Promoter methylation correlates with reduced NDRG2 expression in advanced colon tumour.

Abstract

Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current study to validate our previous microarray data and to identify novel hypermethylated gene promoters.
The validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour tissues from 30 patients with CRC.
Data from our previous genome search have received confirmation in the new set of 8 patients with CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated.
The findings highlight the usefulness of combining gene expression patterns and epigenetic data to identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being associated with a more advanced stage.

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BioMed Central
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BMC Medical Genomics
Open Access
Research article
Promoter methylation correlates with reduced NDRG2 expression
in advanced colon tumour
Ada Piepoli*1, Rosa Cotugno1, Giuseppe Merla2, Annamaria Gentile1,
Bartolomeo Augello2, Michele Quitadamo1, Antonio Merla1, Anna Panza1,
Massimo Carella2, Rosalia Maglietta3, Annarita D'Addabbo3, Nicola Ancona3,
Saverio Fusilli4, Francesco Perri1 and Angelo Andriulli1
Address: 1Gastroenterology Unit and Research Laboratory, "Casa Sollievo della Sofferenza", Hospital, IRCCS, San Giovanni Rotondo, Italy,
2Medical Genetics Service, "Casa Sollievo della Sofferenza", Hospital, IRCCS, San Giovanni Rotondo, Italy, 3Istituto di Studi sui Sistemi Intelligenti
per l'Automazione – CNR, Bari, Italy and 4Health Services, "Casa Sollievo della Sofferenza", Hospital, IRCCS, San Giovanni Rotondo, Italy
Email: Ada Piepoli* - a.piepoli@operapadrepio.it; Rosa Cotugno - r.cotugno@operapadrepio.it; Giuseppe Merla - g.merla@operapadrepio.it;
Annamaria Gentile - a.gentile@operapadrepio.it; Bartolomeo Augello - bartolomeoaugello@gmail.com;
Michele Quitadamo - m.quitadamo@operapadrepio.it; Antonio Merla - tonymerla@hotmail.com; Anna Panza - annapanza82@virgilio.it;
Massimo Carella - m.carella@operapadrepio.it; Rosalia Maglietta - maglietta@ba.issia.cnr.it; Annarita D'Addabbo - dadabbo@ba.issia.cnr.it;
Nicola Ancona - ancona@ba.issia.cnr.it; Saverio Fusilli - s.fusilli@operapadrepio.it; Francesco Perri - f.perri@operapadrepio.it;
Angelo Andriulli - a.andriulli@operapadrepio.it
* Corresponding author
Abstract
Background: Aberrant DNA methylation of CpG islands of cancer-related genes is among the earliest and most
frequent alterations in cancerogenesis and might be of value for either diagnosing cancer or evaluating recurrent
disease. This mechanism usually leads to inactivation of tumour-suppressor genes. We have designed the current
study to validate our previous microarray data and to identify novel hypermethylated gene promoters.
Methods: The validation assay was performed in a different set of 8 patients with colorectal cancer (CRC) by
means quantitative reverse-transcriptase polymerase chain reaction analysis. The differential RNA expression
profiles of three CRC cell lines before and after 5-aza-2'-deoxycytidine treatment were compared to identify the
hypermethylated genes. The DNA methylation status of these genes was evaluated by means of bisulphite
genomic sequencing and methylation-specific polymerase chain reaction (MSP) in the 3 cell lines and in tumour
tissues from 30 patients with CRC.
Results: Data from our previous genome search have received confirmation in the new set of 8 patients with
CRC. In this validation set six genes showed a high induction after drug treatment in at least two of three CRC
cell lines. Among them, the N-myc downstream-regulated gene 2 (NDRG2) promoter was found methylated in all
CRC cell lines. NDRG2 hypermethylation was also detected in 8 out of 30 (27%) primary CRC tissues and was
significantly associated with advanced AJCC stage IV. Normal colon tissues were not methylated.
Conclusion: The findings highlight the usefulness of combining gene expression patterns and epigenetic data to
identify tumour biomarkers, and suggest that NDRG2 silencing might bear influence on tumour invasiveness, being
associated with a more advanced stage.
Published: 3 March 2009
BMC Medical Genomics 2009, 2:11 doi:10.1186/1755-8794-2-11
Received: 20 June 2008
Accepted: 3 March 2009
This article is available from: http://www.biomedcentral.com/1755-8794/2/11
© 2009 Piepoli et al; licensee 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.

