MOLECULAR MEDICINE REPORTS 15: 3055-3061, 2017
Abstract. Abnormal expression of epidermal growth factor
receptor (EGFR) signaling and microRNAs (miRNAs) has
been widely seen in gastric cancer. The present study focused
on the miRNAs that regulate human epidermal growth factor
receptor (HER) activation through mucin 13 (MUC13). The
protein level of MUC13 was demonstrated to be signicantly
increased in gastric cancer tissues compared with normal
tissues by western blot analysis and immunohistochemistry.
TargetScan bioinformatic predictions indicated that miRNA
(miR)-212-3p and miR-132-3p may bind to the 3'-untranslated
region of MUC13. Further investigation revealed that
miR-132-3p was significantly decreased in gastric cancer
tissues compared with normal tissues, whereas miR-212-3p
expression was unaffected. Luciferase assays and western blot
conrmed MUC13 as a target gene of miR‑132‑3p. Inhibition
of miR-132-3p enhanced gastric cancer cell migration through
activation of HER2, extracellular signal-regulated kinase
(ERK) and Akt serine/threonine kinase (Akt) signaling, which
was a similar effect to that of MUC13 overexpression. In
summary, reduction of miR-132-3p may contribute to gastric
cancer proliferation by targeting MUC13.
Introduction
Gastric cancer is one of the most common malignant tumors
worldwide, causing ~738,000 deaths in 2008 (1). Major contrib-
utory factors to gastric cancer include Helicobacter pylori
infection, diet, alcoholic consumption and smoking (2,3).
Receptor tyrosine kinase (RTK) pathways have key roles
in the progression of various tumors (4-6), with aberrant
epidermal growth factor receptor (EGFR) signaling demon-
strated to be particularly common (7); EGF family proteins
have been revealed to be signicantly overexpressed in >60%
of tumors (8,9). The main EGF family members include EGFR
[also termed human epidermal growth factor receptor 1 (HER1)],
HER2 (also termed ErbB2), HER3 (also termed ErbB3) and
HER4 (also termed ErbB4). EGFR and HER2 are often signif-
icantly upregulated in gastric cancer, and are considered to be
well-established oncogenes (10). Mucin 13 (MUC13) has also
been demonstrated to be aberrantly upregulated in various
tumors (11-13). Exogenous expression of MUC13 contributes
to abnormal cell proliferation, motility and tumor growth (13),
and overexpression of MUC13 results in the activation of
HER2, extracellular signal-regulated kinase (ERK) and Akt
serine/threonine kinase (Akt), and the reduction of p53 expres-
sion (12). However, few studies have, thus far, investigated the
expression of MUC13 in gastric cancer.
MicroRNAs (miRNAs) are small non-coding RNAs that
widely control gene expression at the post-transcriptional
level (14-16). Due to the oncogenic or tumor suppressive
roles of miRNAs, abnormal expression can lead to the
initiation, formation and progression of tumors. For example,
numerous miRNAs are differentially expressed in gastric
cancer, including miRNA-199a-3p (miR-199a-3p), miR-429
and miR-34a (14-16). The present study aimed to investigate
miRNAs that regulate the expression of MUC13 in gastric
cancer.
Materials and methods
Patient selection and biopsy collection. In the present study,
biopsies were taken from both tumor tissue and the adjacent
normal tissue of 40 patients receiving adenocarcinoma surgery
of the stomach or esophageal junction. Samples were collected
from July 2012 to November 2014 and written consent was
provided by all individuals. The collection of biopsies was
approved by the Ethics Committee of the First Hospital of
Jilin University (Changchun, China) in accordance with the
Declaration of Helsinki. All biopsy samples were reviewed by
an experienced pathologist to validate the diagnosis.
