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Overexpression of Snail is associated with lymph node metastasis and poor prognosis in patients with gastric cancer

  • Na Ri Shin1, 4,
  • Eun Hui Jeong5,
  • Chang In Choi3, 4,
  • Hyun Jung Moon1, 4,
  • Chae Hwa Kwon1, 4,
  • In Sun Chu6,
  • Gwang Ha Kim2, 4,
  • Tae Yong Jeon3, 4,
  • Dae Hwan Kim3, 4,
  • Jae Hyuk Lee5 and
  • Do Youn Park1, 4Email author
Contributed equally
BMC Cancer201212:521

DOI: 10.1186/1471-2407-12-521

Received: 17 July 2012

Accepted: 12 November 2012

Published: 14 November 2012

Abstract

Background

Epithelial–mesenchymal transition (EMT) plays a significant role in tumor progression and invasion. Snail is a known regulator of EMT in various malignant tumors. This study investigated the role of Snail in gastric cancer.

Methods

We examined the effects of silenced or overexpressed Snail using lenti-viral constructs in gastric cancer cells. Immunohistochemical analysis of tissue microarrays from 314 patients with gastric adenocarcinoma (GC) was used to determine Snail’s clinicopathological and prognostic significance. Differential gene expression in 45 GC specimens with Snail overexpression was investigated using cDNA microarray analysis.

Results

Silencing of Snail by shRNA decreased invasion and migration in GC cell lines. Conversely, Snail overexpression increased invasion and migration of gastric cancer cells, in line with increased VEGF and MMP11. Snail overexpression (≥75% positive nuclear staining) was also significantly associated with tumor progression (P < 0.001), lymph node metastases (P = 0.002), lymphovascular invasion (P = 0.002), and perineural invasion (P = 0.002) in the 314 GC patients, and with shorter survival (P = 0.023). cDNA microarray analysis revealed 213 differentially expressed genes in GC tissues with Snail overexpression, including genes related to metastasis and invasion.

Conclusion

Snail significantly affects invasiveness/migratory ability of GCs, and may also be used as a predictive biomarker for prognosis or aggressiveness of GCs.

Keywords

Stomach Adenocarcinoma Snail Lymph node metastasis Survival

Background

Epithelial–mesenchymal transition (EMT), a developmental process whereby epithelial cells reduce intercellular adhesion and acquire myofibroblastic features, is critical to tumor progression [13]. During EMT, significant changes occur, including downregulation of epithelial markers such as E-cadherin, translocation of β-catenin (i.e., dissociation of membranous β-catenin and translocation into the nuclear compartment), and upregulation of mesenchymal markers such as vimentin and N-cadherin [36]. EMT is induced by repression of E-cadherin expression by EMT regulators such as Snail, Slug, and Twist. The Snail family of zinc-finger transcriptional repressors directly represses E-cadherin in vitro and in vivo via an interaction between their COOH-terminal region and the 5-CACCTG-3 sequence in the E-cadherin promoter [79]. Snail is reportedly important in several carcinomas, including non-small cell lung carcinomas, ovarian carcinomas, urothelial carcinomas, and hepatocellular carcinoma [1013]. Studies have also used immunohistochemical analyses to show the clinical significance of Snail overexpression in gastric adenocarcinoma (GC) [14, 15]. However, few reports on the roles of Snail in GC have included clinicopathological, prognostic, and functional in vitro analyses as well as gene expression results. We therefore evaluated Snail’s effect on invasiveness/migratory ability in gastric cancer cell lines, and also investigated the possibility of Snail being used as a predictive marker for evaluating poor prognosis or tumor aggressiveness in GC patients. We also evaluated the gene expression pattern in 45 GC tissues with Snail overexpression, using cDNA microarrays.

Methods

shRNA lentivirus-mediated silencing and overexpression of Snail in gastric cancer cells

Human gastric cancer cell lines SNU216 and SNU484 were obtained from Korean Cell Line Bank (KCLB) and were authenticated by DNA profiling. These cells cultured in RPMI1640 medium with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (hyClone, Ogden, UT). All cells were maintained at 37°C in 5% CO2. Lentiviral-based RNA knockdown and overexpression were used for silencing and overexpression of Snail. Lentiviruses expressing either non-target or Snail-targeted shRNAs were used for silencing; a PLKO lentiviral vector targeting Snail or an empty PLKO vector were used for overexpression of Snail in the SNU216 and SNU484 cells. Lentivirus stocks were produced using the Virapower™ lentiviral packaging mix using the 293FT cell line according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). SNU216 and SNU484 cells grown to 50% confluence were incubated for 24 h in a 1:1 dilution of virus:media with 5 μg/ml Polybrene. After a 24-h recovery period in complete media without virus, polyclonal stable cell lines were selected and maintained in media containing 5 μg/ml puromycin. Silencing or overexpression of Snail was determined by RT-PCR and western blotting.

Real time RT-PCR analysis of VEGF, MMP11, and Snailin gastric cancer cells

Total cellular RNA was extracted using the TRIzol method (Sigma-Aldrich, St Louis, MO, USA). For RT-PCR analysis, 2-μg aliquots of RNA were subjected to cDNA synthesis with 200 U of MMLV reverse transcriptase and 0.5 μg of oligo(dT)-15 primer (Promega, Madison, WI, USA). Quantitative real-time PCR was performed with the Rotor-Gene™ System (QIAGEN, Hilden, Germany) using AccuPower 2× Greenstar qPCR Master Mix (Bioneer, Daejeon, Korea). cDNA in 1 μl of the reaction mixture was amplified with 0.5 U of GoTaq DNA polymerase (Promega) and 10 pmol each of the following sense and antisense primers: GAPDH 5-TCCATGACAACTTTGGTATCG-3, 5-TGTAGCCAAATTCGTTGTCA-3; Snail 5-CTTCCTCTCCATACCTG-3, 5-CATAGTTAGTCACACCTCGT-3; VEGF 5-TTGCTGCTCTACCTCCACCA-3, 5-GCACACAGGATGGCTTGAA-3; MMP11 5-CTTGGCTGCTGTTGTGTGCT-3, 5-AGGTATGGAGCGATGTGACG-3. The thermal cycling profile was: denaturation for 30 s at 95°C, annealing for 30 s at 52°C (depending on the primers used), and extension for 30 s at 72°C. For semi-quantitative assessment of expression levels, 30 cycles were used for each PCR reaction. PCR products were size-fractionated on 1.0% ethidium bromide/agarose gels and quantified under UV transillumination. The threshold cycle (CT) is defined as the fractional cycle number at which the fluorescence passes a fixed threshold above baseline. Relative gene expression was quantified using the average CT value for each triplicate sample minus the average triplicate CT value for GAPDH. Differences between the control (empty vector) and experiment groups (infected with the lentivirus) were calculated using the formula 2 – ([CT Lenti] – [CT control]) and expressed as a fold change in expression according to the comparative threshold cycle method (2–CT) [16].