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Background
Colorectal cancer (CRC) is the third most common cancer
in men and women, accounting for 11% of all cancer-
related deaths. The majority of cases are diagnosed in
advanced stages when a curative treatment is less likely to
occur and chemotherapy is the only option [1]. The iden-
tification of the molecular, genetic, and epigenetic
changes underlying the adenoma-carcinoma sequence [2]
leading to CRC has been the focus of many researches [3].
It is now widely accepted that sporadic CRC frequently
arises from preneoplastic lesions through the activation of
proto-oncogenes, such as K-ras, and the inactivation of
tumor suppressor genes (TSG), such as APC, p53, DCC,
and the mismatch repair genes [4,5]. Apart from muta-
tions, gene expression may also be modified by altering of
DNA methylation [6]. Two general phenomena have until
now been observed. The first one is global DNA
hypomethylation with decreased 5-methylcytosine con-
tent which results in both enhanced expression of proto-
oncogenes [7] and genomic instability [8]. The second
event is represented by local DNA hypermethylation of
CpG islands, short sequences rich in CpG dinucleotides in
the 5'untranscribed region (5'-UTR). This event occurs in
approximately half of all human genes [9,10] silences spe-
cific TSG, and accelerates cancer formation [11,12]. Treat-
ment with DNA demethylating drugs, such as 5-aza-2'-
deoxycytidine (5-Aza-CdR or Decitabine), was shown to
reverse the hypermethylation and restore expression of
TSG [13]. Therefore, cancer-specific promoter methyla-
tion may by itself serve as a valuable clue to uncover novel
TSG.
In the present study, we aimed to uncover novel targets of
promoter methylation in CRC, by combining gene expres-
sion profile data, already highlighted by our group [14],
with results of demethylating assay and in silico screening
for CpG islands.
Methods
Patients
Peripheral blood, primary tumour and matching normal
tissue samples from a cohort of 30 consecutive CRC
patients undergoing curative surgery at our Institution
were collected. Clinical data, tumour location, and AJCC
staging of these patients are shown in Table 1. Primary
tumour and matching normal tissue samples were
obtained from a second cohort of 8 CRC patients, and
used in a validation assay. Genomic DNA was isolated
from peripheral blood samples using standard tech-
niques. Tissue samples were immediately frozen in liquid
nitrogen and stored at -80°C until nucleic acids extrac-
tion. The study was approved by the Ethics Committee at
our Institution, and all patients gave their informed writ-
ten consent.
RNA extraction from fresh frozen tissue
About 150–200 mg fresh frozen tissues were used to iso-
late total RNA by phenol extraction (TRIzol Reagent, Inv-
Table 1: Clinical data of colorectal cancer (CRC) patients
CRC
n = 30 (%)
Age (yrs):
Mean (± SD)
Sex:
Male/Female
Tumour location:
Ascending colon
Transverse colon
Descending colon
Sigmoid colon
Rectum
Rectum-Sigmoid colon
AJCC stage:
0
I
IIa
IIIb
IIIc
IV
59 ± 14
15/15
7 (23%)
2 (7%)
4 (13%)
8 (27%)
4 (13%)
5 (17%)
1 (3%)
3 (10%)
8 (27%)
3 (10%)
1 (3%)
14 (47%)
Age (yrs):
Mean age <50
CRC:
Familiar/Sporadic
Tumour location:
Ascending colon
Descending colon
Sigmoid colon
Rectum
Rectum-Sigmoid colon
AJCC stage:
I
IIIb
IIIc
IV
41 ± 7
1/8
1 (11%)
2 (22%)
4 (45%)
1 (11%)
1 (11%)
1 (11%)
2 (22%)
1 (11%)
5 (55%)
Age (yrs):
Mean age >50
CRC:
Familiar/Sporadic
Tumour location:
Ascending colon
Transverse colon
Descending colon
Sigmoid colon
Rectum
Rectum-Sigmoid colon
AJCC stage:
0
I
IIa
IIIb
IV
66 ± 9
8/13
6 (29%)
2 (9%)
2 (9%)
4 (19%)
3 (15%)
4 (19%)
1 (5%)
2 (9%)
8 (38%)
1 (5%)
9 (43%)

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itrogen Corporation, Carlsbad, CA, USA) which was
subsequently purified by column chromatography (RNe-
asy Mini Kit, Qiagen, Valencia, CA, USA). RNA integrity
was monitored using MOPS gel electrophoresis.
Cell culture, 5-Aza-CdR Treatments
The HCT116, CaCo2 and SW480 cell lines were pur-
chased from the American Type Culture Collection
(ATCC, Rockville, MD, USA), and maintained in DMEM
medium (Invitrogen Corporation, Carlsbad, CA) supple-
mented with 10% FBS, 100 U/ml penicillin, and 100 μg/
ml streptomycin in a humidified 5% CO2 atmosphere at
37°C. For demethylation studies, cells were seeded at a
density of 1 × 106 cells per 100-mm dish, and incubated
for 24 hrs in a growth media. Subsequently, 5-Aza-CdR
(Merck Chemicals Ltd., Nottingham, UK) was added to
the incubation mixture following two different protocols.
In the acute treatment 1 μM 5-Aza-CdR was added to incu-
bation mixture for 24 hrs; afterwards, the medium was
changed once daily for 3 consecutive days; DNA and RNA
content were checked at 2nd, 4th and 6th days [15,16]. In
the chronic treatment, 2 μM 5-Aza-CdR was added for 24
hrs at day 1st, 3rd and 5th [17]; at each experimental day,
the cells were placed in fresh medium and harvested at
day 6th to isolate DNA and RNA.
qPCR Assay
For qPCR, 1.0 μg of total RNA from CRC cell lines and
normal and tumour tissues of the second cohort of CRC
samples was used with hexamer random primers to run
the first strand cDNA synthesis by the RT-System kit
(Promega Corporation, Madison, WI). Oligonucleotide
sequences were designed by means of the PrimerExpress
program (Applied Biosystems, Applera, Foster City, CA)
with default parameters in every case; whenever possible,
the oligos were designed to span an intron region (Table
2). To ensure specificity, amplicon sequences were
checked by both BLAST and BLAT programs against the
human genome. The efficiency of each oligo pairs was
checked by diluting a series of control cDNAs. All qPCRs
were performed in a 10-μl final volume, in three replicates
per sample, set up in a 384-well plate format with the
Biomek 2000 robot (Beckman Coulter, Inc., Miami, FL).
The assays were run in an ABI 7900 Sequence Detection
System (Applied Biosystems, Applera, Foster City, CA,
USA) with the following amplification conditions: 50°C
for 2 min, 95°C for 10 min, and 50 cycles at 95°C for 15
s and at 60°C for 1 min. Expression of mRNA from candi-
date genes was analysed quantitatively by means of SYBR
Green Real Time PCR (Invitrogen Corporation, Carlsbad,
CA) and raw Ct values calculated with SDS2.0.
In Silico Search and Bisulfite Sequencing Analysis (BSA)
The presence of CpG islands, overlapping the 5'-UTR, was
examined by means of the MethPrimer http://www.uro
gene.org/methprimer/, according to CpG islands defini-
tion.
Bisulfite modification of DNA from colon cancer cell
lines, peripheral blood and frozen tissues of patients was
assayed, as reported by Herman et al. [18]; normal lym-
phocytes (NL) and in vitro methylated DNA (IVD) were
used as negative and positive controls, respectively. In the
assay, 1 μg of DNA was denaturated by treatment with
NaOH at 37°C for 10 min, followed by incubation with
hydroquinone and sodium bisulfite at 50°C for 16–17 h
in the dark. After treatment, DNA was purified using DNA
cleanup kit (Promega Corporation, Madison, WI), incu-
bated with NaOH at 37°C for 15 min, precipitated with
ammonium acetate and 100% ethanol, washed with 70%
ethanol and, finally, re-suspended in 25 μl of distilled
water. DNA methylation patterns in the CpG islands were
determined by BSA using the primers listed in Table 2. The
PCR conditions were 3 min at 94°C, 30 cycles of 94°C for
30 sec, specific annealing temperature for 30 sec, and
72°C for 60 sec. The sequence of the PCR products was
analysed by using Sequencing Analysis 3.4.1 (Applied
Biosystems, Applera, Foster City, CA, USA).
MSP Assay
Qualitative analysis of CpG islands in the promoter
region of the NDRG2, p16, APC, and MLH1 genes in 30
patients of CRC, in cell lines, in NL and IVD was carried
out by MSP assay [18]. The primers for unmethylated and
methylated DNA are listed in Table 2. For the NDRG2 pro-
moter region we used
(NDRG2_UnM/NDRG2_M
NDRG2_M2) to cover the same region sequenced by the
bisulfite assays. PCR reaction was carried out in a 25 μl
mixture containing 0,2 mM each dNTP, 1.5 mM MgCl2,
primers (10 μM each), bisulfite-modified DNA (50 ng),
and 0.75 U of Amplitaq Taq Gold polymerase (Applied
Biosystems, Applera, Foster City, CA) for 35 cycles (95°C
for 12 min, 94°C for 1 min, TA for 1 min, then 72°C for
1 min, followed by a final extension at 72° for 5 min) and
analysed on a 3% agarose gel stained with ethidium bro-
mide. All reactions were run in duplicate to ensure con-
sistent and reproducible results.
two
and
different
NDRG2_UnM2/
primers
Statistical Analysis
We carried out three separate statistical analyses. In the
initial analysis, qPCR data were used to validate our pre-
vious array results [14]. Calculations were made using the
Comparative CT method [19,20]. We used three genes,
that is hEEF1A1, hGAPDH and hHRPT1, to normalize
input cDNA for each sample, with cDNA of normal tissue
used as calibrator. The chi-square method with one degree
of freedom (χ21), calculated by BMDP Statistical Software
(BMDP Statistical Software, Cork Technology Park, Model
Farm Road, Cork, Ireland) [21], was used to asses statisti-

