Introduction
Lung cancer is one of the major causes of
cancer-related mortality worldwide (1). Lung cancer is generally classified
into two major types, small cell lung cancer (SCLC) and non-small
cell lung cancer (NSCLC). The latter is the most common type
accounting for ~80% of lung cancer cases (2). The early diagnosis of lung metastasis
can contribute to the decrease of mortality and morbidity, since
the migration and invasion of cancer cells from the primary tumor
sites into the surrounding tissues promote the progression of
metastasis.
Of note, epithelial to mesenchymal transition (EMT)
was known to promote the tumor cell infiltration into interstitial
stroma by extending microtubule-based protrusions and inhibiting
cell proliferation (3). There is
accumulating evidence that EMT plays an important role in cancer
progression, wound healing, invasion and tissue fibrosis as well as
in embryogenesis (4–6). The EMT process results in the loss of
epithelial cell phenotype and exhibits mesenchymal cell
characteristics. Therefore, epithelial cell type genes such as
E-cadherin, claudins and occludin are downregulated, while
mesenchymal cell type genes including N-cadherin, vimentin and
fibronectin related to the migration, invasion and proliferation
are upregulated during the EMT process (7,8).
However, although EMT transcriptional factors such as Twist, Snail,
Slug and Zinc finger E-box binding homeobox 1 (ZEB1) are aberrantly
overexpressed in a patient with NSCLC as a highly fibrotic
malignancy (9,10), their upstream factors remain
unclear.
ZNF746, which contains a Kruppel-associated box at
its N terminus and a C2HC/C2H2 type zinc finger at its C terminus,
interacts with Parkin as a ubiquitin E3 ligase (11,12) or
as a suppressor of peroxisome proliferator-activated receptor γ
coactivator-1 (PGC-1) in neurodegeneration in Parkinson’s disease
(12). Nevertheless, the role of
ZNF746 has yet to be elucidated in tumorigenesis. Thus, in the
present study, we investigated for the first time whether or not
the inhibition of ZNF746 suppresses the invasion and EMT process in
H460 NSCLC cells.
Materials and methods
Cell culture
H460 NSCLC cells (HTB-177™) were obtained from the
American Type Culture Collection (ATCC). H460 NSCLC cells were
maintained in Roswell Park Memorial Institute (RPMI)-1640 media
(WelGENE, Inc., Daegu, Korea) supplemented with 10% fetal bovine
serum (FBS; Gibco, Carlsbad, CA, USA) and 1% antibiotics at 37°C in
5% CO2.
Invasion assay
To investigate the migratory properties of H460
NSCLC cells, after transfecting with control or small interfering
RNA (siRNA) ZNF746 (Bioneer, Daejun, Korea) (80 nM) using
INTERFERin® reagent (Polyplus, Illkirch, France),
invasion assay was carried out using modified 48-well
microchemotaxis chambers (Neuro Probe Inc., Gaithersburg, MD, USA).
Polyvinylpyrrolidone-free polycarbonate filters (8 mm pore size)
(Neuro Probe, Inc.) were coated with Matrigel, a solubilized
basement membrane matrix. The lower chamber was filled with media
containing 10% FBS as chemoattractant agents. The coated filter and
upper chamber were laid over the lower chamber. The control siRNA
and ZNF746 siRNA (Bioneer, Daejun, Korea)-transfected H460 NSCLC
cells (1×104 cells/25 μl) were seeded onto the upper
chamber wells. After 24 h incubation at 37°C, the filter was fixed
and stained with Diff-Quick (Sysmex, Kobe, Japan) and non-migrated
cells on the upper surface of the filter were wiped off with a
swab. Then, randomly chosen fields were photographed under a
microscope (DFC420C; Leica, Germany) and the number of cells that
migrated to the lower surface was counted.
