Seung-Hyun Bang, Seung-Koo Lee, Asan Institute for Life
Sciences,University of Ulsan College of Medicine, Seoul 138-736,
Kyu-Young Song, Department of Biochemistry, University of
Ulsan College of Medicine, Seoul 138-736, Korea
In-Chul Lee, Department of Pathology, University of Ulsan
College of Medicine, Seoul 138-736, Korea
Supported by a Korea Research Foundation Grant,
Correspondence to: Professor In-Chul Lee, Department of
Pathology, University of Ulsan College of Medicine, 388-1 Poongnap-Dong,
Songpa-Gu, Seoul 138-736, Korea.
Aim: To analyze
the expression profiles of premalignant and/or preclinical lesions
of gastric cancers.
Methods: We analyzed the expression profiles of
normal gastric pit, tubular adenoma and carcinoma in situ using
microdissected cells from routine gastric biopsies. For the DNA
microarray analysis of formalin-fixed samples, we developed a simple
and reproducible RNA extraction and linear amplification procedure
applying two polymerase-binding sites. The amplification procedure
took only 8 h and yielded comparable DNA microarray data between
formalin-fixed tissues and unfixed controls.
comparison with normal pit, adenoma/carcinoma showed 504
up-regulated and 29 down-regulated genes at the expected false
significance rate 0.15%. The differential expression between adenoma
and carcinoma in situ was subtle: 50 and 22 genes were up-,
and down-regulated in carcinomas at the expected false significance
rate of 0.61%, respectively. Differentially expressed genes were
grouped according to patterns of the sequential changes for the 憈endency
analysis in the gastric mucosa-adenoma-carcinoma sequence.
Conclusion: Groups of genes are shown to
reflect the sequential expression changes in the early carcinogenic
steps of stomach cancer. It is suggested that molecular carcinogenic
pathways could be analyzed using routinely processed biopsies.
ã 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Premalignant lesion; Preclinical lesion; Gastric
Lee CH, Bang SH, Lee SK, Song KY, Lee IC. Gene expression profiling
reveals sequential changes in gastric tubular adenoma and carcinoma
in situ. World J Gastroenterol
2005; 11(13): 1937-1945
Gastric cancer is one of the leading cancers worldwide.
Helicobacter pylori (H pylori) infection has been
associated with gastric cancer,
and the associated gastritis is regarded to be a major contributing
factor in carcinogenesis[3,4].
H pylori-infection causes selective neutrophil infiltration
to the proliferative zone of the gastric pits, which puts the
actively regenerating cells under continuous mutagenic pressure and
Acute foveolitis of the proliferative zone often induces extensive
genomic damage in the proliferating cells which may be distinguished
morphologically by the clear cellular changes, i.e. "the
malgun cell changes"[6,
7]. However, the premalignant lesions
and/or carcinogenetic pathways have not been characterized.
Gastric cancers are diverse in the biological behavior as well as
histogenesis. A considerable part of gastric cancers appears to
develop from preexisting adenomas
while de novo carcinogenesis also exists.
They may be distinct not only in histogenesis but also in
clinicopathological behaviors. To prevent and control gastric
cancers, it would be important to figure out the carcinogenetic
steps in both histopathological and molecular levels. The sequential
expression changes from premalignant lesions to early stage cancers
would provide insights into the carcinogenetic pathways and the
detection of molecular targets for a specific treatment.
It has been a goal of oncogenomics to analyze the sequential
expression changes at the premalignant and/or preclinical stage.
Despite widespread application of DNA microarray analysis, it has
remained to be a difficult goal to achieve because early lesions are
often so small and subtle that they are only detected at the
microscopic level convincingly, and consequently, are mostly
available as formalin-fixed, paraffin-embedded samples as remnants
of histopathological examination.
Once distinguished, the early lesions may be microdissected out from
histological sections for the expression profiling analysis.
Formalin-fixation provides excellent histological preparation for
the detection of subtle premalignant lesions. For instance, the
malgun cell change of gastric epithelium in H pylori
gastritis may not be seen on frozen sections or other fixations.