Western blotting

Cells were harvested and disrupted in lysis buffer (1% Triton X-100, 1mM EGTA, 1mM EDTA, 10mM Tris–HCl, pH 7.4 and protease inhibitors). Cell debris was removed by centrifugation at 10,000 × g for 10 min at 4°C. The resulting supernatants were resolved on a 12% SDS-PAGE under denatured reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk at room temperature for 30 min and incubated with primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. The signal was visualized using an enhanced chemiluminescence (Amersham, Buckinghamshire, UK).

Cell migration and Matrigel invasion assay

Gastric cancer cells were harvested with 0.05% trypsin containing 0.02% EDTA (Sigma-Aldrich), and suspended in RPMI at a concentration of 3 × 103 cells/well. Membrane filters (pore size: 8 μm) in disposable 96-well chemotaxis chambers (Neuro Probe, Gaithersburg, MD) were pre-coated for 4 h with 5 mg/ml fibronectin at room temperature. Aliquots (50 μl/well) of the cell suspension were loaded into the upper chambers, and 1% FBS was loaded into the lower chamber. After 24-h incubation, non-migrating cells were removed from the upper chamber with a cotton swab; cells present on the lower surface of the insert were stained with Hoechst33342 (Sigma-Aldrich). Invasive cells were counted under a fluorescence microscope at × 10 magnification.

For the Matrigel invasion assay, 3 × 104 cells/well were seeded in the upper chamber, which was coated with Matrigel (5 mg/ml in cold medium, BD Transduction Laboratories, Franklin Lakes, NJ, USA), and serum-free medium containing 1% FBS or control vehicle was added to the lower chamber. After 24-h incubation, non-migrating cells were removed from the upper chamber with a cotton swab, and cells present on the lower surface of the insert were stained with Hoechst33342 (Sigma-Aldrich). Invasive cells were then counted under a fluorescence microscope at × 10 magnification.

Tissue microarrays, immunohistochemistry, and interpretation of results

A semi-automated tissue arrayer (Beecher Instruments, WI, USA) was used to construct the tissue microarrays. We obtained 3 tissue cores, each 0.6 mm in diameter, from tumor blocks taken from GC patients. Cores were not collected from the more invasive frontal or central areas of the tumors. Slides were baked at 60°C for 30 min, deparaffinized with xylene, and then rehydrated. The sections were subsequently submerged in citrate antigen retrieval buffer, microwaved for antigen retrieval, treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, and then incubated with 1% bovine serum albumin to block non-specific binding. Thereafter, the sections were incubated with rabbit anti-Snail (Abcam, UK) overnight at 4°C. Normal rabbit serum was used as a negative control. After washing, tissue sections were treated with secondary antibody, counterstained with hematoxylin, dehydrated, and mounted. At least 500 tumor cells were counted. The percentage of cells with Snail+ nuclei was expressed relative to the total number of tumor cells counted. Nuclear expression of Snail was graded by classifying the extent of positive nuclear staining as ≤50%, 50–75%, or ≥75%.

Clinicopathological and survival analysis of gastric cancer patients

We studied a cohort of 314 GC patients who each underwent a gastrostomy with lymph node dissection at Pusan National University Hospital (PNUH) between 2005 and 2007. The group comprised 218 men and 96 women with a mean age of 58.3 years (range, 25–83 years). Standard formalin-fixed and paraffin-embedded sections were obtained from the Department of Pathology, PNUH, and the National Biobank of Korea, PNUH. The study was approved by the Institutional Review Board. None of the patients received preoperative radiotherapy and/or chemotherapy. Adjuvant chemotherapy based on 5-FU was administered on patients with stages II, III and IV after curative resection. We assessed several clinicopathological factors according to the Korean Standardized Pathology Report for Gastric Cancer, the Japanese Classification of Gastric Carcinoma (3rd English edition), and the American Joint Committee on Cancer Staging Manual (7th edition), including tumor site, gross appearance and size, depth of invasion, histological classification (i.e., intestinal or diffuse), and lymphovascular invasion [1719]. Clinical outcome for each patient was followed from the date of surgery to the date of death or March 1, 2012. Follow-up periods ranged from approximately 1 to 81.5 months (average, 51.4 months). Cases lost to follow-up or death from any cause other than gastric cancer were censored from the survival rate analysis. Clinicopathological features were analyzed using Student’s t-test, the χ2 test, or Fisher’s exact test to test for differences in Snail expression. Cumulative survival plots were obtained using the Kaplan–Meier method, and significance was compared using the log-rank test. Prognostic factors were identified using the Cox regression stepwise method (proportional hazard model), adjusted for the patients’ age, gender, tumor site, morphologic type (intestinal versus diffuse). Statistical significance was set at P < 0.05. Statistical calculations were performed with SPSS version 10.0 for Windows (SPSS Inc., Chicago, IL, USA).

