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Defining the Regulatory Role of Programmed Cell Death 4 in
Laryngeal Squamous Cell Carcinoma
Journal: Biochemistry and Cell Biology
Manuscript ID bcb-2017-0293.R3
Manuscript Type: Article
Date Submitted by the Author: 16-Feb-2018
Complete List of Authors: XU, YUANTENG; The First Affliated Hospital of Fujian Medical University CHEN, RUIQING; The First Affliated Hospital of Fujian Medical University LIN, GONGBIAO; The First Affliated Hospital of Fujian Medical University FANG, XIULING; The First Affliated Hospital of Fujian Medical University YU, SHUJUAN; The First Affliated Hospital of Fujian Medical University LIANG, XIAOHUA; The First Affliated Hospital of Fujian Medical University
ZHANG, RONG; The First Affliated Hospital of Fujian Medical University
Is the invited manuscript for consideration in a Special
Issue? : N/A
Keyword: Programmed cell death 4, Laryngeal squamous cell carcinoma, Epithelial mesenchymaltransition, miR-21, β-catenin
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Defining the Regulatory Role of Programmed Cell Death 4 in Laryngeal Squamous Cell
Carcinoma
Yuan-Teng Xu1, Rui-Qing Chen
2, Gong-Biao Lin*
1, Xiu-Ling Fang
1, Shu-Juan Yu
1, Xiao-Hua
Liang3, Rong Zhang*
1
1. Department of Otolaryngology, The First Affiliated Hospital of Fujian Medical University,
Fuzhou 350005,Fujian,China
2. Central Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou
350005,Fujian,China
3. Clinical Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou
350005,Fujian,China
*Corresponding author:
Gong-Biao Lin, M.D Ph.D
Department of Otolaryngology, The First Affiliated Hospital of Fujian Medical University
Fuzhou 350005,Fujian,China
E-mail:[email protected]
Rong Zhang, M.D Ph.D
Department of Otolaryngology, The First Affiliated Hospital of Fujian Medical University
,Fuzhou 350005,Fujian,China
E-mail:[email protected]
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Abstract
Purpose: Programmed cell death 4 (PDCD4) is decreased in many malignant tumors. Epithelial
mesenchymal Transition (EMT) endows tumor cells invasive and metastatic properties. Few
studies have elucidated the role of PDCD4 in the regulation of EMT during laryngeal carcinoma.
Methods: Using laryngeal carcinoma tissues, the relationship between PDCD4 and EMT-
associated proteins E-cadherin and N-cadherin was examined. Gene manipulation was utilized to
define the regulatory capacity of PDCD4. Results: We report that PDCD4 and E-cadherin/N-
cadherin expression were significantly altered in carcinoma tissues, their expression was
associated with pathological grade, metastatic state, and clinical stage. The suppression of
PDCD4 (and consequently, E-cadherin) was concomitant with increased proliferation and G2-
phase arrest, decreased apoptosis, and increased cell invasion. PDCD4 up-regulation reversed the
above results. In nude mice, PDCD4 knockdown increased tumor growth and pathological
features, confirming the tumorigenic role of PDCD4. Finally, PDCD4 silencing was associated
with dysregulation of the carcinogenic Wnt/ß-catenin and the STAT3/miR-21 signaling
pathways. Conclusions: This study elucidates a dynamic regulatory relationship between PDCD4
and critical EMT factors, establishing a broad, functional role for PDCD4 in laryngeal carcinoma
which may be propagated by the STAT3/miR-21 pathway. These findings provide new
information on an EMT-associated target which may provide novel therapeutic recourse.