Formalin-fixation and paraffin-embedding also confer the tissue
stability for archival storage, and thus, has remained as the
standard protocol for tissue preparation in pathology laboratories.
However, formalin causes extensive base modifications of nucleic
which make it difficult to recover intact RNA and/or amplification
necessary for the microarray analysis. Thus, a simple and reliable
way of expression profiling of formalin-fixed tissue sections has
been sought for as one of the bridges which would connect medicine
Here, we present the expression profiles of normal gastric mucosa,
adenoma, and carcinoma in situ using microdissected cells
from formalin-fixed, paraffin-embedded sections. For the study, we
have developed a simple RNA preparation/amplification procedure for
the DNA microarray analysis applying two polymerase-binding sites.
The amplification procedure including PCR and an in vitro transcription
took only 8 h and provided comparable correlations with unfixed
counterparts. Depending on the availability of microdissected cells,
the procedure may be extended applying the second RNA polymerase.
Using the procedure, more than 500 genes were detected to express
differentially at the early stage of gastric carcinogenesis. They
were analyzed in groups according to the patterns of sequential
changes in the carcinogenetic pathway. Our data suggested that the
screening for cancer related genes would be facilitated by the
sequential expression changes at the early stage lesions using
archival samples. The procedure and analysis were described in
detail with pertinent information so that it may be reproduced
readily in hospitals as well as research laboratories.
MATERIALS AND METHODS
Cell lines and xenograft tumors
We used xenograft gastric cancer tissues for the development and
fine adjustment of the RNA extraction/amplification method. Human
gastric adenocarcinoma cell lines MKN45 and SNU484 were cultured in
DMEM with 5% PBS. Cells were harvested with trypsinization when they
reached 70% confluency. After being washed with PBS, ten million
cells were resuspended in 0.3 mL PBS, and injected into nude mice
subcutaneously. When the xenograft tumors reached 1 cm in diameter,
mice were killed by cervical dislocation and tumors were harvested.
MKN45 cells grew faster than SNU484 in culture and nude mice. Half
of the tumors were frozen immediately in liquid nitrogen, and the
rest were fixed in 10% buffered-formalin for 10 h at room
temperature. Fixed tissue samples were processed for routine
Tissue samples and microdissection
Ten gastric biopsies having tubular adenomas and/or
well-differentiated adenocarcinomas in situ were selected
randomly from the surgical pathology file of Asan Medical Center,
Seoul, Korea. Tubular adenomas were from eight male and two female
patients ranging from 53 to 69 years old. Carcinomas were from 7
males and 3 females patients ranging from 45 to 74 years old.
Controls were from 10 normal mucosa biopsies which were negative for
H pylori infection. This study was approved by the Clinical
Research Review Board of Asan Medical Center, Seoul, Korea.
Biopsies were fixed immediately in 10% buffered-formalin and
processed routinely. After the histopathological diagnosis,
additional 5 umol/L sections were taken from the paraffin blocks.
For the sectioning and H and E staining, all the solutions were
freshly made using DEPC-treated water, and the slides and
instruments were autoclaved. Cells were microdissected using an
AutoPix laser capture microscope system (Arcturus, Mountain View,
Total RNA was extracted twice from freshly frozen xenograft tissues
using TRIzol reagents (Invitrogen, Carlsbad,
CA) according to the manufacturer's
instructions. For the RNA
extraction from formalin-fixed tissues, deparaffinized sections were
removed from the slides by applying 200 proteinase K buffer [2% SDS,
10 mmol/L Tris-HCl (pH 8.0), 0.1 mmol/L EDTA]. Samples were
transferred into a microcentrifuge tube and incubated at 70 ℃
for 1 h to relieve the formalin-induced modifications. Then, 3
proteinase K (30
mg/mL, Intron biotechnology, Songnam, Korea) was
added, and incubated again at 55 ℃
for 1 h. RNAs were extracted with TRIzol similarly. The extracted
RNAs were precipitated in isopropanol with 5
mg linear acrylamide (Ambion,
Austin, TX), and the RNA pellets were resuspended in 10
nuclease-free water (Ambion). The quality of extracted RNAs was
checked using denaturing agarose gels. The amount of extracted RNA
samples and/or amplified RNA was measured using a RiboGreen RNA
quantitation kit (Molecular probes, Eugene, Oregon) according to the
protocol. Each measurement was
duplicated, and the average values were taken.