Real-time quantitative RT-PCR
(RT-qPCR)
Total RNA was isolated from H460 NSCLC cells with
QIAzol (Invitrogen, Carlsbad, CA, USA). A reverse transcription kit
(Promega, Madison, WI, USA) was used to construct the template
cDNA. RT-qPCR was performed with the LightCycler™ instrument (Roche
Applied Sciences, Indianapolis, IN, USA) according to the
manufacturer’s protocol. The mRNA level of GAPDH was used to
normalize the expression of genes of interest. The primers used
were: E-cadherin forward, 5′-CAAG CTATCCTTGCACCTCAG-3′ and reverse,
5′-GCATCAAGA GAACTCCTATCTTG-3′; matrix metalloproteinase (MMP)1
forward, 5′-CTGGCCACAACTGCCAAATG-3′ and reverse,
5′-CTGTCCCTGAACAGCCCAGTACTTA-3′; MMP2 forward,
5′-TCTCCTGACATTGACCTTGGC-3′ and reverse,
5′-CAAGGTGCTGGCTGAGTAGATC-3′; MMP7 forward,
5′-TGAGCTACAGTGGGAACAGG-3′ and reverse, 5′-TCAT
CGAAGTGAGCATCTCC-3′; MMP9 forward, 5′-TTGACA GCGACAAGAAGTGG-3′ and
reverse, 5′-GCCATTCACGT CGTCCTTAT-3′; Slug forward,
5′-GCGATGCCCAGTCTA GAAAA-3′ and reverse, 5′-GCAGTGAGGGCAAGAAA
AAG-3′; Nanog forward, 5′-CAGCTGTGTGTACTCAATG ATAGATTT-3′ and
reverse, 5′-ACACCATTGCATTTCTTCG GCCAGTTG-3′; octamer-binding
transcription factor 4 (OCT4) forward, 5′-GACAACAATGAAAATCTTCAGG
AG-3′ and reverse, 5′-CTGGCGCCGGTTACAGAACCA-3′; SMAD2 forward,
5′-GTTCCTGCCTTTGCTGAGAC-3′ and reverse, 5′-TCTCTTTGCCAGGAATGCTT-3′;
vimentin forward, 5′-CAACCTGGCCGAGGACAT-3′ and reverse,
5′-ACGCATTGTCAACATCCTGTCT-3′; ZNF746 forward,
5′-GCTGCACACCGGCGAGCGGCCC-3′ and reverse, 5′-CT
TGCGGAGGTGGTCCTTGCG-3′; GAPDH forward, 5′-CCA CTCCTCCACCTTTGAC-3′
and reverse, 5′-ACCCTGTTGC TGTAGCCA-3′.
Immunofluorescence assay
H460 NSCLC cells transfected with control or ZNF746
siRNA (Bioneer) plasmids were fixed with 4% paraformaldehyde and
permeabilized in 0.1% Triton X-100. The fixed H460 NSCLC cells were
then washed with 1X PBS and blocked with 10% normal goat serum
blocking solution (Zymed Laboratories, Carlsbad, CA, USA) for 30
min at room temperature. Fixed cells were incubated with the
specific primary antibodies against ZNF746 (Sigma-Aldrich, St.
Louis, MO, USA), E-cadherin, β-catenin (Cell Signaling Technology,
Inc., Danvers, MA, USA) overnight at 4°C. After washing, the cells
were incubated with Alexa Fluor 594 goat anti-rabbit IgG
(Invitrogen) for 30 min at room temperature. After washing, the
cells were mounted with Vectashield/DAPI (Vector Laboratories,
Inc., Burlingame, CA, USA) and visualized by a Carl Zeiss LSM5
confocal microscope.
Western blotting
Whole cell lysates from H460 NSCLC cells transfected
with control or ZNF746 siRNA plasmids were prepared using lysis
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1%
SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and
protease inhibitor cocktail). The concentration of protein was
measured by using a Bio-Rad DC protein assay kit II (Bio-Rad,
Hercules, CA, USA). The proteins were separated on 8 or 12%
Bis-Tris Gels (Invitrogen). Hybond ECL transfer membrane (GE
Healthcare Bio-Sciences, Piscataway, NJ, USA) was used to transfer
the gel. The membranes were blocked with 5% nonfat dry milk.