Highlights
• PDCD4 is a clinical correlate of EMT-associated cadherins in human LSCC
• PDCD4 regulates LSCC proliferation, apoptosis, growth, migration and
invasiveness
• PDCD4 knockdown demonstrates theanti-tumorigenic role of PDCD4 in vivo
• β-catenin and the miR-21/STAT3 axis are downstream mediators of PDCD4
function
Key Words
Programmed cell death 4, Laryngeal squamous cell carcinoma, Epithelial-mesenchymaltransition,
miR-21,β-catenin
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Abbreviations
abbreviation Full Name
LSCC laryngeal squamous cell carcinoma
FBS Fetal Bovine Serum
DMSO dimethyl sulfoxide
EDTA Ethylene DiamineTetraacetic Acid
GFP Green Fluorescent Protein
GAPDH glyceraldehyde-3-phosphate
dehydrogenase
PDCD4 Programmed cell death 4
EMT epithelial-mesenchymal transition
E-cadherin Epithelial-cadherin
N-cadherin Neuronal-cadherin
β-catenin β-catenin
3´-UTR 3´-Untranslated Regions
miRNAs micro Ribonucleic acids
mRNA Messenger Ribonucleic acid
RNAi Ribonucleic acid interference
shRNA short hairpin RNA
PCR Polymerase Chain Reaction
RT-PCR reverse transcription PCR
PBS phosphate buffer saline
SDS Sodium Dodecyl Sulfate
PAGE Polyacrylamide gel electrophoresis
PI Propidium Iodide
PVDF polyvinylidene fluoride
LV Lentivirus
DEPC Diethypyrocarbonate
STAT3 signal transducer and activator of
transcription 3
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dNTP Deoxyribonucleoside triphosphate
WB Western blot stain
ANOVA Analysis of Variance
UICC Union for International Cancer Control
SPF Specific pathogen Free
ddH2O Double distillated water
ml Milliliter
Μl Microliter
Μg Microgram
Min Minute
sec Second
OD Optical density
rpm revolutions per minute
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Introduction
Laryngeal squamous cell carcinoma (LSCC) is the predominant form of laryngeal
carcinoma and as of 2002, was associated with 160000 new cases/year. The ratio of males to
females affected is 5:1(Thomas et al. 2012). As of 2016, according to the National Cancer
Institute (NCI), laryngeal carcinoma accounts for 0.8% of all new cancer patients and the overall
5-year survival rate was 60.7%(Marioni et al. 2006). Laryngeal carcinoma is typically caused by
smoking and drinking and long-term exposure to coal dust, carbide dust, and chlorine-containing
solvents (associated with supraglottic type)(Shangina et al. 2006). Although the surgical
techniques and chemo and radiotherapies have improved over the past 30 years, the overall
survival rate of patients does not appear to have improved significantly, particularly in advanced
stages.
The prognosis is closely related to local invasion and cervical lymph node metastasis
therefore, it is important to study the processes endowing laryngeal carcinoma cells with their
invasive and metastatic properties. Programmed cell death 4 (PDCD4), is a highly conserved
protein that is widely expressed in normal and cancerous human tissues(Afonja et al. 2004;
Asangani et al. 2008;Hiyoshi et al. 2009;Gaur et al. 2011). Clinical data suggests that high levels
of PDCD4 may be associated with improved prognosis in patients with lung cancer, colon cancer
and ovarian cancer(Chen et al. 2003; Wei et al. 2009 ;Allgayer 2010), implying that PDCD4 acts
as a tumor suppressor in many types of cancers.Indeed, a previous study characterized the
progressive decline of PDCD4 in laryngeal cancer tissues, predictably decreasing according to
tumor differentiation state and cervical lymph node metastasis(Wang et al. 2011).
In recent years, attention has turned to the mechanistic and functional aspects of PDCD4
in tumorigenesis, much of which remains unknown. Some work has detailed the involvement of
PDCD4 in the epithelial-mesenchymal transition (EMT) which is required for cancer cells to
evolve attributes conducive to local invasion of neighboring tissues and long-range metastasis.A
hallmark of EMT is a loss of E-cadherin, a cell surface transmembrane glycoprotein which
interacts with β-catenin to activate cytoskeletal maintenance and epithelial cell adhesion
signaling(Tian et al. 2011).For example, PDCD4 knockdown promoted EMT via the up-
regulation of SNAIL/SLUG- a repressor of E-cadherin and EMT enhancer(Wang et al.
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2013).Additionally, E-cadherin down-regulation has been correlated with laryngeal cancer
recurrence and decreased survival(Cappellesso et al. 2015).
N-cadherin is another adhesion molecule implicated in EMT. Like E-cadherin, N-
cadherin has also demonstrated a relationship with clinical stage and degree of tumor
differentiation in laryngeal carcinoma(Song et al. 2016). Unlike E-cadherin, N-cadherin has been
positively correlated with cancer cell motility and invasive capacity in breast cancer cells(
Nieman et al. 1999;Hazan et al. 2000), prostate cancer(Tanaka et al. 2010), and in lung cancer
cell lines(Zhang et al. 2013). At least part of this function is mediated by N-cadherin induced de-
differentiation of cells, from epithelial to mesenchymal(Hazan et al. 1997).In contrast to E-
cadherin, PDCD4 appears to have an inverse relationship with N-cadherin, as demonstrated by
Wang et al in colon and breast carcinoma cell lines(Wang et al. 2013). Interestingly, N-cadherin
has been previously correlated with E and P-cadherin reduction in prostate cancer cells and the
ratio of N-cadherin to E-cadherin may reflect EMT status(Gravdal et al. 2007). Though the
specific interactions governing the cadherin/EMT axis are unclear, the likely scenario is the
convergence of multiple, EMT-associated pathways. One which has been speculated is the
activation of the STAT3/miR-21 cascade by IL-6 in human bronchial epithelial cells(Luo et al.
