Tài liệu Báo cáo khoa học: MicroRNA-23a promotes the growth of gastric adenocarcinoma cell line MGC803 and downregulates interleukin-6 receptor pdf

MicroRNA-23a promotes the growth of gastric
adenocarcinoma cell line MGC803 and downregulates
interleukin-6 receptor
Li-Hua Zhu
1,2,
*, Tao Liu
1,
*, Hua Tang
1
, Rui-Qing Tian
1
, Chang Su
1
, Min Liu
1
and Xin Li
1
1 Tianjin Life Science Research Center and Basic Medical School, Tianjin Medical University, Tianjin, China
2 Department of Pathobiology, Bioscience Faculty, North China Coal Medical College, Tangshan, China
Introduction
Recent findings have shown that, as regulation factors
of gene expression, microRNAs (miRNAs) are often
overexpressed or downregulated in a number of human
malignancies, and some can also function as tumor
suppressors or oncogenes [1]. miRNA genes are fre-
quently located in cancer-associated genomic regions
or in fragile sites [2]. Previous studies have identified
cancer-specific miRNAs in many types of cancer,
including B-cell chronic lymphoblastic leukemia [3],
lung cancer [4], colorectal cancer [5,6], breast cancer
[7], papillary thyroid cancer [8] and hepatocellular

carcinoma [9]. Gastric cancer is the second most
common cause of cancer deaths worldwide. Previous
studies have revealed several genes related to human
gastric cancer [10,11], but the common molecular mech-
anisms of gastric cancer remain to be elucidated. Gastric
cancer is a complex genetic disease, in which the expres-
sion of many specific genes, known as oncogenes or
tumor suppressors, is abnormally altered. It has been
reported that microRNA-34 (miR-34) is involved in the
p53-directed tumor suppressor network in gastric cancer
[12]. Our previous study showed that miR-27a functions
as an oncogene in gastric adenocarcinoma by targeting
prohibitin [13]. In the current study, we examine the
differential expression of miR-23a in gastric cancer and
normal gastric tissues, and identify that miR-23a can
Keywords
cell growth; gastric adenocarcinoma; IL6R;
miR-23a; target gene
Correspondence
H. Tang, Tianjin Life Science Research
Center and Basic Medical School, Tianjin
Medical University, Tianjin 300070, China
Fax: +86 22 23542503
Tel: +86 22 23542503
E-mail:
*These authors contributed equally to this
work
(Received 11 April 2010, revised 15 June
2010, accepted 12 July 2010)
doi:10.1111/j.1742-4658.2010.07773.x

MicroRNAs are an evolutionarily conserved class of endogenous noncod-
ing RNAs that modulate gene expression at the post-transcriptional level.
Recently, microRNA-23a (miR-23a) has been found to function as a
growth-promoting and antiapoptotic factor in hepatocellular carcinoma
cells. Our previous study showed that miR-23a was significantly upregulat-
ed in gastric adenocarcinoma tissues. In this study, we found that miR-23a
promoted the proliferative potential of gastric adenocarcinoma cell line
MGC803. We also identified IL6R as a direct target gene for miR-23a
using a fluorescent reporter assay. The mRNA and protein levels of IL6R
were both inversely correlated with the miR-23a expression level. Our
results demonstrate that miR-23a can target IL6R and promote the growth
activity of gastric adenocarcinoma cells in vitro. The downregulation of
IL6R by miR-23a may explain why the suppression of miR-23a can inhibit
gastric cancer cell proliferation.
Abbreviations
ASO, antisense oligonucleotide; EGFP, enhanced green fluorescence protein; GAPDH, glyceraldehyde phosphate dehydrogenase;
IL6R, interleukin-6 receptor; miR-23a, microRNA-23a; miRNA, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;
siRNA, small interfering RNA; UTR, untranslated region.
3726 FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS
promote the growth of gastric adenocarcinoma cell line
MGC803 by targeting directly the interleukin-6 receptor
(IL6R) gene product.
Results
miR-23a is overexpressed in gastric
adenocarcinoma
An oligonucleotide microarray was applied to detect
the miRNA profiles in four pairs of gastric adenocarci-
noma tissue samples and matched normal gastric tissue
samples. miR-23a was consistently upregulated in gas-
tric adenocarcinoma tissues (Fig. 1A). This result indi-