Immunohistochemistry. Parafn‑embedded tissues xed in
4% buffered paraformaldehyde were cut into 5 µm sections
and washed three times (5 min per wash) in phosphate-buff-
ered saline (PBS), then incubated in 3% H
2
O
2
for 30 min
at room temperature. Sections were blocked by incubation
Reduction of miR‑132‑3p contributes to gastric
cancer proliferation by targeting MUC13
LIANG HE
1
, LINLIN QU
2
, LIJING WEI
2
, YAN CHEN
1
and JIAN SUO
1
Departments of
1
Gastrointestinal Surgery and
2
Laboratory Medicine,
The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China
Received September 30, 2015; Accepted January 16, 2017
DOI: 10.3892/mmr.2017.6347
Correspondence to: Dr Jian Suo, Department of Gastrointestinal
Surgery, The First Hospital of Jilin University, 71 Xinmin Street,
Changchun, Jilin 130021, P.R. China
E-mail: suojian150930@163.com
Key words: miR-132-3p, gastric cancer, cell migration, MUC13,
HER2
HE et al: miR-132-3p REGULATES GASTRIC CANCER BY TARGETING MUC13
3056
with 10% goat serum in PBS (Origene Technologies, Inc.,
Rockville, MD, USA) for 30 min at 37˚C, then incubated with
the MUC13 primary antibody (1:80; catalog no. ab124654;
Abcam, Cambridge, UK) for 24 h at 4˚C. Sections were
washed with PBS, then incubated with secondary antibody
(biotin-labelled goat anti-mouse immunoglobulin G; 1:200;
catalog no. SP-9000D; Origene Technologies, Inc.) for 1 h at
4˚C. Following washing with PBS, sections were incubated
with horseradish peroxidase conjugated streptavidin (1:200)
for 1 h at room temperature, then with diaminobenzidine/H
2
O
2
for 15 min at room temperature. Following dehydration in
gradient alcohol, and transparentizing in xylene, sections were
mounted with glycerol and observed under a microscope. In
control sections, the primary antibody was replaced with 1%
calf serum (Origene Technologies, Inc.).
Cell culture. Gastric cancer cell line, MKN28 was purchased
from American Type Culture Collection (ATCC, Manassas,
VA, USA) and cultured in RPMI-1640 (GE Healthcare Life
Sciences, Logan, UT, USA) supplemented with 10% fetal
bovine serum (FBS; Invitrogen; Thermo Fisher Scientic,
Inc., Waltham, MA, USA), streptomycin (100 mg/ml) and
penicillin (100 U/ml) at 37˚C in a humidied atmosphere
containing 5% CO
2
. Recent reports have suggested that
the MKN28 gastric carcinoma cell line used in this study
is contaminated with another gastric carcinoma cell line,
MKN74 (17).
RNA extraction. Total RNA was extracted from gastric
tissues or MKN28 cells using TRIzol reagent according to the
manufacturers' instructions (Thermo Fisher Scientic, Inc.,
Waltham, MA, USA).
Reverse transcription‑quantitative polymerase chain reac‑
tion (RT‑qPCR). Total RNA was reverse transcribed using
Takara MicroRNA Reverse Transcription Kit (Takara Bio,
Inc., Otsu, Japan) with specic primers for miR‑132‑3p
(GTC GT ATC CAG TGC AGG GTC CGA GGT ATT CGC ACT
GGA TAC GAC CGA CC) and U6 (GTC GTAT CCA GTG CAG
GGT CCG AGG TAT TCG CAC TGG ATA CGA CAA ATA T).
Subsequently, the P CR amplication was per forme d. 1 mg
of cDNA was used for qPCR using SYBR green Master
mix (Roche Diagnostics, Basel, Switzerland) on a Roche
Lightcycler 480 (Roche Diagnostics) at 95˚C for 10 min
followed by 50 cycles of 95˚C for 10 sec, 55
˚
C for 10 sec, 72˚C
for 5 sec; 99˚C for 1 sec; 59˚C for 15 sec; 95˚C for 1 sec; then
cooling to 40˚C. Relative miRNA expression of miR‑132‑3p
was normalized against the endogenous control, U6, using
the Δ-Δ Cq method (18).
Transient transfection. A total of 6x10
5
cells were equally
seeded in the 6-well plates with 2 ml RPMI-1640 culture
medium containing serum and antibiotics. At the same time,
miR-132-3p mimic, inhibitor, miR negative control or siRNA
targeting MUC1 3 (CCA GCU UGU UG A GGU AG A AGU AGU
A) or non-target control siRNA (Shanghai GenePharma Co.,
Ltd., Shanghai, China) were mixed with HiperFect transfec-
tion reagent (Qiagen GmbH, Hilden, Germany) and incubated
at room temperature for 10 min. The complex was then trans-
fected into MKN28 cells for 48 h.