For the first strand synthesis, 100 pmoL of T7dT primers (100 pmol/mL,
Bioneer, Daejon, Korea) was added to the 10 mL of total RNA, and
incubated at 70 ℃
for 10 min. After primers were let to anneal to RNA templates by
incubating on ice for 10 min, 4
mL 5X first-strand buffer, 0.5
RNase inhibitor (40 U/mL, Promega, Madison, WI), 2
mL 0.1 mol/L DTT,
1 mL dNTPmix (10 mmol/L each, Roche, Mannheim, Germany), and 2
reverse transcriptase (Invtirogen, Carlsbad, CA) were added. The
mixture was incubated at 42 ℃
for 2 h.
For the second strand synthesis, 1 mL RNAse H (2
Carlsbad, CA) was added to the mixture, and incubated at 37 ℃
for 15 min, and at 95 ℃
for 2 min. Then, 1
mL random T3N6 primers (100
5-GCGCGAAATTAACCCTCACTAAAGGGAGANNNNNN-3) were added, incubated at 95
for 2 min, and placed on ice for 10 min. Then, 20 mL 5X
second-strand buffer (Invtirogen, Carlsbad, CA), 2 mL dNTPmix (10 mm
each), nuclease-free water 53.5 mL, 2.5 mL E. coli DNA
polymerase I (10 u/mL, Invitrogen, Carlsbad, CA) were added and
incubated at 16 ℃
for 2 h. The synthesized double-stranded DNA was purified using a
PCR purification kit (Qiagen, Valencia, CA) according to the
protocol. To retrieve DNA
efficiently, samples were eluted twice with 42 mL eluting solution.
Then the double stranded DNA was applied to PCR amplification. To
the elution, 10 mL 10X Advantage 2 PCR buffer (Clontech), 1 mL T7
promoter primers (100 pmol/mL,
5-CGGCCAGTGAATTGTAATACGACTCACT-ATAGGCG-3), 1 mL T3 promoter primers
(100 pmol/mL, 5-GCGCGAAATTAACCCTCACTAAAGGGAGAGGG-3), 2 mL 10 mmol/L
dNTP mix, and 2 mL advantage 2 polymerase mix (Clontech) were added.
PCR reaction was done in a Gene-Amp PCR 9600 system (PE Biosystems,
Foster City, CA) for 1 min at 95 ℃,
20 cycles for 30 s at 95 ℃
for 40 s at 65 ℃
for 5 min at 68 ℃,
for 7 min at 68 ℃.
PCR products were purified using a MinEluteTM
PCR purification kit (Qiagen, Valencia, CA), and were eluted using
10 mL nuclease-free water twice.
Following the PCR amplification, aRNA synthesis (in vitro transcription)
was performed using an AmpliScribeTM
T7 high yield transcription kit (Epicenter, Madison, WI) at 37 ℃
for 5 h in 40 mL of reaction volume. Synthesized aRNA was purified
using a RNeasy Mini kit (Qiagen).
For cDNA microarray analysis, stomach cancer-specific 14K cDNA
microarray chips were applied.
The probe labeling and hybridization were done using the
amine-modified random primer aminoallyl method.
Probes were synthesized from 10 mg unamplified total
RNA or 30 mg aRNA using 2 mg
amine-modified random primer (5-C6dTNNNNN-3, SIGMA Genosys, The
Woodlands, TX). For the xenograft study, SNU484 and MKN45 tumor
samples were labeled with Cy3 and Cy5, respectively. For the
microdissection study, either adenoma or carcinoma was labeled with
Cy3 and hybridized against the same normal control labeled with Cy5.