cated that miR-23a might be involved in the gene
regulation of gastric cancer cells. Previous research has
demonstrated that miR-23a functions as an oncogene
in prostate cancer [14] and is also associated with
hepatocellular carcinoma [15]. Hence, we predicted
that miR-23a might have similar oncogenic activity in
gastric adenocarcinoma.
Alteration of miR-23a affects gastric
adenocarcinoma cell growth in vitro
First, we transfected miR-23a antisense oligonucleo-
tides (ASOs) or pcDNA3 ⁄ pri-23a into MGC803 cells
and confirmed that the expression of miR-23a was
effectually altered (Fig. 1B). Then, MGC803 cells were
transfected with miR-23a ASO or pcDNA3 ⁄ pri-23a.
At 24, 48 and 72 h post-transfection, cell activity was
evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl-tetrazolium bromide (MTT) assay. miR-23a ASO
reduced cell growth activities at both 48 and 72 h after
transfection, whereas pcDNA3 ⁄ pri-23a increased cell
growth activities at the same time points (Fig. 2A).
To detect the effect of miR-23a on the long-term and
independent growth activity of MGC803 cells, a plate
colony formation assay was performed. Compared
with the control group, the colony number of MGC803
cells after transfection with miR-23a ASO was lower
and that for MGC803 cells after transfection with
pcDNA3 ⁄ pri-23a was higher (Fig. 2B). These results
indicate that miR-23a can also promote the long-term
and independent growth activity of MGC803 cells.
IL6R is a candidate target of miR-23a

Many putative miR-23a targets are predicted by various
computer-aided algorithms. However, the predicted
target genes are in large quantity and most have not
been validated experimentally. Therefore, we used a
cDNA microarray to search for downregulated genes in
gastric adenocarcinoma tissue samples. The genes that
were predicted by two of the algorithm programs
(pictar and targetscan release 5.1) and were also
downregulated in our cDNA microarray were selected
as candidate targets of miR-23a. Among these genes,
the tumor suppressor gene IL6R was regarded as a pos-
sible target gene for miR-23a, corresponding to the
model that an oncogenic miRNA promotes tumor
development by targeting and negatively regulating a
tumor suppressor.
The IL6R 3¢ untranslated region (UTR) carries a
putative miR-23a binding site and is negatively
regulated by miR-23a
It is now well known that miRNAs cause mRNA
cleavage or translational repression through imperfect
50
AB
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6

0.4
0.2
0
Array 1
Array 2
Array 3
Array 4
miR-23a miR-27a miR-191
miR-23a
ASO
ASO-NC
*
*
pcDNA3 pri-23a
20
15
10
Fold
miR-23a expression level
5
0
–5
Fig. 1. miR-23a is upregulated in human gastric adenocarcinoma and is effectively altered in MGC803 cells. (A) miR-23a was among the
upregulated miRNAs in human gastric adenocarcinoma as determined by microarray analysis. (B) The miR-23a expression level in MGC803
cells was effectively altered by transfection of miR-23a ASO or pri-23a vector as detected by real-time RT-PCR. U6 snRNA was used for
normalization (*P < 0.05).
L H. Zhu et al. miR-23a promotes gastric cancer growth
FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS 3727
base pairing with the 3¢ UTR of target genes. Further-
more, the 2–8 nucleotides of miRNA, known as the