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cal significance of expression difference for each gene of
the 8 paired samples. The second analysis concerned the
variation of gene expression before and after 5'-Aza-CdR
treatment by means of the T test, calculated by BMDP Sta-
tistical Software. Expression level of the post-treatment
specimen compared to the pre-treatment specimen was
calculated as a log-transformed ratio. A gene was classified
as up-regulated following the 5-Aza-CdR treatment when
relative mRNA expression was greater or equivalent to
1.65-fold in at least one treatment condition in one cell
line. Genes with no change or very low expression levels
in post treatment specimens were no further considered in
the analysis.
The final part of our analysis evaluated the BSA data. The
median number of full CpG islands, present in normal
and tumour tissues, was calculated and compared in
tumour (T) matched normal (N) tissue of the same
patients. The χ21 method was used to asses significant dif-
ference; a number of CpG islands higher than 5 was taken
as statistically significant.
Table 2: Primer Sequences and Conditions for qRT-PCR, Bisulfite sequencing and MSP Analysis
Gene symbol RefSeq mRNAPrimer Forward (5'-->3')Primer Reverse (5'-->3')Size
(bp)
AT†
(°C)
Quantitative Real Time PCR (qPCR) assay
CCATCATGGTATCTGGGAGGTT
CACGGGCTGGCTTTGG
CAAAATGGCCTATCTCAGTATTCCA
GCATGGCATAGTTGGATTCACA
GCCCTTTCAATAGACAGTGTTGAG
ATCGTTGGAACTGATGATGACTTC
AAGAAAGAGAAACGGCAATTCG
CGGTTCAAACACAATAGCAAGTG
AACAAAATCCCAGATGCTGACA
ATCCATGAGATCAATGCAGCAT
CTGAGGGAGACTGCAAATGCT
AAGGAACATTCAAGCACAGCTAATATT
ACGCCGAGCTCAGCAAGA
AATCTGCAATGATGCCAAGGT
CGGCACACAACACTTGGTTT
TTCATTCTCAGTACCAATAAAGCAAGA
AGGAGCCTGAGCTTATGAATGC
CTGACCGAGGCCTTCAAGTACT
AGCCCTTTGTTCCCAAGAAAG
TGGAGCTTACTCTGCAACTGTTTC
CCTGGCTAAGAATGTAGTCATGGTAA
CATTGAAGAATCAGCAGCCAATA
AGCAAGCGAGTCCGCATT
ACATCATTTACAGGGATCTGAAACC
ABCA8
AQP8
CLCA4
HPGD1
PRDX6
SLC26A3
STX12
CSE1L
HSPH1
NEBL1
RFC3
SLC12A2
SOX9
GTF2IRD1
MXI1
NR3C2
SGK1
NDRG2
TPX2
UBE2C
CCNB1
SCNN1B
FOXM1
SGK2
NM_007168
AB013456
NM_012128
NM_000860
NM_004905
NM_000111
NM_177424
NM_001316
NM_00664
HSY1624
NM_002915
NM_001046
NM_000346
ENST00000265755
NM_005962
NM_000901
NM_005627
NM_201535
NM_012112
NM_181799
NM_031966
ENST00000343070
U74613
NM_016276
GCAGGTAATCTTTGCCAAATTTG
CCAGTACGGGAGGAGCATCA
TCGCTTTGGCTTGAAGATTGT
AAGCCTGGACAAATGGCATT
ATCGATGATGGGAAAAGGTAACTT
CAGCATCATGGATTGTTAAGAAAAA
TCATGGCCAAATCTTTAAATATCTGA
GGATGCAATCAGCTTCTGAAAGA
ACCTTTATTTTGGGCTTTTTAGCTT
CATCCTGGGCACTGTAATCGT
ACAGCCTTCCACGAACTTCAA
TGCCATGTAGAGAGCACTAGACACA
CACGAAGGGCCGCTTCT
CCGAGACCCGCTTTCTCTT
GGCTTTTTCTTTCAGCTTCTTCA
GGTTTACTGTTGGATTCCCTTTAAAA
GACGACGGGCCAAGGTT
GGCGAGTCATGCAGGATGA
CCAGCTGAAAAGGTTCCTGAA
CCAAATGCCAGAACCCAACATTGATAGTCC
GCATGCTTCGATGTGGCATA
CCCATCCAGAAGCCAAACTG
CTGCAGAAGAAAGAGGAGCTATCC
TCCTTGCAGAGGCCAAAATC
77
128
68
83
104
90
75
78
79
73
72
86
70
70
75
82
75
66
80
74
83
74
68
87
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Bisulfite Sequencing Analysis (BSA)
TTTTCGAGGGGTATAAGGAGAGTTTATTTT
GTTTGGAATTTTAGTATTTTGGGAG
GGAGAGGGTTTGGGTATGTAA
TATTTTTTTGTAGGGAGTTGGT
TTTTTATTGATTTTTTTTGTAAAAG
NDRG2
CSE1L
HSP1
PRDX6
SOX9
CCAAAAACTCTAACTCCTAAATAAACA
CTCTAACCATACCAACAAACTTCAC
CAAAAAAATAAAATAAACCTAAAAAAC
TAACATCCTTCAAACACTATAAACC
ATACCAAAATTTTAATACCTTCTCC
320
285
194
279
388
53
60
56
56
53
Methylation-Specific PCR (MSP) assay?
AGAGGTATTAGGATTTTGGGTATGA
AGAGGTATTAGGATTTTGGGTACG
GGTAAATTTATTTGGGTATTGA
TAGTGGTAAATTTATTCGGGTATCG
TTATTAGAGGGTGGGGTGGATTGT
TTATTAGAGGGTGGGGCGGATCGC
GTGTTTTATTGTGGAGTGTGGGTT
TATTGCGGAGTGCGGGTC
TTTTGATGTAGATGTTTTATTAGGTTGT
ACGTAGACGTTTTATTAGGGTCGC
NDRG2_UnM
NDRG2_M
NDRG2_UnM_2
NDRG2_M_2
p16-UnM
p16-M
APC-UnM
APC-M
MLH1-UnM
MLH1-M
CCACTAAAAAAACAAAAATCTCACC
GCTAAAAAAACGAAAATCTCGC
CAAAAACAAAATTAACCCTACAAA
CAAAAACGAAATTAACCCTACGA
CAACCCCAAACCCACAACCATAA
GACCCCCGAACCGCGAACCGTAA
CCAATCAACAAACTCCCAACAA
ACCACCTCATCATAACTACCCACA
ACCACCTCATCATAACTACCCACA
CCTCATCGTAACTACCCGCG
125
123
210
214
151
150
108
98
124
115
55
55
54
62
65
68
63
63
60
60
?UnM = unmethylated sequence; M = methylated sequence.
† AT = annealing temperature.