The labeled probes were mixed, and the volume was adjusted to 500 mL
by adding nuclease-free water. The final volume was adjusted to 17 mL by centrifugation (10 000 g) in the Microcon YM-30
(Millipore, Bedford, MA).
For the hybridization, 1 mL poly A (8 mg
/mL, Roche, Mannheim,
Germany), 1 mL Cot-1 DNA (10
mg/mL, Invitrogen, Carlsbad, CA), 1 mL
yeast tRNA (4 mg/mL, Invitrogen, Carlsbad, CA) were added to the
labeled probe, and denatured at 100 ℃
for 2 min, and cooled on ice. The probe was mixed with 20 mL 2
formamide hybridization buffer [50% formamide, 10 SSC, 0.2% SDS],
and applied to the DNA microarray. A glass cover slip was applied,
and the microarray was put in the hybridization cassette (TeleChem
International, Sunnyvale, CA). After being incubated overnight in a
water-bath, microarrays were washed with the first [2 SSC, 0.1% SDS]
and second wash solutions [0.5 SSC, 0.01% SDS] for 5 min,
respectively. Remaining water was removed from the slide by
centrifugation at 800 r/min for 2 min.
The arrays were scanned with a GenePix 4000B scanner (Axon,
Foster City, CA) at 10 mm
resolution. The PMT voltage
settings were varied to obtain the maximum
with <1% probe saturation. The
resulting images were analyzed
using the ImaGeneTM
4.0 (BioDiscovery, Los Angeles, CA) software. Spots having a
signal-to-noise ratio over 1.4 were screened and normalized for the
analysis. Pearson correlation coefficients of the global and
differentially expressed genes were calculated using the SPSS
software (SPSS Inc. Chicago, IL).
SAM analysis was done to detect differentially expressed genes in
microdissected adenomas and carcinomas.
The selected genes were divided into nine groups to show the
patterns of sequential changes among the normal pits, adenoma, and
carcinoma. First, genes were divided into three categories
arbitrarily according to the expression changes in adenomas compared
to the control: up-regulated (>1.4 times, log ratio >0.485),
down-regulated (<1.4 times), and 'unchanged'.
Then, each category was further divided into three groups according
to the expression changes in carcinomas compared to the control:
up-regulated (>1.2 times, log ratio >0.263), down-regulated
(<1.2 times), and 'unchanged'.
Expression profiling of fresh xenografts
Both MKN45 and SNU484 xenograft tumors consisted of solid cell
clusters, being reminiscent of poorly differentiated gastric
adenocarcinomas (data not shown). The expression profiles were
analyzed using unamplified samples, and the data were used as a
control for the development and fine adjustment of the amplification
conditions in the subsequent experiments using formalin-fixed
counterparts. The microarray data including those of microdissected
samples were deposited at the GEO (www.ncbi.nlm.nih.gov/geo/)
(accession numbers GSM20670-5).
The expression profiling of MKN45 and SNU484 xenograft tumors showed
that they had quite distinct expression patterns despite the
histopathological similarity. The up-regulated genes in SNU484 in
comparison with MKN45 included aldehyde dehydrogenase 1 family
member A1 (ALDH1A1), thymosin beta 4 (TMSB4X), activated leukocyte
cell adhesion molecule (ALCAM), collagen type XI alpha 1 (COL11A1),
and plectin 1 (PLEC1), etc. The up-regulated genes in MKN45
tumor included EBNA2 co-activator (p100), regenerating gene type-4 (REG-IV),
S100 calcium binding protein A4 (S100A4), replication initiation
region protein (60 ku) (RIP60), and carcinoembryonic antigen-related
cell adhesion molecule 6 (CEACAM6), etc. Considering that
MKN45 cells grew much faster than SNU484 cells in vitro and ex
vivo, some of the differentially expressed genes might be
related to cell growth and/or aggressiveness. For instance, S100A4
protein up-regulation was shown to associate with metastasis and
poor prognosis of stomach cancer and others.