‘seed region’, have been suggested to be the most
important region for target recognition [16]. Therefore,
we predicted that the IL6R mRNA 3¢ UTR might
contain a miR-23a binding site that is complementary
to the miR-23a seed region. Three binding sites of
miR-23a were found in the 3¢ UTR of IL6R mRNA
(Fig. 3A). To confirm that miR-23a can bind to these
regions and suppress the expression of the target gene,
we constructed an enhanced green fluorescence protein
(EGFP) reporter vector in which the predicated target
regions were inserted downstream of the EGFP coding
region. MGC803 cells were transfected with the repor-
ter vector together with miR-23a ASO or pcDNA3 ⁄
pri-23a. As shown in Fig. 3B, the intensity of EGFP
fluorescence was higher in the miR-23a ASO group
and was lower in the pri-23a group compared with the
control group. Similarly, we constructed another three
EGFP reporter vectors containing the mutations of the
miR-23a binding site (Fig. 4A). It was shown that
miR-23a ASO or pcDNA3 ⁄ pri-23a did not affect the
intensity of EGFP fluorescence in the vector bearing
the mutant of the first miR-23a binding region.
1.0
1.2
0.8
A570
0.4
0.6
0.2
0

0.5
1.0
1.5
90
80
70
60
40
50
30
20
10
0
A570
Number of colonies
0
0.5
1.0
1.5
2.0
A570
0
ASO-NC miR-23a
ASO
24 h
AB
48 h
72 h
*
*

*
*
pcDNA3 pri-23a
ASO-NC miR-23a
ASO
pcDNA3 pri-23a
ASO-NC miR-23a
ASO
pcDNA3 pri-23a
ASO-NC miR-23a
ASO
pcDNA3 pri-23a
*
*
Fig. 2. miR-23a promotes growth activity
of MGC803 cells. MGC803 cells were
transfected with miR-23a ASO or pri-23a
vector, and cell growth activity was
detected through the MTT (A) and colony
formation (B) assays (*P < 0.05).
2521
5′
3′
5′
3′
5′
3′
5′
CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA
3′

UAAAUGUGAAU
ACAAUGUGAAA
GCAAUGUGAUA
IL6R mRNA
miR-23a

2531
20
A
B
18
16
14
12
*
*
10
EGFP intensity
8
6
4
2
0
EGFP
EGFP-IL6R 3′UTR
pri-23a
pcDNA3
miR-23a ASO
ASO-NC
+

–
–
–
–
–
–
+
–
–
–
–
–
+
+
–
–
–
–
+
–
+
–
–
–
+
–
–
+
–
–

+
–
–
–
+
2833 2843 3123 3133
Fig. 3. IL6R is a direct target of miR-23a. (A) The IL6R 3¢ UTR carries three potential miR-23a binding sites. (B) The direct interaction of
miR-23a and IL6R mRNA was confirmed by a fluorescent reporter assay. MGC803 cells were transfected with the EGFP reporter vector
together with miR-23a ASO or pri-23a, and the EGFP intensity was measured (*P < 0.05).
miR-23a promotes gastric cancer growth L H. Zhu et al.
3728 FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS
However, for the other two potential binding sites,
miR-23a ASO or pcDNA3 ⁄ pri-23a affected the inten-
sity of EGFP fluorescence in the vectors bearing either
the wild-type or mutated binding site (Fig. 4B, C).
These observations suggest that miR-23a binds mainly
to the first targeting site of the IL6R mRNA 3¢ UTR
and represses gene expression. These data highlight the
prediction that IL6R is a direct target of miR-23a.
miR-23a negatively regulates IL6R expression at
the mRNA and protein levels
miRNAs can suppress the expression of target genes
through translational repression or degradation of
target transcripts. To assess whether miR-23a has a
functional role in the downregulation of endogenous
IL6R expression, MGC803 cells were transfected with
miR-23a ASO or pcDNA3 ⁄ pri-23a to block or overex-
press miR-23a, respectively, and the expression of IL6R
mRNA was measured by quantitative RT-PCR. When
miR-23a was blocked or overexpressed, IL6R mRNA