Cell viability analysis. To examine cell viability, MKN28 cells
were seeded in 96-well plates at a density of 1.0x10
4
cells/per
well. miR-132 mimics, inhibitors or a scramble/non-targeting
oligo negative control (NC) were transfected into cells at 24,
48, 72 h after seeding of cells. MTT assay was performed as
previously described (19).
Adenovirus vector construction and transfection. The
adenovirus vector (Ad)-MUC13 and Ad-control (Ad-con)
were purchased from the Chinese National Human Genome
Center (Beijing, China). In brief, 6x10
5
cells were equally
seeded in the 6-well plates with 2 ml RPMI-1640 culture
medium containing serum and antibiotics. Following 24 h,
the cells were transfected with Ad-MUC13 or Ad-con at the
density of 100 multiplicity of infection (MOI) for 48 h. The
transfection efciency was calculated as the green uorescent
protein-positive cells/all cells in each eld x100%.
Luciferase target assay. The 3'untranslated region (UTR) of
MUC13 containing the predicted target site for miR-20a-5p,
was cloned into the pmirGLO (Promega Corporation,
Madison, WI, USA) luciferase reporter vector which had been
cleaved at the SacI and XhoI sites. Details of PCR procedures
are described as follows: a heated initial denaturation step
at 95˚C for 10 min, followed by 40 cycles at 95˚C for 15 sec,
55˚C for 45 sec and 72˚C for 30 sec. Prior to conducting the
luciferase reporter assay, 5x10
4
cells per well were seeded
in 24‑well plates in a 500 µl medium and cultured for 18 h.
The cells were transfected with the modied rey luciferase
vector (500 ng/µl) mixed with Vigofect transfection reagent,
according to the manufacturer's protocol. Following a 48 h
continuous exposure, the luciferase activities from rey and
renilla were measured with the Dual-luciferase reporter assay
system (Promega Corporation). Renilla activity was used as
the normalized parameter.
Establishment of MUC13‑expressing MKN28 stable cell line.
MKN28 cells were transfected with pmirGLO-MUC13-3'UTR
or empty vector (pmirGLO) using VigoFect transfec-
tion reagent (Vigorus Biotechnology, Beijing, China).
Individual G418 resistant clones (1 mg/ml; Invitrogen;
Thermo Fisher Scientic, Inc.) were selected and applied for
further study.
Western blotting analyses. Tissue or MKN28 cell protein was
extracted using RIPA buffer (Solarbio Science & Technology
Co., Ltd., Beijing, China). A bicinchoninic protein assay kit
(Pierce; Thermo Fisher Scientic, Inc.) was used to deter-
mine the protein concentration. Equal quantities of protein
(15 µg) were resolved by 10% SDS‑PAGE and transferred
onto a PVDF membrane. The protein was detected with
primary antibodies, MUC13 (catalog no. ab124654; Abcam),
HER2 (catalog no. 4290; 1:1,000), p-ERK (catalog no. 1150;
1:1,000), ERK (catalog no. 9194; 1:1,000), p-Akt (catalog
no. 8200; 1:1,000), Akt (catalog no. 9840; 1:1,000) and
GAPDH (catalog no. 2118; 1:5,000) all obtained from Cell
Signaling Technology, Inc., (Danvers, MA, USA) overnight
at 4˚C. Nonspecific binding was blocked using 8% (w/v)
milk in Tris‑buffered saline with 1% Tween‑20 (TBST;
Beijing SolarBio Science & Technology Co., Ltd.) for 2 h
MOLECULAR MEDICINE REPORTS 15: 3055-3061, 2017
3057
at room temperature. Following several washes with TBST,
the membranes were incubated with horseradish-peroxidase
(HRP)-conjugated goat anti-rabbit and anti-mouse IgG or
HRP-conjugated mouse anti-goat IgG (all 1:5,000; Origene
Technologies, Inc.) for 2 h at room temperature and then
washed. GAPDH was used as the internal control. Signals
were detected with enhanced chemiluminescence according
to the manufacturer's protocol (EMD Millipore, Billerica,
MA, USA). ImageJ software (National Institutes of Health,
Bethesda, MD, USA) was used for density analysis.