cDNA microarray analysis of GC tissues based on Snail overexpression

A total of 45 fresh GC tissues were obtained from the National Biobank of Korea, PNUH, and CNUH; approval was obtained from their institutional review boards. Total RNA was extracted from the fresh-frozen tissues using a mirVana RNA Isolation kit (Ambion Inc., Austin, TX). Five hundred nanograms of total RNA was used for cDNA synthesis, followed by an amplification/labeling step (in vitro transcription) using the Illumina TotalPrep RNA Amplification kit (Ambion) to synthesize biotin-labeled cRNA. cRNA concentrations were measured by the RiboGreen method (Quant-iT RiboGreen RNA assay kit; Invitrogen-Molecular Probes, ON, Canada) using a Victor3 spectrophotometer (PerkinElmer, CT), and cRNA quality was determined on a 1% agarose gel. Labeled, amplified material (1500 ng per array) was hybridized to Illumina HumanHT-12 BeadChips v4.0, according to manufacturer’s instructions (Illumina, San Diego, CA). Array signals were developed by streptavidin-Cy3. Arrays were scanned with an Illumina iScan system. The microarray data were normalized using the quantile normalization method in Illumina BeadStudio software. The expression level of each gene was transformed into a log2 base before further analysis. Excel was primarily used for statistical analyses. Gene expression differences were considered statistically significant if P < 0.05; all tests were 2-tailed. Cluster analyses were performed using Cluster and Treeview [20]. The gene ontology (GO) program (http://david.abcc.ncifcrf.gov/) was used to categorize genes into subgroups based on biological function. Fisher’s exact test was used to determine whether the proportions of genes in each category differed by group. GC tissues were further divided into those with higher (≥75%) and lower (<75%) levels of Snail expression; differential gene expression between the groups was identified. Primary microarray data are available in NCBI’s GEO (Gene Expression Omnibus) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE38024).

Results

Regulation of migration and invasion of gastric cancer cells by Snail

Lentiviral-based RNA knockdown and overexpression approaches were used to determine Snail’s role in invasion and migration of gastric cancer cell lines. SNU216 and SNU484 cells used in this study are established gastric adenocarcinoma cell lines from Korean patients. These cells were infected with a lentivirus expressing either non-target or Snail-targeted shRNAs for silencing. A PLKO lentiviral vector that targeted Snail and an empty PLKO vector were used to induce Snail overexpression in SNU216 and SNU484 cells. Polyclonal stable cell lines were selected using puromycin. Snail expression was determined by RT-PCR and western blotting; stable Snail knockdown (sh-Snail) and Snail overexpression cell lines (OE-Snail) were obtained (Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-521/MediaObjects/12885_2012_Article_3610_Fig1_HTML.jpg
Figure 1

Role of Snail in invasion and migration of gastric cancer cell lines. A. SNU216 (upper panel) and SNU484 (lower panel) cells were infected with lentiviruses expressing either non-target shRNA (shNT) or Snail shRNA on day 0, and then harvested on day 7 post-infection. Snail knockdown was determined by RT-PCR and western blotting; stable cell lines were generated for each of the cell lines (sh-Snail). Silencing of Snail in SNU216 and SNU484 cells induced decreased migration and invasion. B. SNU216 (upper panel) and SNU484 (lower panel) cells were infected with lentiviruses expressing either a lentiviral PLKO vector targeting Snail or an empty PLKO vector (EV) on day 0, and then harvested on day 7 post-infection. The overexpression of Snail was determined by RT-PCR and western blotting; stable cell line was generated for each of the cell lines (O/E-snail). Snail overexpression in SNU216 and SNU484 cells induced increased migration and invasion. C. Snail overexpression induced increased mRNA expression of VEGF and MMP11 in SNU216 and SNU484 cells in real-time RT-PCR analysis. Lower panel indicates representative RT-PCR figures for VEGF, MMP11, Snail, and GAPDH. Data show the mean ± SE of at least 3 independent experiments. * indicates P < 0.05 by Student’s t-test.

To determine Snail’s roles in gastric cancer cell invasion, we measured chemotactic invasion by the cells using the Transwell system with filters pre-coated with Matrigel. To measure migration of gastric cancer cells, we assayed cell migration using a Boyden chamber apparatus. Silencing of Snail by shRNA induced decreased migration and invasion of SNU216 and SNU484 cells, as shown in Figure 1A. In contrast to the Snail silencing results, overexpression of Snail induced increased migration and invasion of SNU216 and SNU484 cells, as shown in Figure 1B. Overexpression of Snail was also associated with increased VEGF and MMP11 (Figure 1C).

Effect of Snail overexpression on tumor aggressiveness and GC patient survival

Positive nuclear staining for Snail at levels of ≤50%, 50–75%, and ≥75% was observed in 13.4% (42/314), 52.2% (164/314), and 34.4% (108/314), respectively, of the 314 GC patients in immunohistochemical analysis. Snail expression was noted in intestinal and diffuse type of GCs (Figure 2A, B). Snail overexpression (≥75% positivity) significantly correlated with tumor size, gross type, depth of invasion, lymphovascular invasion, perineural invasion, and lymph node metastasis (Table 1). Snail overexpression was also associated with increased tumor size (P = 0.028) and excavated gross type (P< 0.001); and increased tumor invasiveness, i.e., higher T stage (P< 0.001) and the presence of perineural invasion (P< 0.001) and lymphovascular tumor emboli (P = 0.002). Increased lymph node metastasis was also related to Snail overexpression (P = 0.002).In accordance with the above data showing the positive relationship between Snail overexpression and GC aggressiveness, Snail overexpression significantly correlated with overall survival among GC patients (P = 0.023) (Figure 2C). A linear relationship was observed between increased nuclear expression of Snail and shortened survival (≤50%: 76.6 ± 2.7 months; 50–75%: 68.5 ± 2.0 months; ≥75%: 63.3 ± 2.8 months). Snail overexpression (≥75% positivity) was identified as an independent predictor of poor prognosis in 314 patients with GC, adjusted for age, sex, histologic classification, and tumor location, using a Cox regression proportional hazard model (P = 0.033; Table 2).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-521/MediaObjects/12885_2012_Article_3610_Fig2_HTML.jpg
Figure 2

Snail expression in gastric adenocarcinoma (GC) tissue samples and KaplanMeir plots of overall survival of 314 GC patients. Snail was mostly expressed in nuclei of GC cells (intestinal type (A), and diffuse type (signet ring) cells (B)) included in tissue array specimens. Some reactive fibroblasts also showed Snail nuclear expression (magnification: ×400). C. Kaplan–Meier analysis of overall survival of GC patients based on Snail expression. A linear relationship between increased Snail nuclear expression and shorter survival was seen among GC patients (P = 0.023). Log-rank test was used to calculate P values.