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Results
qPCR validation of deregulated genes
Among the genes higlighted as significantly deregulated in
our previous microarray study [14], 24 genes were
selected for further validation by using the quantitative
Real-Time PCR (qPCR), based on their cellular function,
such as transport, signal transduction, intracellular and
cell surface signalling, cell cycle, replication-repair of
DNA, and protein folding. (Table 3).
qPCR assay was applied to analyse mRNA expression of
the 24 selected genes, as well as of three control house-
keeping genes (GAPDH, EEF1A1 and HRPT1) in the
tumour and normal tissues taken from new cohort of 8
patients with CRC.
Compared to normal tissue with an expression profile
normalized to 1, in tumour samples 7 genes (ABCA8,
AQP8, CLCA4, HPGD1, PRDX6, SLC26A3, and STX12)
were uniformly under expressed in all 8 CRC patients, 3
genes (MXI1, NDRG2 and SCNN1B) in 7 patients, and 3
other genes (SGK2, NR3C2, and SGK1) in 6 patients. Eight
genes, that is CSE1L, GTF2IRD1, HSPH1, NEBL1, RFC3,
SLC12A2, FOXM1 and SOX9, were specifically over-
expressed in tumour tissues from 7 patients (Fig. 1). The
remaining 3 genes (TPX2, UBE2C, CCNB1) were excluded
from the analysis because of indeterminate qPCR values.
In general, qPCR results were in agreement with the
microarray data.
Gene expression before and after 5-Aza-CdR in colon cell
lines
To investigate the role of methylated CpG islands in the
modulation of gene expression, HCT-116, CaCo2 and
SW480 human colon cancer cell lines were cultured with
different doses of 5-Aza-CdR to induce a demethylation
event, and gene expression levels were measured by
means of qPCR. Two 5-Aza-CdR challenge regimens were
Table 3: List of genes selected from microarrays analysis comparing normal mucosa matched tumour colon tissue. The different
expression in tumoural tissue was showed.
Function/category GeneAccession no.
Microarray Data
P value
Gene description
Expression
Insulin Receptor Signaling
Transport
SGK1
CLCA4
NM_005627.1
NM_012128.2
0.059
0.052
down
down
Serum glucocorticoid regulated kinase (SGK)
chloride channel, calcium activated, family
member 4
solute carrier family 26, member 3
(SLC26A3)
aquaporin 8
sodium channel, nonvoltage-gated 1, beta
(S. Liddle)
ATP-binding cassette, sub-family A (ABC1),
member 8
syntaxin 12
nuclear receptor subfamily 3, group C,
member 2
MAX-interacting protein 1 (MXI1)
N-myc downstream-regulated gene 2
hydroxyprostaglandin dehydrogenase 15-
(NAD)
peroxidase, acidic calcium-independent
phospholipase A2
serumglucocorticoid regulated kinase (SGK)
forkhead box M1 (FOXM1)
GTF2I repeat domain-containing 1
(GTF2IRD1)
Sex determining region Y-box 9
heat shock 105 kD (HSP105B)
solute carrier family 12
restricted expressed proliferation associated
protein
ubiquitin carrier protein E2-C (UBCH10)
CSE1 chromosome segregation 1-like (yeast)
cyclin B1
nebulette protein
(NEBL, actin-binding Z-disc protein)
replication factor C (activator 1) 3 (38 kD)
Transport SLC26A3/DRANM_000111.1
0.044down
Transport
Transport
AQP8
SCNN1B
NM_001169.1
NM_000336.1
0.052
0.046
down
down
Transport (ATP binding)ABCA8NM_007168.1
0.040 down
Protein transport
Receptor activity (mineralcorticoid)
STX12
NR3C2
AI816243
NM_000901.1
0.051
0.049
down
down
Cell cycle (proliferation)
Cell cycle (differentiation)
Prostaglandin metabolism
MXI1
NDRG2
HPGD
NM_005962.1
NM_016250.1
J05594.1
0.053
0.039
0.050
down
down
down
Prostaglandin and leukotriene
Metabolism
Signal tansduction
Transcription factors
Transcription factors
PRDX6 NM_004905.1
0.050down
SGK2
FOXM1
GTF2IRD1
NM_016276.3
NM_021953.1
NM_016328.1
0.038
0.046
0.046
down
up
up
Transcription factor
ATP-binding/proteins folding
Transport solute carrier
Cell cycle (proliferation)
SOX9
HSPH1
SLC12A2
TPX2
NM_000346.1
BG403660
NM_001046
NM_012112.1
0.045
0.043
0.048
0.044
up
up
up
up
Cell cycle (progression)
Cell cycle (proliferation)
Signal Transduction
Focal adhesion
UBE2C
CSE1L/CAS
CCNB1
NEBL
NM_007019.1
NM_001316
Hs.23960
NM_006393.1
0.050
0.052
0.048
0.038
up
up
up
up
Replication and repairRFC3BC000149
0.049 up