Primary antibodies were used for detecting ZNF746 (Sigma-Aldrich),
E-cadherin, β-catenin (Cell Signaling Technology, Inc.), Twist
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and β-actin
(Sigma-Aldrich). Secondary antibody was used with horseradish
peroxidase (HRP)-conjugated secondary antibodies. Enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ, USA) was used.
Results
Silencing of ZNF746 suppresses the
invasive property in H460 NSCLC cells
To investigate the role of ZNF746 in tumorigenesis,
the effect of the silencing of ZNF746 was evaluated on the invasion
of H460 NSCLC cells transfected with ZNF746 siRNA plasmid. Boyden
chamber invasion assay was performed to examine the effect on the
invasive activity of ZNF746. Knockdown of ZNF746 significantly
inhibited the invasive property compared to untreated control in
H460 NSCLC cells (Fig. 1A and B).
Western blot assay showed that ZNF746 was efficiently inhibited by
siRNA transfection (Fig. 1C).
Silencing of ZNF746 attenuates the
expression of MMP1, MMP2 and MMP9, but not MMP7, in H460 NSCLC
cells by RT-qPCR
MMPs promote tumor survival, invasion as well as
metastasis (13). In this regard,
we evaluated whether ZNF746 depletion regulates the expression of
MMPs (MMP1, MMP2, MMP7 and MMP9) in H460 NSCLC cells by RT-qPCR.
The depletion of ZNF746 attenuated the expression of MMP1, MMP2 and
MMP9, but not MMP7 (Fig. 2).
Silencing of ZNF746 upregulates
E-cadherin and β-catenin in H460 NSCLC cells by western blotting
and immunohistochemistry
EMT is a key phenotype by which cancer cells acquire
motility and invasion (4,8). In this regard, to explore whether or
not ZNF746 is associated with EMT in H460 NSCLC cells, we examined
the biomarkers of epithelial and mesenchymal phenotypes after
ZNF746 siRNA knockdown in H460 NSCLC cells by western blotting.
ZNF746 knockdown enhanced the expression of E-cadherin and
β-catenin as epithelial markers, while the expression of Slug was
suppressed as a mesenchymal marker (Fig. 3A). Consistently,
immunohistochemistry revealed that the silencing of ZNF746 reduced
the red color expression for β-catenin and E-cadherin in H460 NSCLC
cells (Fig. 3B).
Silencing of ZNF746 upregulates EMT
molecules at the mRNA level in H460 NSCLC cells and hypoxia
enhances the protein expression of ZNF746 in parallel with
Twist
To verify that ZNF746 regulates EMT molecules at the
mRNA level, RT-qPCR analysis was performed. Expression of
epithelial marker (E-cadherin), mesenchymal marker (vimentin) or
transcriptional factors such as ZEB1, Twist, Snail and Slug, was
evaluated in ZNF746 siRNA-transfected H460 NSCLC cells. ZNF746
knockdown downregulated the expression of Twist, vimentin, ZEB1,
and Slug, but not SMAD2, and Snail, and also upregulated the
expression of E-cadherin in H460 NSCLC cells (Fig. 4A and B).
Hypoxia accelerates the cell invasion (14) and plays an important role in EMT
(15). To confirm that ZNF746 or
Twist can be induced under hypoxia, western blotting was carried
out in H460 NSCLC cells. The expression of ZNF746 was induced from
2 to 8 h in hypoxic H460 NSCLC cells (Fig. 4C). Of note, Twist, one of the EMT
molecules, was also induced from 4 to 8 h in hypoxic H460 NSCLC
cells almost in parallel with ZNF746.
Silencing of ZNF746 significantly
suppresses the expression of Nanog and OCT4 at the mRNA level in
H460 NSCLC cells
Although Nanog and OCT4 are transcriptional factors
in embryonic stem cells (16), they
have been studied in cell metastasis through the EMT process
(17,18). In this regard, we determined the
mRNA expression of OCT4 and Nanog by RT-qPCR following ZNF746 siRNA
transfection. OCT4 and Nanog were significantly suppressed at the
transcriptional level in ZNF746 siRNA-treated H460 NSCLC cells
(Fig. 4D).
Discussion
Lung cancer is generally classified into small cell
lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC).