2013), inhibition of which resulted in suppressed growth of head and neck squamous carcinoma
cells(Zhou et al. 2014; Zhou et al. 2014).
Due to the known relationship ofPDCD4 and tumor progression and invasion and
metastasis, researchers have explored down-regulation techniques to more closely probe the
functional and mechanistic pathways by which it may act. One such study involved gene-editing
techniques to inhibit PDCD4 expression in colon cancer cells. These cells were subsequently
inoculated into the colon wall, where they enhanced metastases in tumor cells(Wang et al.
2013).The role of PDCD4 in the progression of laryngeal squamous cell carcinoma has been
relatively less investigated. Specifically, its role in laryngeal cancer EMT and associated
signaling pathways has not been reported. As such, the purpose of this study was toclarify the
role of PDCD4 gene expression in laryngeal squamous cell carcinoma and to explore the
molecular mechanism of PDCD4 in regulating laryngeal cancer EMT.Additionally, we sought to
investigate changes in cell function, such as growth, apoptosis, invasion and migration. To
accomplish this, carcinoma cells stably expressing either PDCD4-silencing or PDCD4-
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overexpressing plasmids/vectorswere carefully studied. Further, one of these cell lines was
transplanted into nude mice laryngeal carcinoma cells after up-regulation and down-regulation of
PDCD4 in stable-expressing carcinoma cell lines in order to confirm the tumorigenicity of
PDCD4 in vivo.
This study is the first to employ a loss and gain of function approachto define the
regulatory role of PDCD4 in a model of laryngeal squamous cell carcinoma. We found that
PDCD4 expression impacts a broad range of cellular functions and plays a dynamic role in EMT.
Finally, we verified the anti-tumorigenic function of PDCD4 in vivo and identified two signaling
processes that are attached to the PDCD4/EMT axis. These findings shed important light on the
molecular mechanisms of PDCD4 and the EMT axis in laryngeal carcinoma. Additionally, they
provide an experimental basis for the study of future treatment strategies targeting the
PDCD4/EMT pathway.
Methods
Carcinoma tissue acquisition and processing
Eighty laryngeal squamous cell carcinoma surgical resection specimens were acquired from
the First Affiliated Hospital of Fujian Medical University from January 2012 to December
2015.Postoperative pathology was usedtoconfirmlaryngeal squamous cell carcinoma, of which
40 cases were graded as Ⅰ-Ⅱ, 40 cases as grade Ⅲ-IV, and 31 cases determined as cervical
lymph node metastasis. Nosurgical treatment, radiotherapy and chemotherapy, or immunology
and biological therapy had occurred prior to specimen resection. An additional 40 tissues were
collected from adjacent normal tissues for use as controls. Lymph node metastasis was
confirmed by postoperative histopathology. The clinical pathologic features of the patients are
detailed in Table 1.
Assessment of clinicopathologic features
Clinicopathological features of the patients from surgical resection specimens were acquired
including gender, and average age of clinical diagnosis (≥60 and <60 years). According to the
pathological results, specimens were classified as either high/med differentiation and low
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differentiation. Additionally, the tumor site wasclassified as eitherglotticorsupraglottic type.
According to the 2002 UICC diagnostic criteria for laryngeal cancer, specimens were classified
into clinical stages which were subsequently grouped as Ⅰ - Ⅱ or Ⅲ - Ⅳ. Finally, cervical
lymph node metastasis was determined as either positive or negative.
Immunohistochemistry of laryngeal carcinoma and adjacent tissues
The expression of PDCD4, E-cadherin and N-cadherin in laryngeal squamous cell
carcinoma and normal tissues were detected by streptavidin-peroxidase (SP) method.Wax-
embedded specimens were sectioned continuously in 4mm thick sections, with at least six slices
per specimen. After antigen retrieval, the following primary antibodieswere added to each slice
and incubated overnight 4 ° C: anti-PDCD4 (1:1000, rabbit monoclonal, Cell Signaling
Technology, USA), anti-E-cadherin (1:1000, rabbit monoclonal, Cell Signaling Technology,
USA), and anti-N-cadherin (1:1000, rabbit monoclonal, Cell Signaling Technology, USA). The
following day, the biotin-labeled secondary antibody was added (1:200, anti-rabbit IgG, Fuzhou
Maixin Biotechnology Co.) for 10 min at room temperature. Subsequently, slices were incubated
in 50 µl of Streptomyces biotin-peroxidase solution for 10 minutes at room temperature. DAB
solution was then added and slices were observed for 3-10 minutes under a microscope.