Table 1

Relationship between Snail expression and clinicopathological characteristics in 314 patients with gastric cancer

 

Number of patients (N = 314)

Snail Positivity

Pvalue

  

<75%

≥75%

 

Age (years)

 

58.5 ± 10.6

59.1 ± 11.9

0.695

Sex

 Male

218

143

75

0.996

 Female

96

63

33

 

Tumor size

 ≤4.0 cm

192

135

57

0.028

 >4.0 cm

122

71

51

 

Location

 Upper/Middle

167

112

55

0.561

 Lower

147

94

53

 

Invasion depth

 T1

160

127

33

< 0.001

 T2

41

26

15

 

 T3

68

33

35

 

 T4

43

19

24

 

Gross type

 Elevated

77

51

26

< 0.001

 Flat/depressed

131

105

26

 

 Excavated

106

50

56

 

Histological type

 Intestinal

182

123

59

0.609

 Diffuse

122

76

46

 

 Mixed

10

7

3

 

Perineural invasion

 Negative

202

150

52

< 0.001

 Positive

111

55

56

 

Lymphovascular emboli

 Negative

193

139

54

0.002

 Positive

120

66

54

 

Lymph node metastasis

 N0, N1

270

186

84

0.002

 N2, N3

44

20

24

 
Table 2

Multivariate survival analysis with Cox regression model in 314 gastric cancers

Variables

B

SE

HR (95% CI)

P

Age (≤59 versus > 59)

-0.438

0.264

0.645 (0.385-1.082)

0.097

Gender (male versus female)

-0.037

0.267

0.963 (0.571-1.626)

0.889

Site (upper and middle versus lower)

0.635

0.264

1.887 (1.126-3.164)

0.016

 Lauren (intestinal vs diffuse)

-0.537

0.263

0.585 (0.349-0.978)

0.041

Snail (≥75% versus <75%)

-0.528

0.248

0.590 (0.363-0.958)

0.033

Note: B, coefficient; HR, hazard ratio; CI, confidence interval.

Identification of gene expression patterns based on Snail overexpression using cDNA microarrays

cDNA microarrays were used to compare gene expression profiles of 45 GC specimens. We identified 213 genes that were differentially expressed at significant levels (P < 0.05) between GC specimens with higher (≥75%) and lower levels (<75%) of Snail expression (Table 3). Of these 213 genes, 82 were upregulated and 131 were downregulated in the GC specimens with higher levels (≥75%) of Snail expression. We used hierarchical clustering analysis to assess the 213 genes and 45 GC specimens; supervised clustering analysis gave patterns for samples with higher and lower levels of Snail expression clustered into 2 distinct groups, except for one sample with higher levels of Snail expression (Figure 3). To investigate the biological functions involved in discriminating genes, we performed a GO category analysis. Eleven genes were associated with regulating cancer cell–ECM adhesion (P < 0.021) and ECM protein regulation (P < 0.028, Table 4). Most have been implicated in cancer. ONECUT1, ADAMTS, IFNAR2, MSR1, and SORL1 affect migration or metastasis, a process that involves attachment of tumor cells to the basement membrane, degradation of local connective tissue, and penetration and migration of tumor cells through stroma [2125].
Table 3

Genes differentially expressed in GC specimens with higher levels of Snail expression

PROBE_ID

SYMBOL

NAME

Genes upregulated in specimens with higher levels (≥75%) of Snail expression (P< 0.05)

ILMN_2374449

SPP1

Secreted phosphoprotein 1

ILMN_2337923

TPD52L1

Tumor protein D52-like 1

ILMN_1679838

WBP5

WW domain binding protein 5

ILMN_2078592

C6orf105

Androgen-dependent TFPI-regulating protein

ILMN_1714383

TPD52L1

Tumor protein D52-like 1

ILMN_1674817

C1orf115

Chromosome 1 open reading frame 115

ILMN_1813561

SCIN

Scinderin

ILMN_1759818

SORL1

Sortilin-related receptor, L(DLR class) A repeats containing

ILMN_1745686

MFHAS1

Malignant fibrous histiocytoma amplified sequence 1

ILMN_2060115

SORL1

Sortilin-related receptor, L(DLR class) A repeats containing

ILMN_2337263

PKIB

Protein kinase (cAMP-dependent, catalytic) inhibitor beta

ILMN_2173835

FTHL3

Ferritin, heavy polypeptide 1 pseudogene 3

ILMN_1791057

IFNAR2

Interferon (alpha, beta and omega) receptor 2

ILMN_1807114

LOC255620

Similar to unc-93 homolog B1 (C. elegans), transcript variant 1 (LOC255620), mRNA

ILMN_1669393

GGT1

Gamma-glutamyltransferase 1

ILMN_1685798

MAGEA6

Melanoma antigen family A, 6

ILMN_3269395

GGT2

Gamma-glutamyltransferase 2

ILMN_1669833

SH2B2

SH2B adaptor protein 2

ILMN_3238534

LOC100133817

Hypothetical protein LOC100133817

ILMN_2099315

TRPM8

Transient receptor potential cation channel, subfamily M, member 8

ILMN_3298065

LOC729195

Similar to apical early endosomal glycoprotein

ILMN_1717726

FLJ43752

Long intergenic non-protein coding RNA 336

ILMN_1670452

ANKRD20A1

Ankyrin repeat domain 20 family, member A1

ILMN_3201060

LOC100132655

Hypothetical protein LOC100132655

ILMN_3282829

LOC727913

Similar to iduronate 2-sulfatase (Hunter syndrome)

ILMN_2339691

SYVN1

Synovial apoptosis inhibitor 1, synoviolin

ILMN_1785549

SLC30A2

Solute carrier family 30 (zinc transporter), member 2

ILMN_3191898

LOC100129630

Hypothetical LOC100129630

ILMN_1704204

LOC642204

Ankyrin repeat domain-containing protein 26-like

ILMN_1682280

LOC647753

Hypothetical protein LOC647753

ILMN_3201944

LOC646438

Hypothetical LOC646438

ILMN_2233314

SPANXA1

Sperm protein associated with the nucleus, X-linked, family member A1

ILMN_3305980

NS3BP

NS3BP

ILMN_1747850

CRIM2

Kielin/chordin-like protein

ILMN_1700590

LOC645590

Similar to cAMP-dependent protein kinase type I-beta regulatory subunit

ILMN_1766316

FLJ32679

Golgin-like hypothetical protein LOC440321

ILMN_1890741

Hs.552561

Pancreatic islet cDNA clone hbt09690 3, mRNA sequence

ILMN_3308255

MIR33A

MicroRNA 33a

ILMN_1815716

LMLN

Leishmanolysin-like (metallopeptidase M8 family)