was elevated or diminished, respectively, compared with
that in the control group (Fig. 5A), indicating that
miR-23a regulates endogenous IL6R mRNA levels
through a mechanism of mRNA degradation. To con-
firm the results obtained from cell lines, we also detected
the expression of IL6R mRNA in nine other pairs of
tissue samples. Figure 5B shows that, compared with
normal tissue samples, IL6R mRNA was consistently
downregulated in gastric cancer tissue samples. In
addition, knockdown or overexpression of miR-23a also
enhanced or decreased IL6R protein expression, respec-
tively (Fig. 5C).
The effects of miR-23a on the growth of MGC803
cells after IL6R knockdown
Sequence-specific small interfering RNA (siRNA) can
effectively suppress gene expression. Western blot anal-
ysis showed that transfection of the pSilencer ⁄ sh-IL6R
siRNA expression vector into MGC803 cells inhibited
significantly the expression of IL6R (Fig. 6A). As
shown in Fig. 6B, C, inhibition of IL6R expression
promoted gastric adenocarcinoma cell growth com-
pared with the control group, which was concordant
with the overexpression of miR-23a. This suggests that
miR-23a promotes MGC803 cell growth by negatively
regulating IL6R.
Discussion
Gastric cancer causes nearly one million deaths world-
wide per year. Although Helicobacter pylori infection
has been confirmed to be the main risk factor in about
2521A

BC

5′
3′ 5′ 3′ 5′ 5′3′
3′
UAUAAGAGUAU
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
3′ 5′ 3′ 5′ 5′3′
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
3′ 5′ 3′ 5′ 5′3′
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
CCUUUAGGGACCGUUACACUA
ACAAUGUGAAA
GCAAUGUGAUA




5′
3′
UAAAUGUGAAU
ACUAAGAGUAA
GCAAUGUGAUA
IL6R mRNA mut 1

5′
3′
UAAAUGUGAAU
ACAAUGUGAAA GCUAAGAGUUA
IL6R mRNA mut 3
IL6R mRNA mut 2
miR-23a
miR-23a
miR-23a
2531 2833 2843 3123 3133
2521
8
7
6
5
4
EGFP intensity
3
2
1
0
7
6
5
4
EGFP intensity
3
2

1
0
wt
UTR
ASO-
NC
UTR mut 1
#
#
#
#
#
#
*
*
*
*
*
*
UTR mut 2 UTR mut 3
UTR mut 1
UTR mut 2 UTR mut 3
ASO-
NC
ASO-
NC
miR-23a
ASO
miR-23a
ASO

miR-23a
ASO
EGFP
wt
UTR
pcDNA3
pcDNA3
pcDNA3
pri-23a
pri-23a
pri-23a
EGFP
2531 2833 2843 3123 3133
2521
2531 2833 2843 3123 3133
Fig. 4. miR-23a especially interacts with the first potential binding site of the IL6R mRNA 3¢ UTR. (A) The three EGFP reporter vectors bear-
ing mutations of the miR-23a seed region binding site are shown. The arrows indicate the mutated nucleotides. (B) Knockdown of miR-23a
failed to elevate the EGFP intensity in the reporter vector containing a mutation of the first miR-23a binding region, but could still elevate
the EGFP intensity in the reporter vectors containing mutations of the second or third miR-23a binding regions. (C) Similarly, overexpression
of miR-23a failed to suppress the EGFP intensity in the reporter vector containing mutation of the first, opposed to the second or third,
miR-23a binding region (*P < 0.05; #P > 0.05).
L H. Zhu et al. miR-23a promotes gastric cancer growth
FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS 3729
80% or more of gastric cancers, the molecular path-
way leading to the development of gastric cancers
remains unclear. Recently, accumulating evidence has
suggested that miRNAs may regulate diverse biological
processes and may be important in tumorigenesis.
In the analysis of miRNA expression differences in
four pairs of gastric adenocarcinoma tissue samples