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used to obtain expression data under different cellular
conditions: the acute treatment focused on moderate
DNA demethylation to minimize cell viability, and the
chronic treatment to maximize DNA demethylation (see
Additional file 1).
Ten out of the 21 validated genes, i.e. ABCA8, AQP8,
HPGD, PRDX6, SLC26A3, STX12, NDRG2, MXI1, SGK2,
and SCNNB1, were analysed for the impact of their DNA
hypermethylation on epigenetic events (Fig. 2). Other
genes had no epigenetic influence and were, therefore,
excluded from the analysis. Indeed, the underexpression
of NR3C2 and SGK1 has been related to aldosterone reg-
ulation pathway [22], whereas the 14-3-3ε gene modu-
lates the CLCA4 gene by interacting with the calmodulin-
dependent pathway [23]. Demethylation of the 5'-UTRs
Logarithmic expression profile value of twenty-one genes determined by quantitative reverse transcription-PCR by using the Comparative CT method
Figure 1
Logarithmic expression profile value of twenty-one genes determined by quantitative reverse transcription-
PCR by using the Comparative CT method. Three housekeeping genes were used to normalize input cDNA for each
sample with colorectal cancer, with cDNA of normal tissue used as calibrator. Crosses represents mean of triplicate determi-
nations. *P < 0.05; ° P < 0.01; ^ P < 0.001.
ABCA8^
AQP8^
CLCA4^
CSE1L°
FOXSM1°
GTF2ird1*
HPGD°
HSPH1°
MXI1*
NDRG2°
NEBL1°
NR3C2°
PRDX6°
RFC3*
SCNN1B°
SGK1^
SGK2*
SLC26A3^
SLCI2A2^
SOX9°
STX12*
0,000000
1,500000
3,000000
4,500000
6,000000
EXPRESSION PROFILE VALUE
R
R
R
R
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RR
R
R
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R
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R
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R
R
R
R
R
R
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R
R
R
R
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R
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R
R
R
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R
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R
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R
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R
R
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R
R
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R
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R
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R
R
R
R
R
R
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R
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R
R
R
R
R
R
R
R
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R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
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R
R
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R
R