RNA extraction and amplification from formalin-fixed samples
It was reported that sample heating relieved the extensive base
modifications of nucleic acids induced by formalin-fixation.
To make the procedure as simple as possible, we used only heating/proteinase
K treatment to observe how efficient and reproducible the RNA
extraction was. Samples were heated at 70 ℃
for 1 h before the proteinase K treatment for the RNA extraction.
RNAs were extracted from the fixed tissue sections of SNU484 and
MKN45 xenograft tumors using the protocol. The extracted total RNAs
were estimated to be 18.3 and 24.1 pg per fixed cell of MKN45 and
SNU484 tumors, respectively (Table 1). They corresponded to 63.1 and
77.7% of the total RNAs of MKN45 (29 pg) and SNU484 (31 pg) cells in
culture, suggesting that the extraction efficiency from formalin-fixed
cells was comparable to that from fresh counterparts.
The amplification procedure is depicted in Figure 1. The entire
procedure of RNA extraction and one-round amplification took only 8
h. The first strand cDNA was synthesized using T7dT primers as
Then, the second strand were synthesized using T3N6 primers, and 20
cycles of PCR amplification was done using the T3 and T7 promoter
primers as described in the Materials and Methods. The resulting PCR
products were used for the in vitro transcription using
either T7 or T3 polymerase, which yielded similar amplification
rates (data not shown). If the amplified products were not enough
for the microarray analysis, in vitro transcription might be
repeated using either T3 or T7 polymerase, whichever was unused in
the first round.
The amplification results from two xenograft tumors are summarized
in Table 1. To see whether RNAs from formalin-fixed samples were
adequate for the amplification, we first checked the amplification
only in vitro transcription using T7 polymerase. The amounts
of aRNAs of fixed MKN45 and SNU484 tumors were increased 6.43 and
6.08 times, respectively. Provided the mRNA amount was 1% of the
total RNAs, the amplification rates were estimated to be 643 and 608
times, respectively. For the fresh samples, the amplification rates
after the in vitro transcription were estimated to be 761 and
1 066 times for MKN45 and SNU484 tumors, respectively, showing that
the amplification efficiencies of fixed samples were comparable to
those of fresh counterparts. When fixed MKN45 and SNU484 tumors were
processed for a complete round of amplification, the amplification
rates were 210 600 and 225 080 times, respectively. Since 20-40 mg
of aRNA was enough for a DNA microarray depending on the
hybridization methods, it was estimated that less than 1 000 cells
were required for a DNA microarray analysis.
Amplified aRNAs were hybridized on 14K cDNA microarrays, and the
data were compared with those of the unamplified fresh tissue
controls. Upon the filtering, 13 706 and 13 407 spots were left for
the aRNA and controls, respectively. The correlation coefficient of
global gene expression was 0.718. The correlation coefficient was
increased to 0.858, when 500 genes differentially expressed between
SNU484 and MKN45 more than twice were compared.
Table 1 Amplification
efficiency of formalin-fixed tissues
RNA (ng) (1mRNA)
from fresh and formalin-fixed xenograft tumors of MKN45 and SNU484
cells were amplified under the same conditions as described in the
Materials and Methods.
1mRNA : estimated
to be 1/100 of total RNA (ng). 2Cell
number: estimated number of cells from tissue sections required to
obtain the starting RNA (20ng). 3Amplification
folds: aRNA/estimated starting mRNA. 4T7
IVT: in vitro transcription using T7 RNA polymerase. 5PCR-T7
IVT: 20 cycles of PCR followed by T7 IVT.
1 (PDF) Schematic
view of amplification procedure.