Bioinformatic predictions. To determine the potential miRNAs
that target MUC13, bioinformatic prediction was performed
using TargetScan (http://www.targetscan.org).
Cell invasion assay. Invasion of cells were examined using
a Transwell system (Invitrogen; Thermo Fisher Scientific,
Inc.). The MKN28 cells transfected with miR-132 inhibitors
or ad-MUC13 were cultured in the lower chamber with fresh
medium containing 10% FBS. After incubation for 24 h at
37˚C, the cells on the upper chamber was stained with 0.5%
crystal violet and dissolved in 10% acetic acid for measure-
ment of absorbance at 560 nm.
Cell migration assay. The in vitro wound healing assay was
performed as previously described (20). Briey, MKN28 cells
were seeded in 6‑well plates to form a conuent monolayer.
The monolayer was scratched with a sterile 10 µl pipette tip,
and the oating cells were carefully removed by washing with
PBS. Then, the cells were cultured in RPMI-1640 medium
without FBS at 37˚C in a 5% CO
2
atmosphere. The wound
scratches were photographed at 0 and 12 h then scraped to
collect cells.
Statistical analysis. The data are expressed as the mean ± stan-
dard error. The number of independent experiments was
represented by ‘n’. Data were analyzed using SPSS software,
version 13.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was
considered to indicate a statistically signicant difference.
Results
Upregulation of MUC13 in gastric cancer tissues. Initially,
the expression of MUC13 in gastric cancer tissues was exam-
ined. Western blot analysis demonstrated that MUC13 was
signicantly upregulated in gastric cancer tissues compared
with adjacent normal tissues (Fig. 1A). Immunohistochemistry
analysis also demonstrated the enhanced expression of MUC13
in gastric cancer tissues (Fig. 1B).
MUC13 is a target gene of miR‑132‑3p in gastric cancer. To
identify the potential miRNAs that regulate the expression
of MUC13, the TargetScan online prediction program was
used. As demonstrated in Fig. 2A, two putative conserved
binding miRNAs, miR-132-3p and miR-212-3p, were identi-
fied to potentially bind the 3'untranslated region (3'UTR)
of MUC13. The present study determined that miR-132-3p
levels were reduced in gastric cancer tissues compared with
adjacent normal control tissue, whereas miR-212-3p did not
demonstrate signicant a change (Fig. 2B). Subsequently, the
3'UTR of MUC13 was cloned into the pmirGLO plasmid. A
dual luciferase reporter assay demonstrated that miR-132-3p
significantly decreased the relative luciferase units of
pmirGLO-MUC13-3'UTR compared with with the empty
vector pmirGLO (Fig. 2C). Additionally, miR-132-3p mimics
or inhibitors were transfected into MKN28 cells. RT-qPCR
analysis revealed that transfection with miR-132-3p mimics
markedly enhanced the level of miR-132-3p, whereas trans-
fection with miR‑132‑3p inhibitors signicantly reduced the
level of miR-132-3p (Fig. 2D). Overexpression of miR-132-3p
significantly increased the level of miR-132-3p, however
decreased the protein level of MUC13 (Fig. 2E). By contrast,
inhibition of miR-132-3p reduced the level of miR-132-3p,
however increased the expression of MUC13 (Fig. 2E). These
data indicated that reduction of miR-132-3p led to enhanced
MUC13 expression in gastric cancer cells.
Overexpression of MUC13 increases the activation of HER
signaling and cell invasion and migration in MKN28 cells.
To explore the role of MUC13 on gastric cancer progression,
cell invasion and migration were analyzed. As demonstrated
in Fig. 3A, the transfection efficiency of Ad-MUC13 or
Ad‑con was nearly 100% (Fig. 3A). No alterations of MUC13
protein levels were observed following simple transfection of
blank adenovirus vectors (data not shown). A Transwell assay
demonstrated that overexpression of MUC13 signicantly
Figure 1. Expression of MUC13 was increased in gastric cancer tissues
compared with normal tissues. (A) Western blot analysis.
*
P<0.05 vs. control.
(B) Immunohistochemistry analysis. n=5 independent tissues. MUC13,
mucin 13.