ILMN_1654945

DNMT3A

DNA (cytosine-5-)-methyltransferase 3 alpha

ILMN_2256050

SERPINA1

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1

ILMN_1759487

EGFLAM

EGF-like, fibronectin type III and laminin G domains

ILMN_1760410

LOC653086

Similar to RAN-binding protein 2-like 1 isoform 2

ILMN_1668969

MIXL1

Mix paired-like homeobox

ILMN_3279757

LOC100132532

Hypothetical protein LOC100132532

ILMN_1715372

CAMKK1

Calcium/calmodulin-dependent protein kinase kinase 1, alpha

ILMN_1731370

C9orf84

Chromosome 9 open reading frame 84

ILMN_1679049

COLEC12

Collectin sub-family member 12

ILMN_1676011

LOC642561

Similar to FXYD domain-containing ion transport regulator 6

ILMN_1815442

LOC652875

Similar to Protein KIAA0685

ILMN_1737213

LOC653641

Golgin A6 family, member C

ILMN_1793529

LOC389031

Myosin

ILMN_1709319

C13orf39

Methyltransferase like 21C

ILMN_2284930

FLJ40296

Proline rich 20A

ILMN_1678310

TXNRD3IT1

Thioredoxinreductase 3 neighbor

ILMN_1806052

UNC119

unc-119 homolog (C. elegans)

ILMN_2242345

LPAL2

Lipoprotein, Lp(a)-like 2, pseudogene

ILMN_1687725

C17orf41

ATPase family, AAA domain containing 5

ILMN_1886395

Hs.574341

Soares_multiple_sclerosis_2NbHMSP Homo sapienscDNA clone IMAGp998G11618; IMAGE:126826, mRNA sequence

ILMN_3308612

MIR149

MicroRNA 149

ILMN_1811103

PCDHGB5

Protocadherin gamma subfamily B, 5

ILMN_1736104

LOC645218

Hypothetical LOC645218

ILMN_1824307

Hs.571901

Full-length cDNA clone CS0DF20YK03 of Fetal brain of Homo sapiens

ILMN_1803871

RHO

Rhodopsin

ILMN_3237314

LOC732402

Similar to butyrate-induced transcript 1

ILMN_1714191

LOC652682

Similar to Y46G5A.1a

ILMN_3246580

LOC730429

e3 ubiquitin-protein ligase UBR5-like

ILMN_3229028

LOC728586

hCG1981531

ILMN_3239734

LOC100134822

Uncharacterized LOC100134822

ILMN_1769785

SH3MD4

SH3 domain containing ring finger 3

ILMN_3309864

MIR449B

MicroRNA 449b

ILMN_1653927

SNORD83A

small nucleolar RNA, C/D box 83A

ILMN_3200648

LOC151174

uncharacterized LOC151174

ILMN_1652023

AGFG2

ArfGAP with FG repeats 2

ILMN_1749776

LOC642816

Similar to hypothetical protein LOC284701

ILMN_1671985

LOC646829

Hypothetical protein LOC646829

ILMN_1684499

LOC650373

Similar to deubiquitinating enzyme 3

ILMN_1676452

ADAMTS14

ADAM metallopeptidase with thrombospondin type 1 motif, 14

ILMN_1723855

LOC390427

Similar to TBP-associated factor 15 isoform 1

ILMN_1658019

LOC648447

Hypothetical protein LOC648447

ILMN_3227291

LOC728701

Hypothetical LOC728701

ILMN_1767469

LOC650781

Hypothetical protein LOC650781

Genes downregulated in specimens with higher levels (≥75%) of Snail expression (P< 0.05)

ILMN_1796946

ALLC

Allantoicase

ILMN_3248008

LOC442308

Tubulin, beta class I pseudogene

ILMN_3230623

FLJ40039

Uncharacterized LOC647662

ILMN_1676596

LOC642263

Hypothetical LOC642263

ILMN_3165745

ERCC-00084

Synthetic construct clone NISTag41 external RNA control sequence

ILMN_3242420

HCG8

HLA complex group 8

ILMN_1783827

LOC649397

Similar to Tripartite motif protein 44 (DIPB protein) (Mc7 protein)

ILMN_3244733

LOC100131898

Hypothetical protein LOC100131898

ILMN_3195376

LOC100130092

Similar to MAPRE1 protein

ILMN_2123683

FLJ43763

Uncharacterized LOC642316

ILMN_1730601

FAM194A

Family with sequence similarity 194, member A

ILMN_1652015

LOC647451

Similar to heat shock protein 90Bf

ILMN_1784349

LOC647191

Similar to Kinase suppressor of ras-1 (Kinase suppressor of ras) (mKSR1) (Hb protein)

ILMN_3251375

WBP11P1

WW domain binding protein 11 pseudogene 1

ILMN_1911713

Hs.550068

UI-E-EJ1-ajn-i-16-0-UI.s1 UI-E-EJ1 Homo sapienscDNA clone UI-E-EJ1-ajn-i-16-0-UI.3, mRNA sequence

ILMN_1888057

Hs.554470

nc63e05.r1 NCI_CGAP_Pr1 Homo sapienscDNA clone IMAGE:745952, mRNA sequence

ILMN_3229818

LOC729828

Misc_RNA (LOC729828), miscRNA

ILMN_1654987

HCG2P7

HLA complex group 2 pseudogene 7

ILMN_1683453

FRAS1

Fraser syndrome 1

ILMN_1840493

Hs.112932

ag03b01.s1 Soares_testis_NHTHomo sapienscDNA clone IMAGE:1056169 3, mRNA sequence

ILMN_1860820

Hs.126468

tm27h01.x1 Soares_NFL_T_GBC_S1 Homo sapienscDNA clone IMAGE:2157841 3, mRNA sequence

ILMN_3227213

LOC728940

Hypothetical LOC728940

ILMN_3247774

LOC100134235

Similar to hCG1642820

ILMN_1902571

Hs.557622

tw46h08.x1 NCI_CGAP_Ut1 Homo sapienscDNA clone IMAGE:2262783 3 similar to contains PTR5.b2 PTR5 repetitive element, mRNA sequence