Expression profiling of microdissected gastric lesions
We, then, analyzed the expression profiles of normal gastric
epithelium, adenoma, and adenocarcinoma in situ using
microdissected cells from 'routine'
gastric biopsies. For the sequential analysis of early lesions only
the carcinomas without stromal invasion were included. For the
normal control, pit epithelia were taken only from the proliferative
zone in order to minimize the 'contamination'
by genes related to nonspecific cell proliferation in adenomas and
carcinomas (Figure 2A). To avoid cells with DNA damage, any
epithelial cells showing the malgun cell change were excluded.
Adenomas had glands which were rather regular in size and
orientation, which were much more than the normal glandular
distribution (Figure 2B). Epithelial cells were uniformly columnar
and well oriented. Nuclei were hyperchromatic with a high
nucleus/cytoplasm ratio. Carcinomas consisted of glands which were
mildly irregular in size and arrangement in comparison with those of
adenomas (Figure 2C). Cells were not particularly different from
adenoma cells in size, but nuclei were ovoid and nucleoli were more
(PDF) Histologic view (left) and microdissected cells (right)
of the normal gastric mucosa. (A) adenoma; (B) and carcinoma in
situ; (C) (HE stain. x20). Compared to adenomas, carcinoma in
situ had only mild glandular complexity.
For the experiment, a total of 10 000 cells from 10 adenomas and/or
carcinomas and 20 000 normal control cells were microdissected and
pooled, respectively. After the amplification, 135.2, 77.3, and 77.9
ug of aRNAs were obtained from the normal pit, adenoma, and
carcinoma, respectively. Microarray hybridization was duplicated for
adenomas and carcinomas, respectively, using the same control of
normal pit epithelium.
The DNA microarray data were analyzed using the SAM method.
At the expected false significance rate of 0.15%, 504 up-regulated
and 29 down-regulated genes were detected in adenoma and carcinoma
(Figure 3A). Genes with differential expressions are summarized in
Tables 2 and 3. The difference of expression between adenoma and
carcinoma was subtle. Fifty up-regulated and 22 down-regulated genes
were detected in carcinomas compared to adenomas at the expected
false significance rate of 0.61% (Figure 3B).
(PDF) SAM analysis of gastric adenoma/carcinoma
microdissected from formalin-fixed biopsies. A: At the expected
false significance rate of 0.15%, 504 up-regulated and 29
down-regulated genes in adenoma/carcinoma vs normal control
(Tables 2 and 3); B: At the expected false significance rate of
0.61%, 50 up-regulated and 22 down-regulated genes in carcinomas vs
Table 2 Up-regulated
protein A (17 ku)
(RNA) II (DNA directed) polypeptide C, 33 ku
glucosidase II alpha subunit
3-monooxygenase/tryptophan 5-monooxygenase activation protein,
(Asp-Glu-Ala-His) box polypeptide 9
cycle progression 2 protein
binding clathrin assembly protein
(C-X-C motif) ligand 5
seq X - hypothetical protein MGC3103
helix-loop-helix ubiquitous kinase
factor pathway inhibitor (lipoprotein-associated
antigen (p24): tetraspanin superfamily
finger protein 276
Tat interactive protein, 60 ku
precursor cell expressed, developmentally down-regulated 5
factor acetylhydrolase, isoform Ib, beta subunit 30 ku
5-phosphate isomerase A (ribose 5-phosphate epimerase)
guanine nucleotide exchange
binding protein S1, serine-rich domain
nuclear matrix-associated protein
kinase, AMP-activated, gamma 1 non-catalytic subunit
group box 1
rejection antigen (gp96) 1
associated protein-1 (ubiquitin carboxy-terminal hydrolase)
related, matrix associated, actin dependent regulator of
chromatin, subfamily d, member 2
nuclear ribonucleoprotein M
protein complex 3, beta 1 subunit
resistance related protein CRR9p
factor 2 (spasmolytic protein 1)
5, subtypes A and C, tracheobronchial/gastric
islet-derived family, member 4
to calpain 8
panbronchiolitis critical region
factor 1 (breast cancer, estrogen-inducible sequence expressed
carrier family 25 (mitochondrial deoxynucleotide carrier),
galactoside-binding, soluble, 3
stem cell antigen
voltage-gated channel, Isk-related family, member 3
in gastric cancerGDDR
(or cysteine) proteinase inhibitor, clade H (heat shock
protein 47), member 1, (collagen binding protein 1)
finger protein 265
P450, family 2, subfamily S, polypeptide 1
