adenocarcinoma cell line MGC803 and downregulatesinterleukin-6 receptor Li-Hua Zhu1,2,*, Tao Liu1,*, Hua Tang1, Rui-Qing Tian1, Chang Su1, Min Liu1and Xin Li1 1 Tianjin Life Science Rese
Trang 1adenocarcinoma cell line MGC803 and downregulates
interleukin-6 receptor
Li-Hua Zhu1,2,*, Tao Liu1,*, Hua Tang1, Rui-Qing Tian1, Chang Su1, Min Liu1and Xin Li1
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: htang2002@yahoo.com
*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 IL6Rby 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.
Trang 2promote 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
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 ASO ASO-NC
*
*
pcDNA3 pri-23a
20 15 10
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).
Trang 3base 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
A5700.40.6
0.2
0
0.5
1.0
1.5
90 80 70 60
40 50
30 20 10 0
0
0.5
1.0
1.5
2.0
0
ASO-NC miR-23a
ASO
24 h
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 ′
CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA
3 ′
miR-23a
2531
20
A
B
18 16 14 12
*
*
10
8 6 4 2 0 EGFP EGFP-IL6R 3 ′UTR pri-23a pcDNA3 miR-23a ASO ASO-NC
+ – – – – –
– + – – – –
– + + – – –
– + – + – –
– + – – + –
– + – – – +
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).
Trang 4However, 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 exoverex-pression 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
2521
A
5 ′
3 ′ UAUAAGAGUAU
CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA CCUUUAGGGACCGUUACACUA
3 ′ CCUUUAGGGACCGUUACACUA 5 ′ 3 ′ CCUUUAGGGACCGUUACACUA 5 ′ 3 ′ CCUUUAGGGACCGUUACACUA 5 ′
3 ′ CCUUUAGGGACCGUUACACUA 5 ′ 3 ′ CCUUUAGGGACCGUUACACUA 5 ′ 3 ′ CCUUUAGGGACCGUUACACUA 5 ′
ACAAUGUGAAA GCAAUGUGAUA
5 ′ UAAAUGUGAAU ACUAAGAGUAA GCAAUGUGAUA 3 ′
IL6R mRNA mut 1
5 ′ UAAAUGUGAAU ACAAUGUGAAA GCUAAGAGUUA 3 ′ IL6R mRNA mut 3
IL6R mRNA mut 2 miR-23a
miR-23a
miR-23a
2521
8
7
6
5
4
3
2
1
0
7 6 5 4
3 2 1 0
wt
UTR
ASO-NC
UTR mut 1
#
#
#
#
#
#
*
*
*
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
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 bindbear-ing 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).
Trang 580% 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
*
*
1.2
1.0
0.8
0.6
0.4
0.2
0
0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
90 80 70 60 50 40 30 20 10 0
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0
0.6 0.8 1.0
0.4 0.2 0
pSilencer/
sh-IL6R
pSilencer/
NC
24 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
3
2
1
0
1.2
1.0
0.8
0.6
0.4
0.2
0
1.2 1.4 1.6 1.8
1.0 0.8
0.6 0.4 0.2 0
ASO-NC miR-23a
ASO
Normal
IL6R
GAPDH
Cancer
pcDNA3 pri-23a
ASO-NC miR-23a ASO pcDNA3 pri-23a
ASO-NC miR-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).
Trang 6It 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 usprofil-ing 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
IL6Rexpression 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
Trang 7Cell 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 37C in a humidified
chamber supplemented with 5% CO2 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
5 ngÆlL)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 94C for 1 min, 50 C
for 1 min and 72C for 1 min The SYBR Green Mix Taq
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 lg of
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: 94C for 4 min, followed
by 40 cycles of 94C 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 LabWorks 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· 103 cells per well in 100 lL of cell culture medium and incu-bated at 37C 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) at 37C 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 37C and 5% CO2
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 http://pictar.mdc-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
Trang 83¢ 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 4C Membranes were washed and
incubated with horseradish peroxidase-conjugated secondary
antibody Protein expression was assessed by enhanced
chemiluminescence and exposure to chemiluminescent film
LabWorks 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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