ILMN_2384405

RTBDN

Retbindin

ILMN_3234879

LOC653786

Otoancorinpseudogene

ILMN_1914891

Hs.334272

RST40254 Athersys RAGE Library Homo sapienscDNA, mRNA sequence

ILMN_3272356

LOC100129315

Hypothetical protein LOC100129315 (LOC100129315), mRNA

ILMN_3230388

LOC100130855

Hypothetical protein LOC100130855( LOC100130855), mRNA

ILMN_1656553

LOC653160

Hypothetical protein LOC653160, transcript variant (LOC653160), mRNA

ILMN_1700935

HAS2

Hyaluronan synthase 2

ILMN_1733783

LOC652790

Similar to anaphase promoting complex subunit 1

ILMN_2209221

DMRT1

Doublesex and mab-3 related transcription factor 1

ILMN_1815118

ZNF554

Zinc finger protein 554

ILMN_3293210

LOC100131031

Similar to hCG2041190 (LOC100131031), mRNA

ILMN_1703222

FRS2

Fibroblast growth factor receptor substrate 2

ILMN_1732807

GPRC6A

G protein-coupled receptor, family C, group 6, member A

ILMN_1875332

Hs.545527

he15g04.x1 NCI_CML1 Homo sapienscDNA clone IMAGE:2919216 3 similar to contains element PTR5 repetitive element

ILMN_3235789

BPY2C

Basic charge, Y-linked, 2C

ILMN_3203116

LOC100131961

Misc_RNA (LOC100131961), miscRNA

ILMN_2198802

FAM22G

Family with sequence similarity 22, member G

ILMN_1858700

Hs.538558

zh20c06.s1 Soares_pineal_gland_N3HPG Homo sapienscDNA clone IMAGE:412618 3, mRNA sequence

ILMN_1873107

Hs.282800

AV649053 GLC Homo sapienscDNA clone GLCBPH07 3, mRNA sequence

ILMN_1891673

Hs.164254

hb73c02.x1 NCI_CGAP_Ut2 Homo sapienscDNA clone IMAGE:2888834 3, mRNA sequence

ILMN_3206632

LOC643802

u3 small nucleolarribonucleoprotein protein MPP10-like

ILMN_1883034

Hs.546089

RST29145 Athersys RAGE Library Homo sapienscDNA, mRNA sequence

ILMN_2373335

LIG3

Ligase III, DNA, ATP-dependent

ILMN_3239639

CD200R1L

CD200 receptor 1-like

ILMN_1870857

Hs.148168

Barstead spleen HPLRB2 Homo sapienscDNA clone IMAGp998L113601 ; IMAGE:1425178, mRNA sequence

ILMN_1813909

CRSP2

Mediator complex subunit 14

ILMN_1891885

Hs.332843

qg83a07.x1 Soares_NFL-T_GBC_S1 Homo sapienscDNA clone IMAGE:1841748, mRNA sequence

ILMN_3235126

LOC100133558

Similar to hCG1642170

ILMN_1677186

MGC52498

Family with sequence similarity 159, member A

ILMN_3252608

HCRP1

Hepatocellular carcinoma-related HCRP1

ILMN_1652871

PLSCR5

Phospholipid scramblase family, member 5

ILMN_1698894

OR5AS1

Olfactory receptor, family 5, subfamily AS, member 1

ILMN_1705828

RICTOR

RPTOR independent companion of MTOR, complex 2

ILMN_1683046

OR6Y1

Olfactory receptor, family 6, subfamily Y, member 1

ILMN_2114812

ONECUT1

One cut homeobox 1

ILMN_1770248

PDLIM2

PDZ and LIM domain 2 (mystique)

ILMN_1784272

CD1E

CD1e molecule

ILMN_1755635

FLJ33534

Hypothetical protein FLJ33534 (FLJ33534), mRNA

ILMN_1799067

TRY1

Protease, serine, 1 (trypsin 1)

ILMN_1693448

LOC643811

Similar to FERM domain containing 6

ILMN_1723323

HCG4

HLA complex group 4 (non-protein coding)

ILMN_1865604

Hs.253267

60270330F1 NCI_CGAP_Skn3 Homo sapienscDNA clone IMAGE:4800534 5, mRNA sequence

ILMN_3308698

MIR1276

MicroRNA 1276

ILMN_1714014

LOC644491

NMDA receptor regulated 2 pseudogene

ILMN_2114185

C1orf104

RUSC1 antisense RNA 1 (non-protein coding)

ILMN_1911044

Hs.540915

nf66b06.s1 NCI_CGAP_Co3 Homo sapienscDNA clone IMAGE:924851 3, mRNA sequence

ILMN_1748543

STRC

Stereocilin

ILMN_1675221

DGKZ

Diacylglycerol kinase, zeta

ILMN_1726263

LOC653748

Similar to dipeptidylaminopeptidase-like protein 6 (dipeptidylpeptidase VI) (dipeptidylpeptidase 6) (dipeptidyl peptidase VI-like protein) (dipeptidylaminopeptidase-related protein) (DPPX)

ILMN_1817113

Hs.547985

UI-H-BI0p-abm-h-10-0-UI.s1 NCI_CGAP_Sub2 Homo sapienscDNA clone IMAGE:2712450 3, mRNA sequence

ILMN_1793525

KIR2DS3

Killer cell immunoglobulin-like receptor, two domains, short cytoplasmic tail, 3

ILMN_2415617

C10orf72

V-set and transmembrane domain containing 4

ILMN_1746277

MLLT4

Myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 4

ILMN_1678246

LOC644001

Hypothetical protein LOC644001

ILMN_3257856

LOC100130938

Hypothetical LOC100130938 (LOC100130938), mRNA

ILMN_1865630

Hs.116333

Soares_testis_NHTHomo sapienscDNA clone IMAGp998A031828, mRNA sequence

ILMN_2152028

LOC642452

Hypothetical LOC642452 (LOC642452), mRNA

ILMN_3244579

LOC649330

Heterogeneous nuclear ribonucleoprotein C-like

ILMN_1905832

Hs.564127

UI-E-DW1-ahc-g-05-0-UI.r1 UI-E-DW1 Homo sapienscDNA clone UI-E-DW1-ahc-g-05-0-UI.5, mRNA sequence

ILMN_1897251

Hs.547715

UI-E-EJ0-ahv-e-11-0-UI.s1 UI-E-EJ0 Homo sapienscDNA clone UI-E-EJ0-ahv-e-11-0-UI 3, mRNA sequence