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of some genes with a concomitant increase in mRNA
expression was documented in cell lines (Fig. 2). For
HPGD and NDRG2 genes a 1.4 and 1.3-fold increase in
CaCo2, a 7.0 and 1.6-fold increase in HCT116, and a 4.1
and 1.3-fold increase in SW480, respectively, was
observed. PRDX6 gene expression increased 1.5 and 3.0-
fold in Caco2 and HCT116 cells, respectively. MXI1
showed a 1.8 and 5.0-fold increase in Caco2 and SW480
cells, respectively (Fig. 2).
In silico search verification and Bisulfite Sequencing Analysis (BSA)
Two of the demethylated genes had CpG islands overlap-
ping their putative promoter regions at in silico confirma-
tion. By means of the MethPrimer software, we found 16
CpGs islands located between nucleotides 20,563,460 and
20,564,147 in NDRG2 genes (Fig. 3A and Additional file 2),
and 26 CpGs islands located immediately at the 5' of the
transcription start site and exon 1 (nucleotides 171,170,862
and 171,713,160) in the PRDX6 gene (Fig. 3A).
The methylation status of promoter regions of these puta-
tive tumour-suppressor genes was assessed by BSA in
untreated cell lines, in tumour matched to normal tissues
of one patient, in vitro methylated DNA (IVD) and in nor-
mal lymphocytes (NL). PRDX6 showed dense methyla-
tion only in IVD; NDRG2 showed a significant
methylation in cell lines and in tumour tissue compared
to normal tissue, suggesting a potential epigenetic regula-
tion of the gene (Fig. 3B and Additional file 3). Full meth-
ylation in all 16 CpG sites of the NDRG2 gene was found
in HCT116 and CaCo2 cell lines, and partial methylation
at the 3th CpG site in the SW480 cell line (Fig. 3B).
Histograms depict expression levels of AQP8, HPGD, PRDX6, MXI1, NDRG2, SCNNB1 and SGK2 genes in CaCo2 cell line (A), HCT116 cell line (B) and SW480 cell line (C) before and after exposure to 5-Aza-CdR determined by quantitative real-time PCR using the Comparative CT method
Figure 2
Histograms depict expression levels of AQP8, HPGD, PRDX6, MXI1, NDRG2, SCNNB1 and SGK2 genes in CaCo2
cell line (A), HCT116 cell line (B) and SW480 cell line (C) before and after exposure to 5-Aza-CdR determined
by quantitative real-time PCR using the Comparative CT method. 2^DCt indicates the ratio between the values of CT
normalized to three housekeeping genes and compared to cDNA of untreated cells used as calibrator. Error bars indicate
standard deviation from triplicate experiments. p16 gene was used as positive control in the experiments. Statistical signifi-
cance threshold p-value (p < 0.001) for both acute and chronic treatment are shown. Asterisks indicate P < 0.05 values and
double asterisks indicate P < 0.001 values.
??
?????????????????????
?
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???
???????
?
(A)
CaCo2
(B) HCT116
(C) SW480

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Quantitation of NDRG2 methylation in paired tumour and
normal tissue samples of CRC patients
To determine whether hypermethylation of the NDRG2
gene could be ascertained in primary CRC (Table 1), BSA
of 30 primary colon tumour tissues matched to normal
tissues was performed. When compared to their paired
normal tissues, a relative increase of methylation in
tumours was observed in 19 of 30 (63%) CRC patients
(Fig. 4), but the increase was significant in only 3 patients
(χ2 > 5, df = 1, p < 0.05). In four tissue pairs, the relative
methylation was apparently decreased, likely due a low
sensitivity of the detection method. In the remaining
patients figures were unchanged.
Methylation-specific PCR (MSP) assay in colon cancer cell
lines and primary CRC samples
MSP was performed to examine the methylation status of
CpG islands identified in the NDRG2 gene (see Addi-
tional file 3). The methylation status of the gene was com-
pared with that observed in three usually hypermethilated
genes (p16, APC, and MLH1) in 3 colon cancer cell lines
(HCT116, CaCo2 and SW480) and in 30 paired tumour-
normal tissues. CpG methylation in NDRG2 was detected
in all cell lines and in 8 of the 30 (27%) colorectal cancer
patients (Table 4). No methylation was detected in 30
samples from normal tissue. Hypermethylation of APC,
p16, and MLH1 genes in tumour tissue was found in 3
(10%), 4 (13%) and 6 (20%) patients, respectively, but
not in normal colonic tissue (Table 5).
When relating the NDRG2 methylation status to clinical
pathologic features, no association with age, gender,
tumour site, and MSI status was observed. Conversely, a
significant correlation was found between the NDRG2
methylation and the AJCC stage of the cancer (Z test, p <
0,05) (Table 4).
CpG islands present in putative promoter regions of the two genes of interest
Figure 3
CpG islands present in putative promoter regions of the two genes of interest. A. Promoter structure of NDRG2
gene (the sequence are shown in Additional file 2). The black arrows correspond to NDRG2 primers for BSA assay, the grey
arrows correspond to NDRG2_M_2 and NDRG2_M primers for MSP assays. B. CpG islands present in NDRG2 (numbered
from 1 to 16) and PRDX6 (numbered from 1 to 26) genes obtained by MethPrimer software. Methylation status of CpG sites in
vitro methylated DNA (IVD), normal lymphocytes (NL), three colon cancer cell lines, and in normal (N) and tumor (T) tissue of
one patient with colorectal cancer. Methylated and unmethylated cytosine residues are indicated with filled and small circles
while open circles denote partially methylated sites.
A
B

NDRG2
20,564,147
(-2900)
20,563,460
(-2600)




IVD
NL
HCT116
CACO2
SW480
(N)
(T)
1 2 3 4 56789 1011 1213 14 15 16
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PRDX6 171,170,862
171,713,160