HE et al: miR-132-3p REGULATES GASTRIC CANCER BY TARGETING MUC13
3058
enhanced cell invasion capacity (Fig. 3B). Furthermore, a
scratch assay demonstrated that MKN28 cell migration was
enhanced by MUC13 overexpression (Fig. 3C). The signaling
downstream of MUC13 was also examined. As demonstrated
in Fig. 3D, overexpression of MUC13 obviously enhanced
the level of HER2, and the phosphorylation of ERK and Akt
(Fig. 3D).
Knockdown of MUC13 partially reverses miR‑132‑3p
inhibition‑induced MKN28 cell invasion and migration. To
explore whether miR-132-3p exerts its role through MUC13,
miR-132-3p inhibitors were transfected into MKN28 cells.
As demonstrated in Fig. 4A, inhibition of miR‑132‑3p signi-
cantly enhanced the protein level of MUC13, and enhanced the
level of HER2, and ERK and Akt phosphorylation. Notably, an
Figure 2. Reduction of miR-132-3p led to enhanced MUC13 expression in gastric cancer cells. (A) TargetScan prediction programs indicated that miR-132-3p
and miR-212-3p may bind to the 3'UTR of MUC13. (B) Reverse transcription-quantitative polymerase chain reaction was performed to determine the level
of miR-132-3p and miR-212-3p in gastric cancer tissues (GC) (n=5 independent samples) and normal control (NC). (C) Luciferase assay indicated that
miR-132-3p decreased the relative luciferase activity of pmirGLO-MUC13-3'UTR. (D) RT-qPCR analysis of miR-132-3p levels following transfection
of miR‑132‑3p mimics or inhibitors. (E) Transfection of miR‑132‑3p mimics signicantly decreased the protein level of MUC13 in MKN28, and transfection
of miR‑132‑3p inhibitors signicantly decreased the protein level of MUC13.
*
P<0.05,
**
P<0.01,
***
P<0.001 vs. control. MUC13, mucin 13; UTR, untranslated
region; miR, microRNA; RLU, relative luciferase unit; NC, negative control.
MOLECULAR MEDICINE REPORTS 15: 3055-3061, 2017
3059
siRNA targeting MUC13 was selected to suppress the expres-
sion of MUC13. miR-132-3p inhibition reduced the effects of
MUC13 siRNA on HER2 expression, and ERK and Akt acti-
vation (Fig. 4B). Additionally, the effect was on cell invasion
and migration was also determined. miR-132-3p inhibitors
reduced the effect of MUC13 knockdown on cell invasion and
migration (Fig. 4C and D).
Discussion
Mucins are considered as potential oncogenes and possible
therapeutic targets in various malignancies (21-23). As a
high-molecular-weight transmembrane glycoprotein, MUC13
is reported to be frequently overexpressed in various epithe-
lial carcinomas, including gastric, colorectal and ovarian
cancers (24). MUC13 includes three EGF-like domains and
a cytoplasmic domain with phosphorylation sites, which
trigger the activation of HER2 signaling (24). The current
study examined MUC13 expression in gastric cancer tissues
and detected that MUC13 protein levels were signicantly
increased compared with adjacent normal tissues.
A previous study reported that overexpression of MUC13
signicantly enhanced the activation of HER2, ERK and
Akt (13). To validate the role of MUC13 in gastric cancer
Figure 3. Overexpression of MUC13 prompted MKN28 cell invasion and migration through activation of HER signaling. (A) Transfection efciency of
Ad‑MUC13 or Ad‑con. (B) Transwell assay demonstrated that overexpression of MUC13 signicantly enhanced MKN28 cell invasion capacity. (C) Scratch
assay demonstrated that MKN28 cell migration was signicantly enhanced when MUC13 was overexpressed. (D) Overexpression of MUC13 obviously
enhanced the expression of HER2, and the phosphorylation of ERK and Akt. n=3 independent experiments,
*
P<0.05,
**
P<0.01 vs. control. Ad, adenovirus;
con, control; MUC13, mucin 13; HER2, human epidermal growth factor receptor 2; p-, phosphorylated; ERK, extracellular signal-regulated kinase; Akt, Akt
serine/threonine kinase.