ILMN_1782800

LOC651410

Hypothetical protein LOC651410

ILMN_1732554

ZNF346

Zinc finger protein 346

ILMN_1674014

LOC653878

Similar to Cytosolic acyl coenzyme A thioester hydrolase, inducible (Long chain acyl-CoA thioester hydrolase) (Long chain acyl-CoA hydrolase) (CTE-I) (CTE-Ib)

ILMN_1911501

Hs.543905

xi89f08.x1 NCI_CGAP_Mel3 Homo sapienscDNA clone IMAGE:265999 3, mRNA sequence

ILMN_1878305

Hs.262789

xk07d09.x1 NCI_CGAP_Co20 Homo sapienscDNA clone IMAGE:2666033 3, mRNA sequence

ILMN_1858245

Hs.156566

Soares_testis_NHTHomo sapienscDNA clone IMAGp998M073519, mRNA sequence

ILMN_1704313

GSTCD

Glutathione S-transferase, C-terminal domain containing

ILMN_1707398

ESRRB

Estrogen-related receptor beta

ILMN_3307954

L3MBTL4

l(3)mbt-like 4 (Drosophila)

ILMN_1851244

Hs.59368

UI_H_BI1_aex-h-12-0-UI.s1 NCI_CGAP_Sub3 Homo sapienscDNA clone IMAGE:2720903 3, mRNA

ILMN_1828556

Hs.541581

nac23e12.x1 Lupski_sciatic_nerveHomo sapienscDNA clone IMAGE:3394270 3, mRNA sequence

ILMN_1692894

LOC654042

Similar to dehydrogenase/reductase (SDR family) member 4 like 2

ILMN_1893728

Hs.377660

Homo sapienscDNA FLJ26242 fis, clone DMC00770

ILMN_1667005

LOC652676

Similar to similar to hypothetical protein FLJ36144

ILMN_3241607

LOC100132106

Hypothetical LOC100132106

ILMN_1797503

GOLGA8G

Golgin A8 family, member G

ILMN_1828034

Hs.154513

ik89c11.z1 Human insulinomaHomo sapienscDNA clone IMAGE:6027645 3, mRNA sequence

ILMN_1886816

Hs.544491

qq31a07.x1 Soraes_NhHMPu_S1 Homo sapienscDNA clone IMAGE:1934100 3, mRNA sequence

ILMN_1847950

Hs.505398

wq87c02.x1 NCI_CGAP_GC6 Homo sapienscDNA clone IMAGE:2479010 3, mRNA sequence

ILMN_1734479

ACCN3

Acid-sensing (proton-gated) ion channel 3

ILMN_1675025

H2BFM

H2B histone family, member M

ILMN_2073279

SIM1

Single-minded homolog 1 (Drosophila)

ILMN_1910185

Hs.98563

zw57h03.s1 Soares_total_fetus_Nb2HF8_9w Homo sapienscDNA clone IMAGE:774197 3, mRNA sequence

ILMN_3251491

UQCRB

Ubiquinol-cytochrome c reductase binding protein

ILMN_2180315

ATG4D

ATG4 autophagy related 4 homolog D (S. cerevisiae)

ILMN_1885583

Hs.542934

Homo sapienscDNA FLJ26431 fis, clone KDN01390

ILMN_1743301

MSR1

Macrophage scavenger receptor 1

ILMN_1809820

LOC648963

Similar to retinitis pigmentosa 1-like 1

ILMN_1869348

Hs.460114

UI-E-EJ0-ahv-d-07-0-UI.s1 UI-E-EJ0 Homo sapienscDNA clone UI-E-EJ0-ahv-d-07-0-UI 3, mRNA sequence

ILMN_1711332

TFEC

Transcription factor EC

ILMN_2228538

IRAK1BP1

Interleukin-1 receptor-associated kinase 1 binding protein 1

ILMN_1756455

IL5RA

Interleukin 5 receptor, alpha

ILMN_1719202

ZNF174

Zinc finger protein 174

ILMN_1847029

Hs.553290

HESC3_84_D06.g1_A036 Human embryonic stem cells Homo sapienscDNA clone IMAGE:7483454 5, mRNA sequence

ILMN_1740217

HACE1

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1

ILMN_1787464

LOC651296

Similar to RAB, member of RAS oncogene family-like 2B isoform 1

ILMN_1734096

DCLRE1A

DNA cross-link repair 1A

ILMN_2391333

CYP20A1

Cytochrome P450, family 20, subfamily A, polypeptide 1

ILMN_2226314

DBR1

Debranching enzyme homolog 1 (S. cerevisiae)

ILMN_2379560

CDC14B

CDC14 cell division cycle 14 homolog B (S. cerevisiae)

ILMN_2078466

DZIP1L

DAZ interacting protein 1-like

ILMN_1653039

LOC642934

Hypothetical protein LOC642934 (LOC642934), mRNA

ILMN_2044293

KBTBD7

Kelch repeat and BTB (POZ) domain containing 7

ILMN_1809951

ZNF200

Zinc finger protein 200

ILMN_1760280

NXT1

NTF2-like export factor 1

ILMN_1657796

STMN1

Stathmin 1

ILMN_1793578

ZFP37

Zinc finger protein 37 homolog (mouse)

https://static-content.springer.com/image/art%3A10.1186%2F1471-2407-12-521/MediaObjects/12885_2012_Article_3610_Fig3_HTML.jpg
Figure 3

Supervised clustering analysis of 45 gastric adenocarcinoma (GC) specimens and 172 genes. Hierarchical clustering was used for 45 GC specimens and 213 genes. Data are shown in a matrix format, with rows representing individual genes and columns representing tissues. Each cell in the matrix represents the expression level of a gene featured in an individual tissue. Red and green cells reflect GCs with higher (≥75%) and lower (<75%) levels of Snail expression, respectively. Matrix patterns for specimens clustered into 2 distinct groups, except for one sample with higher levels of Snail expression.

Table 4

Cellular functions of selected genes that are differentially expressed in GC specimens that overexpress Snail

Probe ID

Gene acronym

Gene name

Accession No.

