasia–dysplasia–invasive adenocarcinoma sequence in esophagus (41). In our previous study, a SNP in the
pre-miR-196a was associated with susceptibility of ESCC risk in this
Chinese Han population (42). These data provided some evidence that
miR-196a could play some roles in esophageal cancer.
SNP rs6573 located in 3′UTR of RAP1A flanking the binding site
for miR-196a and SNP A to C change interfere in the interaction of
miR-196a binding to RAP1A 3′UTR, resulting in higher constitutive
expression of RAP1A. Because we suggest RAP1A probably function
as an oncogene in the development of ESCC, SNP A to C change would
be expected to promote the development of ESCC, which is consistent
with the results of association study that individuals carrying the rs6573
C allele had an increased risk for the development and metastasis of
ESCC.
Conclusion
In conclusion, we found that SNP rs6573 in the miR-196a binding site of RAP1A is associated with the risk of ESCC. This SNP
C allele may prevent miR-196a from binding to RAP1A mRNA,
resulting in altered regulation of RAP1A expression and increased
ESCC risk and metastasis. RAP1A could promote esophageal cancer cell migration and invasion through MMP2 (Supplementary
Figure 3, available at Carcinogenesis Online). Our results provide
a new insight into ESCC tumorigenesis and metastasis and have
potential implications in individual treatment and prevention of this
disorder.
Supplementary material
Supplementary Tables 1–3 and Figures 1–3 can be found at http://
carcin.oxfordjournals.org/
Funding
National Natural Science Foundation of China (30971603) and
Natural Science Foundation Project of CQ CSTC (2009BA5013).‍
Acknowledgements
The authors would like to acknowledge the patients and healthy controls participated in this study.
Conflict of Interest Statement: None declared.
References
1.Jemal, A. et al. (2011) Global cancer statistics. CA. Cancer J. Clin., 61,
69–90.
2.Stahl, M. et al.; ESMO Guidelines Working Group. (2010) Esophageal
cancer: Clinical Practice Guidelines for diagnosis, treatment and follow-up.
Ann. Oncol., 21 Suppl 5, v46–v49.
3.Ye, Q. et al. (2011) MUC1 induces metastasis in esophageal squamous cell
carcinoma by upregulating matrix metalloproteinase 13. Lab. Invest., 91,
778–787.
4.Esquela-Kerscher, A. et al. (2006) Oncomirs - microRNAs with a role in
cancer. Nat. Rev. Cancer, 6, 259–269.
5.Berezikov, E. et al. (2005) Phylogenetic shadowing and computational
identification of human microRNA genes. Cell, 120, 21–24.
6.Pillai, R.S. (2005) MicroRNA function: multiple mechanisms for a tiny
RNA? RNA, 11, 1753–1761.
7.Zamore, P.D. et al. (2005) Ribo-gnome: the big world of small RNAs.
Science, 309, 1519–1524.
8.Carthew, R.W. (2006) Gene regulation by microRNAs. Curr. Opin. Genet.
Dev., 16, 203–208.
9.Kong, K.L. et al. (2012) MicroRNA-375 inhibits tumour growth and
metastasis in oesophageal squamous cell carcinoma through repressing
insulin-like growth factor 1 receptor. Gut, 61, 33–42.
10. Chen, Z.L. et al. (2011) microRNA-92a promotes lymph node metastasis of human esophageal squamous cell carcinoma via E-cadherin. J. Biol.
Chem., 286, 10725–10734.
11. Ryan, B.M. et al. (2010) Genetic variation in microRNA networks: the
implications for cancer research. Nat. Rev. Cancer, 10, 389–402.
12. Liu, Z. et al. (2011) A functional variant at the miR-184 binding site in
TNFAIP2 and risk of squamous cell carcinoma of the head and neck.
Carcinogenesis, 32, 1668–1674.
13. Zhang, S. et al. (2012) REV3L 3’UTR 460 T>C polymorphism in microRNA target sites contributes to lung cancer susceptibility. Oncogene.
14. Sethupathy, P. et al. (2008) MicroRNA target site polymorphisms and
human disease. Trends Genet., 24, 489–497.
15. Xiong, F. et al. (2011) Genetic variation in an miRNA-1827 binding site
in MYCL1 alters susceptibility to small-cell lung cancer. Cancer Res., 71,
5175–5181.
16. Teo, M.T. et al. (2012) The role of microRNA-binding site polymorphisms
in DNA repair genes as risk factors for bladder cancer and breast cancer
and their impact on radiotherapy outcomes. Carcinogenesis, 33, 581–586.
17. Hattori, M. et al. (2003) Rap1 GTPase: functions, regulation, and malignancy. J. Biochem., 134, 479–484.
18. Kometani, K. et al. (2004) Rap1 and SPA-1 in hematologic malignancy.
Trends Mol. Med., 10, 401–408.