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HCT116
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(N)
(T)
??
|++|++||||||++||++||++ ++|||||++||++||++||||||||||||++||||++|++||||||||||++||||||||++||||++||++ |++||++|++|++||++|||++|++||||++
-2900 -2800-2600-2400-2200-18000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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Discussion
Carcinogenesis is a complex event characterized by the
progressive development of genetic and epigenetic aberra-
tions which ultimately result in loss of physiological con-
trol of cell growth and differentiation. The two most
important epigenetic mechanism are represented by the
DNA methylation, the conversion of cytosine into
methyl-cytosine catalyzed by the DNA methyltransferase
And histone modifications [24]. Changes in the DNA
methylation pattern may occur everywhere in the DNA
molecule. Global DNA hypomethylation generally occurs
in centromeric repeats and repetitive sequences and con-
tributes to carcinogenesis by causing chromosomal insta-
bility, reactivation of transposable elements, and loss of
imprinting [25]. Hypermethylation is especially frequent
in CpG islands, i.e. short DNA sequences rich in CpG
dinucleotides, mostly located in the 5'-untranslated
region (5'-UTR) of genes [24]. When CpG islands are
heavily methylated, transcriptional gene silencing gener-
ally occurs. Although the fine mechanisms of regulation
of the "epigenetic" machinery are still poorly understood,
the DNA methylation may switch on or off several genes
and, in particular, those regulating important biological
phenomena, such as cell growth and differentiation [25].
In normal cells, this epigenetic mechanism is involved in
several physiological events, such as the inactivation of X
chromosome in female cells, silencing either paternal or
maternal alleles of "imprinted" genes, and transcriptional
blocking of exogenous integrated genes potentially dan-
gerous for the cell life. However, aberrant DNA methyla-
tion is also relatively common in cancer cells and is likely
to play an important role in cancer initiation and progres-
sion [26].
Since the pioneer studies of Baylin et al. [27], it has been
widely recognized that cancer cells are characterised by
two opposite events: a global hypomethylation which
results in either up-regulation of proto-oncogenes and
induction of genomic instability, favouring both uncon-
trolled cell growth [9] and mutations, and CpG islands
hypermethylation of other genes, the so-called tumour-
suppressor-genes (TSG), which contributes to loss of the
negative control of the cell cycle [12]. Searching for up-
regulated oncogenes and down-regulated TSG is impor-
tant in basic science, especially when an epigenetic mech-
anism (hypomethylation or hypermethylation) is
suspected. In fact, oncogenes and TSG not only may eluci-
date the highly complex molecular derangement in cancer
cells, but also may be used as potential targets for new
therapeutic approaches. DNA methylation is a reversible
phenomenon which can be modulated by specific agents.
An example is represented by demethylating drugs which
can globally reduce the DNA methylation level of TSG
promoters, restoring their normal activity. Interestingly,
some in vitro experiments have shown that cancer cell
lines reverted to normal phenotype after treatment with
demethylating agent.
The current study was carried out with a three-step design.
First, we specifically looked at up- and down-regulated
genes not yet firmly associated with colon carcinogenesis,
and selected 24 genes for validation with qPCR. A straight
Methylation analysis of the NDRG2 promoter in 30 tumour and normal tissue pairs, evaluated by the bisulfite sequencing anal-ysis
Figure 4
Methylation analysis of the NDRG2 promoter in 30 tumour and normal tissue pairs, evaluated by the bisulfite
sequencing analysis. Data are expressed as the difference in methylation status between tumour and normal tissue.
Difference of relative Methylation (T- N)
4-
2-
0
2
4
6
8
16 1 1 1 62126
p= 0.023
p= 0.025 p= 0.012
Number of tissue pair

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correlation between results obtained from qPCR and
those from DNA microarray was found, implying that
DNA microarray technology is a reliable tool to search for
new genes significantly deregulated in cancer [28]. Sec-
ond, we selected 10 of 21 genes (ABCA8, AQP8, HPGD,
PRDX6, SLC26A3, STX12, NDRG2, MXI1, SGK2, and
SCNNB1) as possible targets of epigenetic modifications
in colon cancer, and after treatment with a demethylating
agent, seven of them showed a significant increase of
mRNA expression (AQP8, HPGD, PRDX6, MXI1,
SCNNB1, SGK2 and NDRG2). From an in silico screening,
only 2 genes (PRDX6 and NDRG2) were considered as
possible candidates for the presence of CpG islands in
their 5'-UTR. For the excluded genes, additional mecha-
nisms of transcriptional regulation were hypothesized to
be responsible for their differential expression. Third, to
evaluate the methylation status of PRDX6 an NDRG2
genes in normal and cancer tissues, as well as in colon
cancer cell lines, bisulphite sequencing analysis was used.
In the PDRX6 gene the methylation status was not differ-
ent from that observed in normal tissue. In the NDRG2
gene a significant methylation status either in colon can-
cer cell lines and in tumour tissue compared to normal tis-
sue was observed. The underexpression of the PRDX6
protein responsible for the red-ox regulation of the cell,
was found to be correlated with loss of function of
NKX3.1 gene, known as TSG [29].
Table 4: Comparison of the clinicopathological features of 30 CRC patients according to the presence of NDRG2 methylation
Number of CRC
with NDRG2 methylation (%)
Number of CRC
without NDRG2 methylation (%)
p value
Age (yrs):
Age <50
Age >50
Gender
Male
Female
CRC:
Familiar
Sporadic
Tumour location:
Proximal colon
Distal colon
4 (13.3)
4 (13.3)
5 (16.6)
17 (56.6)
ns
4 (26.7)
4 (26.7)
11 (73.3)
11 (73.3)
ns
2 (25.0)
6 (27.3)
6 (75.0)
16 (72.7)
ns
2 (22.2)
6 (28.6)
7 (77.7)
15 (71.4)
ns
AJCC stage:
0
I
IIa
IIIb
IV
0 1 (4.8)
2 (9.5)
7 (33.3)
3 (14.3)
8 (38.1)
ns*
1 (12.5)
1 (12.5)
0
6 (75.0) < 0,05^
MSI status1
High
Low
Stable
1 (25.0)
1 (33.4)
6 (27.3)
3 (75.0)
2 (66.7)
17 (72.7)
ns
Fisher exact test (2-tail); *Pearson Chi-square; ^Z test only for IV AJCC' stage
1Microsatellite instability (MSI) was determined by the mobility shift of PCR products using CC-MSI kit (AB Analitica s.r.l., Padova, Italy, http://
www.abanalitica.com), that include the Bethesda panel microsatellite (BAT25, BAT26, D5S346, D17S250 and D2S123) and other four
mononucleotide microsatellite loci (NR21, NR24, BAT40 and TGFβRII), in tumours. Tumours showing instability in four or more markers were
classified as high MSI, those showing it in two marker as low MSI, and those showing no instability as microsatellite-stable.
Table 5: Promoter gene methylation rates in tumour and normal tissue from patients with colorectal cancer (CRC) sorted by tumour
location
Proximal colon CRC (n = 9)
Tumour
Distal colon CRC (n = 21)
Tumour Normal Normal
APC
p16
MLH1
00
0
0
3 (14%)
3 (14%)
4 (19%)
0
0
0
1 (11%)
2 (22%)