Pvalue

Cancer cell–ECM adhesion

ILMN_1759487

EGFLAM

EGF-like, fibronectin type III, and laminin G domains (↑)

NM_182801

0.005

ILMN_2114812

ONECUT1

One cut homeobox 1 (↓)

NM_004498

0.002

ILMN_2374449

SPP1

Secreted phosphoprotein 1 (↑)

NM_000582

0.004

ECM protein regulation

ILMN_1676452

ADAMTS14

ADAM metallopeptidase with thrombospondin type 1 motif, 14 (↑)

NM_080722

0.005

ILMN_1759487

EGFLAM

EGF-like, fibronectin type III, and laminin G domains (↑)

NM_182801

0.005

ILMN_1683453

FRAS1

Fraser syndrome 1 (↓)

NM_020875

0.003

ILMN_1791057

IFNAR2

Interferon (alpha, beta, and omega) receptor 2 (↑)

NM_207585

0.001

ILMN_1756455

IL5RA

Interleukin 5 receptor, alpha (↓)

NM_000564

0.004

ILMN_1747850

CRIM2

Kielin/chordin-like protein (↑)

NM_199349

0.005

ILMN_1743301

MSR1

macrophage scavenger receptor 1 (↓)

NM_002445

0.002

ILMN_2374449

SPP1

secreted phosphoprotein 1 (↑)

NM_000582

0.004

ILMN_2256050

SERPINA1

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (↑)

NM_000295

0.002

ILMN_2060115, ILMN_1759818

SORL1

Sortilin-related receptor, L(DLR class) A repeats-containing (↑)

NM_003105

0.003 <0.001

NOTE: ↑, upregulation; ↓, downregulation.

Discussion

Snail is reportedly a key regulator of tumor progression and metastasis via increased MMP expression and tumor invasion [26, 27]. Similarly, we found that upregulated Snail expression increased gastric cancer cell invasion/migration, whereas downregulated Snail expression decreased gastric cancer cell invasion/migration. Yang et al. reported that Snail overexpression in hepatocellular carcinoma cell lines induced increased invasiveness/metastasis [13]. In addition, Kosaka et al. reported that Snail knockdown was associated with decreased invasive capacity of a urothelial carcinoma cell line, supporting our results [12]. We also found that Snail overexpression induced increased expression of VEGF and MMP11, which are known markers of tumor invasion and metastasis. Jin et al. also reported that Snail knockdown by antisense Snail was associated with inhibited MMP activity, demonstrating the importance of regulating MMP activity in cancer metastasis.10 Furthermore, Peinado et al. reported that I MDCK cells with Snail overexpression had increased angiogenesis and VEGF [28]. We also observed increased VEGF in gastric cancer cells with Snail overexpression.

The clinical importance of Snail in various carcinomas, including non-small cell lung carcinomas, ovarian carcinomas, urothelial carcinomas, hepatocellular carcinoma, and breast cancer, is well known, as is the poor prognosis associated with Snail overexpression [1013, 29]. However, only limited immunohistochemical data have been available on Snail expression in GC, with no comprehensive clinical and functional analysis of Snail expression in GC patients. Kim et al. reported immunohistochemical data indicating that Snail expression was an independent indicator of prognosis in tissue microarray specimens [14]. Rye et al. reported that the combination of Snail, vimentin, E-cadherin, and CD44 was also significantly associated with poor prognosis in gastric cancer [15]. In contrast, no significant correlation between tumor stage and Snail expression was noted in upper gastrointestinal tract adenocarcinoma, including cancers of the esophagus, cardia, and stomach [30]. In our study, overexpression of Snail (≥75% nuclear Snail expression) was significantly associated with tumor progression, lymph node metastases, lymphovascular invasion, perineural invasion, and poor prognosis in GC patients. Recently, He et al. reported Snail to be an independent prognostic predictor of patient survival among gastric cancer patients; this is in agreement with our data [31]. Although 5-FU based adjuvant chemotherapy for advanced or metastatic gastric adenocarcinoma was usually performed in our cohort, further work is required to reveal exact significance of Snail expresssion as predictor of chemotherapy response in gastric adenocarcinoma. For the practical use of Snail as a tissue biomarker in predicting lymph node metastasis and poor prognosis, we defined a cut-off value of 75% positive nuclear expression for Snail overexpression. There are wide variations in cut-off values for Snail overexpression in different types of cancer; for example, 75% is used in non-small cell lung carcinoma [11], 100 (score of mean percentage × intensity, range 0–300) is used in urothelial carcinomas [12], and 50% is used in hepatocellular carcinoma [13]. For gastric cancers, cut-off values of 10% [14] and 5% [15] positive nuclear expression of Snail have been reported. Further work is required to determine a practical consensus cut-off value for Snail overexpression.

A total of 213 genes that were differentially expressed among GC samples with higher (≥75%) and lower levels of Snail expression were clustered into 2 distinct groups: those associated with regulation of cancer cell–ECM adhesion, and those associated with ECM protein regulation, such as ONECUT1[21], ADAMTS[22], IFNAR2[23], MSR1[24], and SORL1[25]. These functions indicate that Snail greatly affects cancer cell migration and metastasis by regulating attachment of tumor cells to basement membranes, degradation of local connective tissue, and penetration and migration of tumor cells through stroma.

Conclusions

In this study, we showed that Snail overexpression induced increased migration and invasion in gastric cancer cell lines. Snail overexpression was also significantly associated with tumor progression, lymph node metastases, lymphovascular invasion, perineural invasion, and poor prognosis in GC patients. We identified 213 genes that were differentially expressed in GC tissues that overexpressed Snail, including genes related to metastasis and invasion by tumor cells. Our results indicate that Snail is crucial in controlling progression and metastasis of gastric cancer. Thus Snail may be used as a predictive biomarker for evaluating prognosis or aggressiveness of GCs.

Notes

Declarations

Acknowledgements

This study was supported by grant 0920050 from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea.

Authors’ Affiliations

(1)
Department of Pathology, Pusan National University Hospital and Pusan National University School of Medicine
(2)
Department of Internal Medicine, Pusan National University Hospital and Pusan National University School of Medicine
(3)
Department of Surgery, Pusan National University Hospital and Pusan National University School of Medicine
(4)
BioMedical Research Institute, Pusan National University Hospital
(5)
Department of Pathology, Cheonam National University
(6)
Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology

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  32. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/521/prepub

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© Shin et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.