miR-26b suppresses tumor proliferation and metastasis by targeting

Int J Clin Exp Pathol 2016;9(7):6945-6951
www.ijcep.com /ISSN:1936-2625/IJCEP0027117
Original Article
miR-26b suppresses tumor proliferation and metastasis
by targeting matrix metalloproteinases
14 in neuroblastoma
Meiji Chen, Weimin Feng, Feng Wei
Department of Pediatrics, Eastern District of The First Affiliated Hospital, Sun Yat-sen University, Guangzhou,
China
Received March 2, 2016; Accepted June 27, 2016; Epub July 1, 2016; Published July 15, 2016
Abstract: Increasing reports suggested that microRNAs (miRNAs) act as key regulators in multiple cancers. miR-26b
functions as a tumor suppressor in some malignancies. However, its role in neuroblastoma (NB) remains poorly
understood. Herein, we found that miR-26b is downregulated in NB tissues. Enhanced overexpression of miR-26b
significantly suppressed NB cell proliferation, migration, and invasion. Matrix metalloproteinase 14 (MMP14) was
found to be a direct target of miR-26b in NB cells. Rescue experiments indicated that the suppressive effect of miR26b on cell proliferation, migration, and invasion is partially mediated by inhibiting MMP14 expression. Collectively,
the present study indicated that miR-26b might inhibit NB cell viability and motility partially by targeting MMP14.
This newly identified function of miR-26b provides novel insights into neuroblastoma and may foster therapeutic
applications.
Keywords: Neuroblastoma, MicroRNAs, miR-26b, MMP14
Introduction
Neuroblastoma (NB) is one of the most common extracranial childhood tumors, accounting
for 7% of childhood malignancies and >15% of
all childhood cancer deaths [1]. NB is characterized by heterogeneous clinical behaviors,
ranging from spontaneous regression to rapid
progression or resistance to multimodal treatment, such as surgery, chemoradiotherapy, and
immunotherapy [2]. Despite recent therapeutic
improvements in NB treatment, the overall
5-year survival rate remains poor, 73% of NB
patients have already developed malignant
lesions at the time of diagnosis [3, 4]. Therefore,
a better understanding of the detailed mechanisms might be helpful to find new therapeutic
targets and strategies for the treatment of NB.
MicroRNAs (miRNAs), a class of small non-coding RNAs with 22 to 25 nucleotides in length,
are able to inhibit gene expression at the posttranscriptional or translational levels through
forming base pairs with their targets, usually in
the 3’-untranslated region (3’-UTR) [5, 6]. miRNAs play essential roles in a variety of biological
and pathological processes, including cell proliferation, migration, invasion, apoptosis, and
metastasis [7]. In cancer, the function of miRNAs is dependent on their mRNA targets, so
they can act as tumor suppressors or as oncogene [8]. For example, Friedman et al suggested that miR-101 acted as a tumor suppressor to
modulate the cancer epigenome by repressing
the polycomb group protein EZH2 [9]. Jiang et al
showed that miR-155 functions as an OncomiR
in breast cancer by targeting the suppressor of
cytokine signaling 1 gene [10]. Tsai et al indicated that miR-122 function as a tumor suppressor that regulated intrahepatic metastasis
of hepatocellular carcinoma [11].
In the present study, we identified the important molecular mechanism by which miR-26b
exerted its tumor suppressive effects on NB
cell proliferation, migration and invasion, which involved the direct targeting the 3’-UTR of
MMP14.
miR-26b target MMP14 in NB
Figure 1. miR-26b was down-regulated in NB tissues.
miR-26b expression was determined by quantitative
real-time PCR (qRT-PCR) in human NB tissues and
adjacent non-tumor tissues. *P<0.05 compared with
adjacent non-tumor tissues.
plus (Applied Biosystems). miRNA was extracted using All-in-one microRNA extraction kit
(GeneCopoeia). Primers for MMP14: sense,
5’-TCGGCCCAAAGCAGCAGCTTC-3’ and anti-sense, 5’-CTTCATGGTGTCTGCATCAGC-3’. Primers
for GAPDH: sense, 5’-AACGGATTTGGTCGTATTG-3’, and anti-sense, 5’-GGAAGATGGTGATGGGATT-3’. Primers for miR-26b and U6 were purchased from GeneCopoeia (Carlsbad). The
expression of MMP14 was normalized with
GAPDH, and the expression of miR-26b was
normalized with U6.