gradient 2 (Xenepus laevis) homolog
To analyze the genes according to the patterns of sequential
expression changes, we further divided the genes into nine groups as
described in the Materials and Methods (Figure 4). Groups 1, 2, and
3 included genes that were up-regulated in adenomas, and kept on
being up-regulated, maintained, and down-regulated in carcinomas
(47, 178, and 117 genes), respectively. Groups 4, 5, and 6 included
genes with minimal to mild variations in adenomas, which were then
up-regulated, maintained, and down-regulated in carcinomas (57, 73,
and 2 genes), respectively. Groups 7, 8, and 9 included genes which
were down-regulated in adenomas, and then, up-regulated, maintained,
and kept on being down-regulated in carcinomas (28, 10, and 9
genes), respectively. It was expected that such a 'tendency
analysis' at multiple points of carcinogenetic steps would help
recognize groups of genes with a distinct biological significance at
the early stage of gastric carcinogenesis. As discussed later, many
genes associated with cancers were included in the groups that were
expected to implicate in the carcinogenesis significantly. Many
novel genes with unknown functions were also included in each group.
(PDF) Selected genes of 9 groups showing the patterns of
sequential expression changes. N: normal, A: adenoma, C: cancer.
We analyzed the expression profiles of early lesions of gastric
cancer using formalin fixed biopsies. Our goal was to develop a
simple and practical procedure for the RNA extraction and
amplification that may be applied to the reproducible analysis of
formalin-fixed samples in hospitals and research laboratories. It
has been suggested that the RNA extracted from formalin-fixed
tissues using various methods could be used for quantitative
analysis in many fields of biological research[18-25].
Our data suggested that the simple procedure could improve not only
the extraction efficiency but also the quality of RNA that were good
enough for the DNA microarray analysis.
Our strategy for the amplification was to introduce two RNA
polymerase binding sites that might be used for both PCR and in
vitro transcription (s). We applied T7dT primers and random T3N6
primers to the first and second strand DNA synthesis, respectively.
The T3N6 primers yielded a similar reproducibility but a better
efficiency in comparison with T3N9 primers, which were shown to
reproduce similar results after repeated rounds of amplifications
(data now shown). Our 2 binding sites strategy reduced the
unnecessary preparation time for each step so that the entire
procedure of PCR and one round in vitro transcription took
only 8 h. When it was necessary, the second round of in vitro
transcription might be added using either T3 or T7 RNA polymerase,
whichever was not used in the first round. Alternatively, two
consecutive rounds of in vitro transcriptions might be done
omitting the PCR step, although it would be more time-consuming. In
any case, the flexibility is of a critical advantage when the
availability of cells is limited. Because the bias induced by the
random primer hybridization would have already been reflected in the
PCR products, the second round amplification was not expected to
introduce significant errors.
The PCR was applied to the linear amplification for DNA microarray
Iscove et al.
limited the elongation time so that only
extreme 3 ends of similar length were amplified. Aoyagi et al.
did the in vitro transcription first, and then, applied
adaptors to the cDNAs for the PCR amplification. In contrast, our
method was simple and did not require any additional procedure for
the PCR. We allowed sufficient elongation time to assure a complete
cycle for each cDNA. PCR amplification was also applied to the
simultaneous amplification of multiple genes.
Formalin-fixed tissues produced comparable data with those of the
unfixed control (global correlation coefficient 0.718).
Interestingly, the correlation coefficients increased considerably
when only differentially expressed genes were analyzed. The
increased correlation may suggest that the amplification of abundant
genes is relatively privileged. Anyway, it is an encouraging finding
for the practical application, because the aim of most DNA
microarray screening is to detect the differentially expressed genes
rather than global gene profiling.