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Using these approaches, the NDRG2 gene was selected for
further analysis because: (i) it was suppressed in all colon
cancer cell lines, (ii) its expression may be up-regulated in
all cell lines by 5Aza-CdR treatments, and (iii) it is
involved in important biological process such as cell
growth [30], differentiation [31] and apoptosis [32]. The
NDRG2 gene is a new member of the N-myc downstream-
regulated gene (NDRG) family, that is located on chromo-
some 14q11.2 and encodes for a 41 kDa protein. It has
been proposed that the NDRG2 gene is a candidate TSG,
and its expression is low or undetectable in several pri-
mary tumour and tumour cell lines [30,33,34]. Liu et al.
[35] revealed that the down-regulation reported in cancer
be driven by promoter methylation, mutation, and
genomic deletion of the NDRG2 gene. Recently, it has
been shown that expression of the NDRG2 protein is
modulated by the insulin-stimulated Akt-dependent
phosphorylation [36]. Several studies have suggested that
the NDRG2 mRNA is down-regulated or undetectable in
a number of human primary cancers, such as squamous
cell carcinoma, pancreatic cancer [37], glioblastoma [30],
and cancer cell-lines. Recently, Zhang et al. [38] have dem-
onstrated that c-Myc represses NDRG2 gene expression via
Miz-1-dependent interaction with NDRG2 core promoter
region, and this inverse regulatory relationship induces
cell differentiation and proliferation.
The MSP assay was used to check for NDRG2 methylation
status in 30 primary colon tumour tissues compared to
normal colonic mucosal samples. After sorting colon can-
cer patients by age, gender, tumour site, and MSI status,
no statistically significant association was observed
between these features and the NDRG2 methylation. Nev-
ertheless, there was a trend towards NDRG2 methylation
status with an advanced tumour stage of the CRC samples,
with significant value detected in patients with AJCC stage
IV (p < 0.05). These results are in agreement with those
reported in other cancer types [34,39] where NDRG2
expression is reduced in high-grade compared to low-
grade tumours. In particular, Lorentzen et al. [39] sug-
gested that in CRC samples the down-regulation of
NDRG2 expression occurs during the progression from
adenoma to carcinoma.
Conclusion
In conclusion, we showed that NDRG2 expression is fre-
quently suppressed in colon cancer cell lines in conjunc-
tion with aberrant DNA methylation, and that the loss of
expression of this gene could be related to advanced colon
tumour stage.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AP, FP and AA wrote the manuscript with edits from all
co-authors. RC designed and performed the methylation
experiment. GM and BA designed and performed the
qPCR experiment. MC designed the microarray patterns.
RM, AD, NA performed the statistical analysis of microar-
ray. SF performed the statistically analysis. MC deposited
the data in Array Express. AP and FP conceived the project.
All authors have read and approved the manuscript.
The microarray data are accessible through ArrayExpress
accession number E-MTAB-57.
Additional material
Acknowledgements
We thank Dr. Pierluigi Di Sebastiano for colon cancer sample collection;
Dr. Mirco Fanelli for critical reading of the manuscript. This work was sup-
Additional File 1
Supplementary Table. In table are shown the expression value (2^DCt),
error standard (SE), t-test and p-value of ABCA8, AQP8, HPGD,
PRDX6, SLC26A3, STX12, ENACB1, SGK2, MXI1, NDRG2 and p16
genes determined by quantitative real-time PCR, using the Comparative
CT, before and after exposure to 5-Aza-CdR. 2^DCt indicates the ratio
between the values of CT normalized to three housekeeping genes and
compared to cDNA of untreated cells used as calibrator. Two 5-Aza-CdR
challenge regimens (acute and chronic treatment) were used to obtain
expression data.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1755-
8794-2-11-S1.pdf]
Additional File 2
Sequence of NDRG2 promoter gene. Sequence of NDR2 gene promoter
region located between 20,564,147 and 20,563,460 nucleotides. The
300-bp region contains 16 CpG (boxes, numbers are indicated above) was
analyzed by both bisulfite-sequencing and methylation specific PCR and
the position of the primers (see Table 2) are indicated by horizontal black
and grey arrows, respectively. Primers NDRG2_M_2 (grey arrows) was
downstream and amplify a fragment of 124-bp.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1755-
8794-2-11-S2.pdf]
Additional File 3
Bisulfite-sequencing assay (BSA) and Methylation-specific PCR (MSP).
A) Demostration of NDRG2 promoter methylation by bisulfite-sequenc-
ing from: in vitro methylated DNA (IVD), normal lymphocytes (NL),
CaCo2 cell line, normal (N) and tumour (T) tissue of one patient. Note
methylation of 4 depict CpG islands (CpG sites 13–16). B) Methylation-
specific PCR of NDRG2 gene in two colon cancer cell lines (HCT116 and
CaCo2), in normal lymphocyte (NL) and in vitro methylated DNA
(IVD). U, primers specific for unmethylated DNA; M, primers specific for
methylated DNA.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1755-
8794-2-11-S3.pdf]

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ported by "Ministry of Italian Health" grants RC0502GA07 and
RC0604GA52, through Research Unit of Gastroenterology, "Casa Sollievo
della Sofferenza" IRCCS, San Giovanni Rotondo (FG), Italy.
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Pre-publication history
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