Luciferase reporter assay
22 paired of NB and matched adjacent nontumor tissues from patients were obtained
from Eastern District of the First Affiliated
Hospital. The diagnoses of these samples were
verified by pathologists. The collection of human tissue samples was approved and supervised by the Ethics Committee of Sun Yat-sen
University.
pGL3 plasmid encoding a luciferase reporter
gene was purchased from Promega. Recombinant plasmid of pGL3-MMP14 3’-UTR (Wt)
or pGL3-MMP14 3’-UTR (Mut) was constructed in our laboratory. HEK-293 cells were plated
in a 24-well plate and cotransfected with either
miR-26b mimics or miRNA control, either pGL3MMP14 3’-UTR (Wt) or pGL3-MMP14 3’-UTR
(Mut), and 2 ng of pRL-TK (Promega) by using
Lipofectamine 2000. The pRL-TK vector was
used as an internal control to correct the differences in both transfection and harvest efficiencies. HEK-293 cells were collected 48 h after
transfection and analyzed using the DualLuciferase Reporter Assay System (Promega).
Cell culture and transfection
Cell proliferation analysis
The human neuroblastoma cell line SHSY5Y
and the HEK-293 cell lines were purchased
from the Institute of Biochemistry and Cell
Biology (Shanghai, China). All cells were cultivated in RPMI1640 medium supplemented
with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) and maintained at 37°C
in a humidified 5% CO2 incubator. miR-26b
mimics and control mimics were purchased
from RiboBio (Guangzhou, China). pcDNA3MMP14 was obtained from Applied Biosystems
(Foster). Transfections of miRNA mimics or pcDNA3-MMP14 were carried out using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s protocol.
Cell viability was measured using the MTT
assay. The viability of SHSY5Y cells transfected
with miR-26b or control mimics was detected
every 24 h after transfection. 100 μl MTT (10
mg/mL) was added to each well of the 96-well
plates and incubated for 4 h, and then the reaction was terminated by DMSO. The optical density (OD) at 490 nm was detected using a
Microplate Reader (Bio-Tek).
Materials and methods
Patients
RNA extraction and quantitative real time PCR
(qRT-PCR)
Total RNA was extracted by TRIzol Reagent
(Invitrogen). SYBR Premix Ex Taq (Takara) was
used to detect gene expression on ABI Stepone
6946
Cell invasion and migration assays
Transfected SHSY5Y cells were seeded in the
upper compartments of the 8 um Boyden
chamber (BD Biosciences). Migration and invasion assays were carried out with coated
Matrigel (invasion) or uncoated Matrigel (migration). After incubation for 48 h, cells migrated
and invaded through the lower chamber were
fixed with methanol, stained with 0.5% Giemsa,
and photographed under the microscope
(Olympus).
Int J Clin Exp Pathol 2016;9(7):6945-6951
miR-26b target MMP14 in NB
Figure 2. Ectopic expression of miR-26b repressed viability and motility of NB cells. A. Expression of miR-26b was
determined in SHSY5Y cells after transfection of miR-26b mimics or negative controls (miR-NC). B. The cell viability
of SHSY5Y cells were determined by MTT assay. C. The cell migration ability of SHSY5Y cells were determined by
Transwell migration assay. D. The cell invasion ability of SHSY5Y cells were determined by Transwell invasion assay.
*P<0.05 compared with control.
Western blot
Transfected cells were washed twice with PBS
and proteins were extracted with SDS lysis buffer (Beyotime), and separated by 10% SDSPAGE gel. Protein samples were transferred to
PVDF membrane (Millipore), and were probed
with primary antibodies against MMP14
(Abcam). Membranes were incubated at 4°C
overnight, followed by incubation with AP-conjugated secondary antibodies and detected by
ECL (Thermo Scientific).
Statistical analysis
All data were presented as mean ± SD and analyzed by using SPSS 16.0. Two-tail Student’s t
test and ANOVA were performed to determine
6947
the differences. P<0.05 was considered statistically significant.
Results
miR-26b was down-regulated in NB tissues
To investigate whether miR-26b is involved in
the progression of neuroblastoma, we firstly
compared the expression of miR-26b between
NB tissues and adjacent non-tumor tissues.