and matched normal tissue samples, several candidate
miRNAs emerged that may be involved in gastric
adenocarcinoma. In these differentially expressed
miRNAs, we presumed that miR-23a was a signifi-
cant miRNA because of its higher fold of upregula-
tion in gastric adenocarcinoma tissues. Although the
extent of upregulation of miR-23a in the four pairs
of gastric tissues showed great variance because of
the different malignancies as well as individual differ-
ences, the dysregulation of miR-23a in gastric cancer
was consistent. Recently, it has been reported that
the miR-23a_27a_24 cluster functions as a growth-
promoting and antiapoptotic factor in human hepato-
cellular carcinoma cells [15]. Moreover, a recent study
has suggested that miR-27a functions as an onco-
genic miRNA in gastric adenocarcinoma cells by the
direct regulation of prohibitin [13]. As the coding
genes of miR-23a and miR-27a are located in the
same cluster, we determined whether miR-23a also
functions as an oncogenic miRNA in gastric adeno-
carcinoma cells.
IL6R
GAPDH
A
C
B
pSilencer/
NC
pSilencer/NC
*

*
Relative IL6R protein level
1.2
1.0
0.8
0.6
0.4
0.2
0
A570
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
Number of colonies
90
80
70
60
50
40
30
20
10
0

A570
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
A570
0.6
0.8
1.0
0.4
0.2
0
pSilencer/
sh-IL6R
pSilencer/
NC
24 h
*
*
48 h 72 h
pSilencer/
sh-IL6R
pSilencer/
NC
pSilencer/
sh-IL6R

pSilencer/
NC
pSilencer/
sh-IL6R
pSilencer/sh-IL6R
pSilencer/NC
pSilencer/sh-IL6R
Fig. 6. Knockdown of IL6R showed concordant effects with miR-23a overexpression in MGC803 cells. (A) Western blot analysis showed
that expression of IL6R was successfully suppressed by IL6R siRNA. (B, C) IL6R was knocked down in MGC803 cells, and cell growth activ-
ity was detected through the MTT (B) and colony formation (C) assays (*P < 0.05).
9
A
C
B
*
*
*
*
8
7
6
5
4
Relative IL6R mRNA level
3
2
1
0
1.2
1.0

0.8
Relative IL6R mRNA level
0.6
0.4
0.2
0
1.2
1.4
1.6
1.8
1.0
0.8
Relative IL6R protein level
0.6
0.4
0.2
0
123 45 6 789
ASO-NC
miR-23a
ASO
Normal
IL6R
GAPDH
Cancer
pcDNA3 pri-23a
ASO-NCmiR-23a
ASO
pcDNA3 pri-23a
ASO-NCmiR-23a

ASO
pcDNA3 pri-23a
Fig. 5. The expression level of IL6R was
inversely correlated with the level of
miR-23a. (A) When miR-23a was blocked or
overexpressed, the level of IL6R mRNA was
subsequently elevated or diminished
compared with the level in the control
group. (B) Compared with normal tissue
samples, IL6R mRNA was consistently
downregulated in gastric cancer tissue
samples. (C) When miR-23a was blocked or
overexpressed, the level of IL6R protein
was subsequently elevated or diminished
compared with the level in the control group
(*P < 0.05).
miR-23a promotes gastric cancer growth L H. Zhu et al.
3730 FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS
It is assumed that the overexpressed miRNAs in
cancers may function as oncogenes. Hence, we inferred
that miR-23a might be a growth-promoting factor in
gastric adenocarcinoma. Both MTT and colony forma-
tion assays confirmed the active role of miR-23a in the
growth promotion of malignant cells.
The fundamental function of miRNAs is to regulate
their targets by direct cleavage of mRNA or by inhibi-
tion of protein synthesis. On the one hand, computa-
tional algorithms have been widely used to predict
miRNA targets. On the other, gene expression profil-
ing analysis using cDNA microarrays is a strong tool