19. Huang, M. et al. (2012) EGFR-dependent pancreatic carcinoma cell metastasis through Rap1 activation. Oncogene, 31, 2783–2793.
20. Bailey, C.L. et al. (2009) Activation of Rap1 promotes prostate cancer
metastasis. Cancer Res., 69, 4962–4968.
21. Wang, L.D. et al. (2010) Genome-wide association study of esophageal
squamous cell carcinoma in Chinese subjects identifies susceptibility loci
at PLCE1 and C20orf54. Nat. Genet., 42, 759–763.
22. Abnet, C.C. et al. (2010) A shared susceptibility locus in PLCE1 at 10q23
for gastric adenocarcinoma and esophageal squamous cell carcinoma. Nat.
Genet., 42, 764–767.
23. Altschuler, D.L. et al. (1998) Mitogenic and oncogenic properties of the
small G protein Rap1b. Proc. Natl. Acad. Sci. U.S.A., 95, 7475–7479.
24. Gao, L. et al. (2006) Ras-associated protein-1 regulates extracellular
signal-regulated kinase activation and migration in melanoma cells: two
processes important to melanoma tumorigenesis and metastasis. Cancer
Res., 66, 7880–7888.
25. Singh, L. et al. (2003) The high-risk human papillomavirus type 16 E6
counters the GAP function of E6TP1 toward small Rap G proteins. J. Virol.,
77, 1614–1620.
26. De Falco, V. et al. (2007) RET/papillary thyroid carcinoma oncogenic signaling through the Rap1 small GTPase. Cancer Res., 67, 381–390.
27. Zhang, Z. et al. (2006) Rap1GAP inhibits tumor growth in oropharyngeal
squamous cell carcinoma. Am. J. Pathol., 168, 585–596.
28. Tsygankova, O.M. et al. (2007) Downregulation of Rap1GAP contributes
to Ras transformation. Mol. Cell. Biol., 27, 6647–6658.
29. Tsygankova, O.M. et al. (2010) Downregulation of Rap1GAP in human
tumor cells alters cell/matrix and cell/cell adhesion. Mol. Cell. Biol., 30,
3262–3274.
2153
K.Wang et al.
30. Fralix, K.D. et al. (2003) Rap1 reverses transcriptional repression of TGFbeta type II receptor by a mechanism involving AP-1 in the human pancreatic cancer cell line, UK Pan-1. J. Cell. Physiol., 194, 88–99.
31. Zuo, H. et al. (2010) Downregulation of Rap1GAP through epigenetic
silencing and loss of heterozygosity promotes invasion and progression of
thyroid tumors. Cancer Res., 70, 1389–1397.
32. Yajnik, V. et al. (2003) DOCK4, a GTPase activator, is disrupted during
tumorigenesis. Cell, 112, 673–684.
33. Kataoka, M. et al. (1996) Matrix metalloproteinase 2 and 9 in esophageal
cancer. Int. J. Oncol., 8, 773–779.
34.Mitra, R.S. et al. (2008) Rap1GAP promotes invasion via induction
of matrix metalloproteinase 9 secretion, which is associated with poor
survival in low N-stage squamous cell carcinoma. Cancer Res., 68,
3959–3969.
35. Yekta, S. et al. (2004) MicroRNA-directed cleavage of HOXB8 mRNA.
Science, 304, 594–596.
36. Debernardi, S. et al. (2007) MicroRNA miR-181a correlates with
morphological sub-class of acute myeloid leukaemia and the expression of its target genes in global genome-wide analysis. Leukemia, 21,
912–916.
2154
37. Thorsteinsdottir, U. et al. (2002) Overexpression of the myeloid leukemiaassociated Hoxa9 gene in bone marrow cells induces stem cell expansion.
Blood, 99, 121–129.
38. Hoffman, A.E. et al. (2009) microRNA miR-196a-2 and breast cancer: a
genetic and epigenetic association study and functional analysis. Cancer
Res., 69, 5970–5977.
39. Luthra, R. et al. (2008) MicroRNA-196a targets annexin A1: a microRNAmediated mechanism of annexin A1 downregulation in cancers. Oncogene,
27, 6667–6678.
40. Bloomston, M. et al. (2007) MicroRNA expression patterns to differentiate
pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis.
JAMA, 297, 1901–1908.
41. Maru, D.M. et al. (2009) MicroRNA-196a is a potential marker of progression during Barrett’s metaplasia-dysplasia-invasive adenocarcinoma
sequence in esophagus. Am. J. Pathol., 174, 1940–1948.
42. Wang, K. et al. (2010) A functional variation in pre-microRNA-196a is
associated with susceptibility of esophageal squamous cell carcinoma risk
in Chinese Han. Biomarkers, 15, 614–618.
Received May 20, 2012; revised July 15, 2012; accepted July 26, 2012