Our data showed that the expression of miR26b was significantly decreased in NB tissues
compared with matched adjacent non-tumor
tissues (Figure 1, P<0.05). These data suggested that miR-26b may contribute to the progression of NB.
Int J Clin Exp Pathol 2016;9(7):6945-6951
miR-26b target MMP14 in NB
Figure 3. MMP14 was a target of miR-26b in NB cells. A. MMP14
contained potential binding sites of miR-26b. B. HEK293 cells
were co-transfected with miR-26b mimics or miR-NC and wildtype (Wt) or mutant (Mut) 3’-U TR of MMP14. Dual luciferase
activity was used. C, D. SHSY5Y cells were transfected with miR26b mimics or miR-NC. The mRNA and protein expression level
of MMP14 was detected by qRT-PCR and Western blot 48 h later.
*P<0.05 compared with control.
Ectopic expression of miR-26b inhibited NB
cell viability and motility
To examine the role of miR-26b in NB cell progression, SHSY5Y cells were transfected with
miR-26b mimics or miR-NC and the transfection efficient was determined by qRT-PCR. We
found that miR-26b expression was significantly increased in SHSY5Y cells after transfected
with miR-26b mimics (Figure 2A, P<0.05). MTT
assay revealed that over-expression of miR26b significantly inhibited the proliferation ability of SHSY5Y cells (Figure 2B, P<0.05). In addition, transwell migration and invasion assays
suggested that overexpression of miR-26b also
remarkably suppressed the in vitro migration
and invasion abilities of SH-SY5Y cells (Figure
2C and 2D, P<0.05). These findings indicated
that miR-26b could suppress the development
and progression of NB.
miR-26b directly targeted MMP14 and inhibited its expression in NB cells
To screen the function target of miR-26b in NB
cells, TargetScan 6.2 was used to screen the
6948
target gene of miR-26b. MMP14 was predicted
to be a target of miR-26b in NB cells (Figure
3A). We constructed luciferase reporter plasmids to contain the putative sequences (Wt) for
MMP14 and their corresponding mutant
sequences (Mut) as controls. Luciferase activity assay showed that miR-26b significantly
inhibited the luciferase activity of the Wt 3’-UTR
but not that of Mut 3’-UTR of MMP14 (Figure
3B, P<0.05). In addition, overexpression of
miR-26b significantly inhibited MMP14 mRNA
and protein levels (Figure 3C and 3D, P<0.05).
These results suggested that MMP14 might be
a direct target of miR-26b in NB cells.
MMP14 overexpression attenuated the suppressive effect of miR-26b
Further experiments were performed to explore
whether overexpression of MMP14 could attenuate the suppressive effect of miR-26b. The
effect of MMP14 plasmids was detected by
qRT-PCR (Figure 4A, P<0.05). MTT assay
revealed that supplement of MMP14 by pcDNA3-MMP14 could significantly attenuate the
Int J Clin Exp Pathol 2016;9(7):6945-6951
miR-26b target MMP14 in NB
Figure 4. miR-26b inhibited NB progression by targeting MMP14. A. The mRNA expression of MMP14 in SHSY5Y
cells after transfection of pcDNA3-MMP14 (MMP14) or empty vector (Control). B. MTT assay was used to detect
proliferation of SHSY5Y cells co-transfected with miR-26b mimics, pcDNA3-MMP14 or empty vector. C. Transwell
migration assay was used to detect invasion of SHSY5Y cells co-transfected with miR-26b mimics, pcDNA3-MMP14
or empty vector. D. Transwell invasion assay was used to detect invasion of SHSY5Y cells co-transfected with miR26b mimics, pcDNA3-MMP14 or empty vector. *P<0.05 compared with control; #P<0.05 compared with miR-26b
mimics group.
suppressive effect of miR-26b in NB cell proliferation (Figure 4B, P<0.05). In addition, in vitro
cell migration and invasion showed that overexpression of MMP14 significantly attenuated
the suppressive effect of miR-26b in NB cell
motility (Figure 4C and 4D, P<0.05). These
results suggested that miR-26b might suppress NB progression by targeting MMP14
(Figure 5).
Discussion
cantly decreased in NB tissues and ectopic
expression of miR-26b remarkably inhibited the
proliferation, migration and invasion of NB
cells. By using luciferase activity assay and
Western blot, we found that MMP14 was a
direct target of miR-26b. Overexpression of
miR-26b suppressed cell growth and motility,
whereas overexpression of MMP14 antagonized this effect. Our findings illustrated the
possible role of miR-26b and MMP14 in the
progression of NB.