to identify miRNA targets. As expression regulation at
the mRNA level may be a common mechanism for
miRNA function [17], it is more convenient to monitor
transcriptional changes using a high-throughput micro-
array approach [18]. In order to predict exactly the
functional targets for miR-23a, we combined the bioin-
formatic assay with a cDNA microarray assay [19].
The genes that were both predicted in the two com-
puter-based databases and were downregulated in the
cDNA microarray were selected as candidate targets
for miR-23a. This strategy enhanced the precision of
target prediction. To confirm the postulation, we
checked the regulatory effects of miR-23a on the
expression of IL6R by RT-PCR and western blot, and
found an inverse correlation between miR-23a and
IL6R expression at both the mRNA and protein levels.
To confirm the direct regulation of IL6R by miR-23a,
we used an EGFP-IL6R 3¢ UTR reporter vector bearing
the potential miR-23a binding site in the fluorescent
reporter, and found an increase in EGFP intensity after
blocking miR-23a and a decrease in EGFP intensity
after overexpressing miR-23a. Furthermore, another
three reporter vectors containing mutations of the
miR-23a binding site were used in the fluorescent
reporter assay. The reporter vector containing a muta-
tion of the first binding site showed no response to
altered miR-23a expression, suggesting that the first
binding site in the 3¢ UTR of IL6R mRNA mainly
reacted with miR-23a. Moreover, the inhibition of
IL6R by specific siRNA promoted gastric adenocarci-

noma cell growth, which was consistent with the results
of the overexpression of miR-23a, and suggested a
critical role of IL6R in miR-23a-mediated cell growth
regulation.
IL6R is an evolutionarily conserved antiproliferative
protein and may function as a tumor suppressor
through interaction with IL6. A previous study has
indicated that exogenous IL6 ⁄ IL6R slows PC-3 and
LNCaP cell growth, demonstrating its antiproliferative
activity [20]. Another study has demonstrated the
induction of antiapoptotic regulators by IL6 ⁄ IL6R in
both naive and activated T-cell populations [21]. In
gastric cancer, IL6 contains polymorphisms [22]. The
expression of IL6 is involved in gastric cancer invasion
and lymph node and ⁄ or hepatic metastasis, and can be
used as a prognostic factor for survival [23]. These
observations indicate an important role of the
IL6 ⁄ IL6R complex in gastric cancer. In this study, we
found that the inhibition of IL6R, which may be
caused by redundant miR-23a, promoted gastric ade-
nocarcinoma cell growth. This function may be associ-
ated with the potential antiproliferative activity of
IL6R. This study provides a potential mechanism of
IL6R post-transcriptional regulation by miR-23a.
Moreover, some miRNAs were located in a transcript
cluster and could have synergistic biological functions.
For example, the miR-17-92 cluster displays oncogenic
activity in B-cell lymphoma [24], Burkitt’s lymphoma
[25] and human lung cancer [26]. Given that miR-
23a ⁄ 24 ⁄ 27a was also in a transcript cluster and that

miR-27a also showed oncogenic activity in human gas-
tric cancer [13], we are led to presume that this cluster
might have a synergistic function that needs to be elu-
cidated in further studies.
Collectively, our studies demonstrate that miR-23a
potently promotes the growth of the gastric adeno-
carcinoma cell line MGC803, providing the first proof-
of-concept that there is a potential link between the
tumor promoter miR-23a and gastric adenocarcinoma
cell proliferation. More importantly, the mechanism of
miR-23a-mediated promotion of gastric adenocarci-
noma proliferation might be related to the direct
modulation of the downstream target IL6R, a gene
regulating cell proliferation. Our findings suggest a
potential regulatory pathway in which the upregulated
expression of miR-23a causes the downregulation of
IL6R by binding to its first conserved binding site,
which then leads to the development of gastric adeno-
carcinoma.
Materials and methods
Human cancer tissue samples
Fresh-frozen human gastric adenocarcinoma tissue samples
and matched normal gastric tissue samples were obtained
from the Tumor Bank Facility of Tianjin Medical University
Cancer Institute and Hospital and the National Foundation
of Cancer Research. All of the tumor types were confirmed
by pathologic analysis. The experiments were undertaken
with the understanding and written consent of each subject.
The study methodologies conformed to the standards set by
the Declaration of Helsinki, and were approved by the local