Our data suggested that the subtle differential expression between
adenoma and carcinoma in situ could be detected convincingly.
DNA microarray analysis was based on competitive hybridization, and
so far, most studies have been designed to compare the expression
profiles of two distinct lesions that may or may not be directly
related. Our approach of multi-point comparison may provide a unique
opportunity for the "tendency
analysis", which could be helpful for the screening of biologically
significant genes in the carcinogenesis and progression of diseases.
Groups 1, 2, and 3 included genes that were up-regulated in
adenomas, and kept on being up-regulated, maintained, and
down-regulated in carcinomas. They included many genes that were
up-regulated in cancers and implicated in the carcinogenesis,
suggesting that our data were quite reproducible. Group 1 included
many genes which were implicated in carcinogenesis and/or
up-regulated in cancers: junction plakoglobin (gamma catenin: JUP),
squamous cell carcinoma antigens recognized by T cell 3 (SART3),
DEAH (Asp-Glu-Ala-His) box polypeptide 9 (DHX9), mucin 4 (MUC4),
ribosomal protein L15 (RPL15), microphthalmia-associated
transcription factors(MITF), and fusion (involved in t (12;16) in
malignant liposarcoma) (FUS). JUP and MITF have been implicated in
the Wnt pathway, which is one of the main carcinogenic pathways[28,29].
Groups 2 and 3 included A-Raf (v-raf murine sarcoma 3611 viral
oncogene homolog 1: ARAF1), v-akt murine thymoma viral oncogene
homolog 2 (AKT2), v-jun sarcoma virus 17 oncogene homolog (JUN),
glioblastoma amplified sequence (GBAS), tumor rejection antigen 1
(TRA1), tumor protein D52-like 2 (TPD52L2), hypoxia up-regulated 1
(HYOU1), heparan sulfate proteoglycan 2 (HSPG2), polo-like kinase (PLK),
high-mobility group box 1(HMGB1), and tumor protein, translationally-controlled
1 (TPT1). In addition, nuclear proteins such as nucleolin, nucleolar
protein family A, member 1 (NOLA1), nucleoporin 153 ku (NUP153),
nucleoporin (Nup37), and lamin B receptor (LBR) were also included.
The up-regulation of nucleolar proteins was compatible with the
continuous enlargement of nucleoli in adenoma and carcinomas.
In group 4, genes up-regulated in carcinoma but not in adenoma,
villin 2 (VIL2) and S100A4 were included. Villin 2 plays a key role
in cell surface structure adhesion, migration, and organization, and
has been reported to be associated with invasiveness of esophageal
S100A4 was also up-regulated in MKN45 cells in comparison with
SNU484 cells, and was shown to associate with metastasis and poor
prognosis of stomach cancers and others.
Nucleolin was also included in this group. Group 6 consisted of two
genes that were down regulated in carcinoma: progesterone receptor
membrane component 2 (PGRMC2) and acyl-coenzyme A dehydrogenase, C-2
to C-3 short chain (ACADS).
Groups 7, 8, and 9 included down-regulated genes in gastric cancers.
Group 7 included mucin 5 subtypes A and C, tracheobronchial/gastric
(MUC5AC) and trefoil factor 1 (TFF1), and group 8 included fatty
acid binding protein 1 (FABP1). TFF1 has been reported to be
down-regulated in most gastric cancers.
It should be noted that FABP1 is one of the tamoxifen-target
proteins, the block of which might be related to the anti-cancer
Group 9 included trefoil factor 2 (TFF2) which was down-regulated in
gastric cancer (GDDR). GDDR was reported to be a down-regulated gene
in stomach cancer.
It is a transmembrane protein homologous to carbonic anhydrase-like
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expression analysis at multiple points in a pathogenic pathway
facilitates the detection of biologically relevant genes in the
development and progression of diseases. The protocol may be applied
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Editor Wang XL and Guo SY Language
Editor Elsevier HK