In this study, we explored the expression of
miR-26b in NB patients. Our data showed that
the expression levels of miR-26b were signifi-
Accumulating evidence revealed the important
function of miRNAs in NB development and progression. For example, Liu et al showed that
6949
Int J Clin Exp Pathol 2016;9(7):6945-6951
miR-26b target MMP14 in NB
miR-150-5p could inhibit hepatoma cell migration and invasion by targeting MMP14 [24]. In
this study, we found that MMP14 could also be
regulated by miR-26b in NB, supporting its
oncogenic role in NB progression.
Figure 5. Schematic miR-26b suppressed NB cell
progression via targeting MMP14.
miR-451 inhibited NB cell proliferation, invasion and migration by targeting macrophage
migration inhibitory factor [12]. Wu et al indicated that miR-362-5p inhibited proliferation
and migration of NB cells by targeting phosphatidylinositol 3-kinase-C2β [13]. On the other
hand, several miRNAs function as oncogenes in
NB. For example, Qu et al reported that miR558 promoted tumorigenesis and aggressiveness of NB cells through activating the transcription of heparanase [14]. Li et al found that
miR-421 promoted proliferation and migration
of NB cells by targeting tumor suppressor
menin [15].
Recent studies showed that miR- 26b acted as
a tumor suppressor in some cancers. For example, Li et al suggested that miR-26b inhibited
hepatocellular carcinoma cell proliferation,
migration, and invasion by targeting EphA2
[16]. Li et al revealed that miR-26b suppressed
the metastasis of non-small cell lung cancer by
targeting MIEN1 via NF-κB/MMP-9/VEGF pathways [17]. Zheng et al showed that miR-26b
inhibited osteosarcoma cell migration and invasion by down-regulating PFKFB3 expression
[18]. Our study expanded the tumor suppressive role of miR-26b in NB progression.
Matrix metalloproteinases 14 (MMP14) is a
“master switch” proteinase with a C-terminal
sequence that acts as membrane-anchoring
domain, and is a key enzyme involved in ECM
degradation and invasion of tumor cells [19].
MMP14 is constitutively increased in several
cancers, such as gastric cancer, tongue squamous cell carcinoma and renal cancer [20-22].
MMP14 contributes to cancer progression and
was regulated by many miRNAs. For example,
Chang et al found that miR-133b inhibited cell
migration and invasion by targeting MMP14 in
glioblastoma [23]. Li et al demonstrated that
6950
In conclusion, this study identify a functional
link between miR-26b and MMP14 expression
in NB, indicating that miR-26b may serve as a
potential therapeutic target for the treatment
of NB.
Acknowledgements
Supported by Science and Technology Planning Project of Guangdong Province, China
(No.201510010148).
Disclosure of conflict of interest
None.
Address correspondence to: Dr. Meiji Chen, Department of Pediatrics, Eastern District of The First
Affiliated Hospital, Sun Yat-sen University, Guangzhou 510070, China. E-mail: meijichen83@163.com
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
Siegel R, Naishadham D and Jemal A. Cancer
statistics, 2013. CA Cancer J Clin 2013; 63:
11-30.
Cheung NK and Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer 2013; 13: 397411.
Park JR, Eggert A and Caron H. Neuroblastoma:
biology, prognosis, and treatment. Hematol
Oncol Clin North Am 2010; 24: 65-86.
Diede SJ. Spontaneous regression of metastatic cancer: learning from neuroblastoma.
Nat Rev Cancer 2014; 14: 71-72.
Bartel DP. MicroRNAs: genomics, biogenesis,
mechanism, and function. Cell 2004; 116:
281-297.
Bartel DP. MicroRNAs: target recognition and
regulatory functions. Cell 2009; 136: 215233.
Esquela-Kerscher A and Slack FJ. Oncomirs—
microRNAs with a role in cancer. Nature
Reviews Cancer 2006; 6: 259-269.
Shenouda SK and Alahari SK. MicroRNA function in cancer: oncogene or a tumor suppressor? Cancer Metastasis Rev 2009; 28: 369378.