ethics committee.
L H. Zhu et al. miR-23a promotes gastric cancer growth
FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS 3731
Cell culture and transfection
Human gastric adenocarcinoma cell line MGC803 was
maintained in RPMI1640 (GIBCO BRL, Grand Island,
NY, USA) supplemented with 10% fetal bovine serum,
100 IUÆmL
)1
of penicillin and 100 lgÆmL
)1
of streptomy-
cin. The cell line was incubated at 37 °C in a humidified
chamber supplemented with 5% CO
2
. Transfection was
performed with Lipofectamine 2000 Reagent (Invitrogen,
Carlsbad, CA, USA) following the manufacturer’s protocol.
Briefly, cells were trypsinized, counted and seeded in plates
on the day before transfection to ensure suitable cell conflu-
ency on the day of transfection. Oligonucleotides and plas-
mids were used at final concentrations of 200 nm and
5ngÆlL
)1
, respectively, both in antibiotic-free Opti-MEM
medium (Invitrogen). The transfection efficiency was moni-
tored by cyanine-5 oligonucleotides.
Isolation of RNAs
Total RNA extraction of cells or tissue samples was
performed with the mirVana miRNA Isolation Kit (Ambion,

Austin, TX, USA) according to the manufacturer’s instruc-
tions. Large (larger than 200 nucleotides) and small (smaller
than 200 nucleotides) RNAs were separated and purified in
this procedure. The integrity of the large RNA was con-
firmed by 1% denatured agarose gel electrophoresis.
miRNA microarray and cDNA microarray analyses
The miRNA microarray and cDNA microarray analyses
were performed as described previously [13].
Quantitative RT-PCR
For the detection of the miR-23a level in MGC803 cells
transfected with miR-23a ASO or pcDNA3 ⁄ pri-23a, stem-
loop quantitative RT-PCR [27] was performed. PCR primers
were designed as follows: miR-23a forward, 5¢-ATCAC
ATTGCCAGGGATTTCC-3¢; miR-23a reverse, 5¢-CCAG
TGCAGGGTCCGAGGT-3¢; U6 forward, 5¢-TGCGG
GTGCTCGCTTCGGCAGC-3¢; U6 reverse, 5¢-CCAGTGC
AGGGTCCGAGGT-3¢. PCR cycles were as follows: 94 °C
for 4 min, followed by 40 cycles of 94 °C for 1 min, 50 °C
for 1 min and 72 °C for 1 min. The SYBR Green Mix TaqÔ
Kit (TaKaRa, Otsu, Shiga, Japan) was used to trace the
amplified DNA. All primers were purchased from AuGCT
Inc. (Beijing, China).
For the detection of IL6R gene expression levels, 5 lgof
large RNA extracted from cells or tissue samples was reverse
transcribed to cDNA using the M-MLV reverse transcriptase
(Promega, Madison, WI, USA). The cDNA was used for the
amplification of IL6R genes and an endogenous control gene
glyceraldehyde phosphate dehydrogenase (GAPDH) via
PCRs. PCR cycles were as follows: 94 ° C for 4 min, followed
by 40 cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C

for 1 min. PCR primers were as follows: IL6R sense,
5¢-CCGAGATCTGGCTTTTACTTAAACCG-3¢; IL6R
antisense, 5¢-CAGGAATTCACTTGCTCTGTCACCC-3¢;
GAPDH sense, 5¢-GCGAATTCCGTGTCCCCACTGCC
AACGTGTC-3¢; GAPDH antisense, 5¢-GCTACTCGAGT
TACTCCTTGGAGGCCATGTGG-3¢. The PCR products
were resolved on a 1% agarose gel. LabWorksÔ Image
Acquisition and Analysis Software (UVP, Upland, CA,
USA) was used to quantify band intensities. All primers were
purchased from AuGCT Inc.
MTT assay
MGC803 cells were seeded in 96-well plates with 3 · 10
3
cells per well in 100 lL of cell culture medium and incu-
bated at 37 °C for 24 h. The cells were then transfected
with miR-23a ASO (5¢-GGAAATCCCTGGCAATGTG
AT-3¢), control oligonucleotides (5¢-GTGGATATTGTT
GCCATCA-3¢), pcDNA3 ⁄ pri-23a or pSilencer ⁄ sh-IL6R
siRNA expression vector. After incubation for 24, 48 and
72 h, the cells were incubated with 20 lL of MTT (at a
final concentration of 0.5 mgÆmL
)1
)at37°C for 4 h. The
medium was removed and the precipitated formazan was
dissolved in 100 lL of dimethylsulfoxide. After shaking for
10 min, the absorbance at 570 nm was detected using a
lQuant Universal Microplate Spectrophotometer (Bio-tek
Instruments, Winooski, VT, USA).
Plate colony formation assay
MGC803 cell growth activity was determined by colony