The lung is a common metastatic site from other parts of the body
and primary lung cancer usually metastasizes to the brain, bones,
liver and adrenal glands (19).
Metastases represent the end products of a multistep
cell-biological process known as the invasion-metastasis cascade in
several types of cancer (20). In
the present study, the role of ZNF746 was examined in the invasion
and EMT in H460 NSCLC cells.
In the present study, invasion assay using Boyden
chamber revealed that ZNF746 knockdown significantly reduced the
number of the invaded H460 NSCLC cells. It is well documented that
several MMP family members such as MMP1 (21), MMP2 (22), MMP7 (23) and MMP9 (24) are involved in invasion and
metastasis. RT-qPCR analysis showed that the silencing of ZNF746
attenuated the expression of MMP1, MMP2 and MMP 9, but not MMP7, in
H460 NSCLC cells, indicating that the ZNF746 knockdown can suppress
the invasion of H460 NSCLC cells, since ZNF746 may mediate
invasion-metastasis cascades.
Accumulating evidence suggests that EMT is a
critical event in tumor invasion and metastasis (3,4,25).
Thus, activation of the EMT is known to endow the invasive and
metastatic properties upon cancer cells for the successful
colonization of distal target organs (26). Previous studies also revealed that
EMT is crucial in drug resistance to EGFR inhibitor in NSCLC
(27) and promotes the cell
invasion and metastatic property. NSCLC cells expressing epithelial
phenotype are more sensitive to EGFR inhibition than NSCLC lines
expressing mesenchymal phenotype such as vimentin (28–30).
Here, western blotting showed that E-cadherin and β-catenin as
epithelial phenotypes were upregulated, while Slug as a mesenchymal
phenotype was downregulated in ZNF746 siRNA vector-transfected H460
NSCLC cells compared to untreated control, indicating that the
silencing of ZNF746 inhibits invasion-metastasis cascades via
regulation of EMT molecules. Accordingly, mRNA expression of
E-cadherin was upregulated, while that of vimentin or Slug, Twist,
ZEB was also attenuated at the mRNA level in ZNF746
siRNA-transfected H460 NSCLC cells, strongly demonstrating that the
silencing of ZNF746 favorably regulates EMT molecules at the mRNA
and protein levels in H460 NSCLC cells.
Hypoxia plays an important role in the progression
and metastasis of cancer (31).
Also, hypoxia inducible factor (HIF) α regulates transcriptional
regulators of EMT such as Twist (32)and ZEB1 (33–35).
In several cancer cell lines, N-cadherin, vimentin and fibronectin
are upregulated in hypoxia exposure (32). Similarly, ZNF746 was induced by
hypoxia in parallel with Twist expression in H460 NSCLC cells. In
addition, several studies showed that transcriptional marker Nanog
and OCT4 promoted malignancy and metastasis in lung adenocarcinoma
(18), melanoma (16) and colorectal (17) cancer. Here, ZNF746 siRNA-transfected
H460 NSLC cells suppressed the expression of Nanog and OCT4 in H460
NSCLC cells, indicating that ZNF746 knockdown may possibly regulate
lung cancer progression and metastasis mediated by Nanog and
OCT4.
In summary, the silencing of ZNF746 suppressed the
invasion by inhibition of MMP1, MMP2 and MMP9, upregulated
E-cadherin and β-catenin as well as downregulated Slug at the
protein level, consistently enhanced E-cadherin and attenuated the
expression of EMT key transcriptional factors such as vimentin,
Slug, Twist and ZEB, suppressed the expression of Nanog and OCT4 in
H460 NSCLC cells. Also, ZNF746 was induced by hypoxia in parallel
with Twist expression in H460 NSCLC cells. Overall, our findings
suggest that ZNF746 may be a critical target molecule in the
invasion and EMT process in H460 NSCLC cells. Nevertheless, animal
studies should be performed in the future to further validate our
in vitro evidence.
Acknowledgements
This study was supported by the National Research
Foundation of Korea (NRF) grant funded by the Korean government
(MEST) (no. 2012-0005755).
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