Friedman JM, Liang G, Liu CC, Wolff EM, Tsai
YC, Ye W, Zhou X and Jones PA. The putative
tumor suppressor microRNA-101 modulates
Int J Clin Exp Pathol 2016;9(7):6945-6951
miR-26b target MMP14 in NB
[10]
[11]
[12]
[13]
[14]
[15]
[16]
the cancer epigenome by repressing the polycomb group protein EZH2. Cancer Res 2009;
69: 2623-2629.
Jiang S, Zhang HW, Lu MH, He XH, Li Y, Gu H,
Liu MF and Wang ED. MicroRNA-155 functions
as an OncomiR in breast cancer by targeting
the suppressor of cytokine signaling 1 gene.
Cancer Res 2010; 70: 3119-3127.
Tsai WC, Hsu PWC, Lai TC, Chau GY, Lin CW,
Chen CM, Lin CD, Liao YL, Wang JL and Chau
YP. MicroRNA122, a tumor suppressor microRNA that- regulates intrahepatic metastasis of
hepatocellular carcinoma. Hepatology 2009;
49: 1571-1582.
Liu G, Xu Z and Hao D. MicroRNA‑451 inhibits
neuroblastoma proliferation, invasion and migration by targeting macrophage migration inhibitory factor. Mol Med Rep 2016; 13: 225360.
Wu K, Yang L, Chen J, Zhao H, Wang J, Xu S and
Huang Z. miR-362-5p inhibits proliferation and
migration of neuroblastoma cells by targeting
phosphatidylinositol 3-kinase-C2β. FEBS Lett
2015; 589: 1911-1919.
Qu H, Zheng L, Pu J, Mei H, Xiang X, Zhao X, Li
D, Li S, Mao L and Huang K. miRNA-558 promotes tumorigenesis and aggressiveness of
neuroblastoma cells through activating the
transcription of heparanase. Hum Mol Genet
2015; 24: 2539-2551.
Li Y, Li W, Zhang JG, Li HY and Li YM. Downregulation of tumor suppressor menin by miR421 promotes proliferation and migration of
neuroblastoma. Tumour Biol 2014; 35: 1001110017.
Li H, Sun Q, Han B, Yu X, Hu B and Hu S. MiR26b inhibits hepatocellular carcinoma cell proliferation, migration, and invasion by targeting
EphA2. Int J Clin Exp Pathol 2015; 8: 47824790.
6951
[17] Li D, Wei Y, Wang D, Gao H and Liu K. MicroRNA26b suppresses the metastasis of non-small
cell lung cancer by targeting MIEN1 via NFkappaB MMP-9/VEGF pathways. Biochem
Biophys Res Commun 2016; 472: 465-70.
[18] Zheng WD, Zhou FL and Lin N. MicroRNA-26b
inhibits osteosarcoma cell migration and invasion by down-regulating PFKFB3 expression.
Genet Mol Res 2015; 14: 16872-16879.
[19] Egeblad M and Werb Z. New functions for the
matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002; 2: 161-174.
[20] Dong Y, Chen G, Gao M and Tian X. Increased
expression of MMP14 correlates with the poor
prognosis of Chinese patients with gastric cancer. Gene 2015; 563: 29-34.
[21] Jia LF, Wei SB, Mitchelson K, Gao Y, Zheng YF,
Meng Z, Gan YH and Yu GY. miR-34a inhibits
migration and invasion of tongue squamous
cell carcinoma via targeting MMP9 and
MMP14. PLoS One 2014; 9: e108435.
[22] Catania JM, Chen G and Parrish AR. Role of
matrix metalloproteinases in renal pathophysiologies. Am J Physiol Renal Physiol 2007; 292:
F905-911.
[23] Chang L, Lei X, Qin Y, Zhang X, Jin H, Wang C,
Wang X, Li G, Tan C and Su J. MicroRNA‑133b
inhibits cell migration and invasion by targeting matrix metalloproteinase 14 in glioblastoma. Oncol Lett 2015; 10: 2781-2786.
[24] Li T, Xie J, Shen C, Cheng D, Shi Y, Wu Z, Zhan
Q, Deng X, Chen H, Shen B, Peng C, Li H and
Zhu Z. miR-150-5p inhibits hepatoma cell migration and invasion by targeting MMP14.
PLoS One 2014; 9: e115577.
Int J Clin Exp Pathol 2016;9(7):6945-6951