formation analysis. Twenty-four hours after transfection,
cells were harvested and seeded at 100 cells per well in
12-well plates. Plates were incubated at 37 °C and 5% CO
2
in a humidified incubator for 2 weeks. During colony
growth, the culture medium was replaced every 3 days.
Colonies were counted under a microscopic field at · 100
magnification. Each assay was performed in triplicate.
Bioinformatic method
The miRNA targets predicted by computer-aided algorithms
were obtained from PicTar (the Rajewsky Lab, Berlin,
Germany. - berlin.de/ cgi-bin/new_PicTar_
vertebrate.cgi) and TargetScan Release 5.1 (Whitehead Insti-
tute for Biomedical Research, Cambridge, MA, USA, http://
www.targetscan.org).
EGFP reporter assay
MGC803 cells were transfected in 48-well plates with 0.2 lg
of IL6R EGFP reporter vector with wild-type or mutated
miR-23a promotes gastric cancer growth L H. Zhu et al.
3732 FEBS Journal 277 (2010) 3726–3734 ª 2010 The Authors Journal compilation ª 2010 FEBS
3¢ UTR. Cells were also cotransfected with 20 pmol of
miR-23a ASO or 0.2 lg of pcDNA3 ⁄ pri-23a per well. The
assay was normalized with 0.05 lg of red fluorescence pro-
tein expression vector pDsRed2-N1 (Clontech, Mountain
View, CA, USA). Forty-eight hours after transfection, cells
were lysed with lysis buffer (0.15 m NaCl, 0.05 m Tris ⁄ HCl
pH 7.2, 1% Triton X-100, 0.1% SDS). The fluorescence
intensities of EGFP and red fluorescence protein were
detected with a Fluorescence Spectrophotometer F-4500
(Hitachi, Tokyo, Japan).

Western blot analysis
To determine the levels of protein expression, cells were trans-
fected, lysed with RIPA lysis buffer and the proteins were
harvested 48 h later. Proteins were resolved on a 10% SDS
denatured polyacrylamide gel, transferred onto a nitrocellu-
lose membrane, blocked with 5% skimmed milk, and then
probed with the relevant primary antibodies to IL6R and
GAPDH overnight at 4 °C. Membranes were washed and
incubated with horseradish peroxidase-conjugated secondary
antibody. Protein expression was assessed by enhanced
chemiluminescence and exposure to chemiluminescent film.
LabWorksÔ Image Acquisition and Analysis Software was
used to quantify the band intensities. The antibody to IL6R
was purchased from Saier Inc. (Tianjin, China), and all others
were obtained from Sigma-Aldrich (St Louis, MO, USA).
Statistical analysis
The data were expressed as the means ± standard devia-
tion (SD) and statistical analysis utilized a two-tailed Stu-
dent’s t-test. Statistical significance was set at P £ 0.05.
Acknowledgements
We thank the Tumor Bank Facility of Tianjin Medical
University Cancer Institute and Hospital and the
National Foundation of Cancer Research for provid-
ing human gastric tissue samples. We also thank the
College of Public Health of Tianjin Medical University
for technical assistance with fluorescence detection.
This work was supported by the National Natural
Science Foundation of China (NO: 30873017) and
the Natural Science Foundation of Tianjin (NO:
08JCZDJC23300 and 09JCZDJC17500).

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