Counterstain was performed by hematoxylin dye.
Quantitative assessment of immunohistochemical examinations
Quantitative evaluation of immunoreactivity was based on the number of colored cells and
color strength. Specific score criteria were as follows: random selection of cells at high
magnification on a microscope, positive expression of cells in view expressed as percentage of
total (such as <5% =0 point, 5% to 25%= 1 point, 26% to 50% =2 points, 51% to 75% =3 points,
and 76% and above= 4 points). Approximate intensity of total staining was subsequently scored
according to the cell coloring strength of 0-3 points, (no colorimeter =0 points, light yellow= 1
point, yellow= 2 points, and brown =3 points). The two scores of each slice were then multiplied
and divided into the following categories:0 = negative, 1-4= weak positive, 5-8 =positive, 9-12 =
strong positive. For simplicity, both negative and weak positive results were considered
“negative” and both positive and strong positive results were considered “positive”.
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Culture of Hep-2 and SNU-899 cells
The human laryngeal carcinoma cell line Hep-2 was purchased from ATCC (American Type
Culture Collection) and SNU-899 cells were acquired. Human immortalized epidermal (Hacat)
cells were stored in our laboratory. Hep-2 and SNU-899 cells were cultured in 10 ml RPMI1640
complete culture medium in100 mm Petri dishesand incubated in a 5% CO2 incubator at 37 ° C.
Hacat cells were cultured in DMEM medium. Cells were passaged at 80-90% confluence using1
ml of trypsin containing 0.25% EDTA and gentle mechanical dissociation of cell clusters.
Extraction of cells was performed by digestion with 1 ml of trypsinsolution containing 0.25%
EDTA. After cell retraction was observed, RPMI1640 complete culture medium containing 10%
fetal bovine serum was added to terminate the digestion and the cells were homogenized in 3 ml
ofsterilepasteurpipette. The digested cells were collected into a 15 ml centrifuge tube and
centrifuged at 1500 rpm for 5 min. The supernatant was discarded and cells were transferred into
1.8ml cryopreservation tubes and stored at -70 ℃.
Establishment of laryngeal carcinoma Hep-2 cell line with stable silencing of PDCD4 gene
The plasmid used in this experiment was designed and synthesized by Shanghai Ji Kai
Gene Chemical Technology Co., Ltd., and GV248 was used as vector plasmid. The gene
structure was shown in Supplementary Figure 2A. Four short hairpin RNAs (shRNA) and one
negative control sequence were designed according to the PDCD4 mRNA SEQ ID NO:
NM_014456 published by NCBI (see Supplementary Figure 2B). The target sequence of the
negative control shRNA-NC was a random sequence: 5 '-TTCTCCGAACGTGTCACGT-3',
which is not homologous with any human gene sequence. The shRNA are abbreviated as
RNAi1-4 (chronologically) and shRNA-NC.
Hep-2 cells were re-suspended by trypsin digestion and seeded ontoa 60 mm culture dish
(~6x105 cells) with DMEM. DNA plasmid and transfection liquid Lipofectamine 3000
TM were
mixed at a ratio of 1:3 and incubated for 40 min at room temp to allow the plasmid and the
transfection solution to form a complex. After 24hr incubation, plasmid DNA and transfection
solutionwas added to Hep-2 cells followed by gentle shaking. After 48 h transfection, cells were
placed under fluorescence microscope to observe the transfection efficiency of the cells. A
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subgroup of each experimental condition was cultured with Puromycin at a concentration of 0.5
µg / ml for 2-3 weeks.
Construction of lentivirus expression vector, viral packaging, lentivirus titer determination
The lentivirus packaging system used involved three plasmids: the vector (GV358) carrying
the gene of interest, helper1.0 and helper2.0, the virus-supporting plasmids. The GV358
lentiviral vector contains the basic components of HIV 5'LTR and 3'LTR as well as other
auxiliary components. Vector plasmid (GV358) element sequence contained the following: Ubi-
MCS-3FLAG-SV40-EGFP-IRES-puromycin, cloning siteAgeI / AgeI, labeling / resistance
marker 3FLAG (tag), EGFP, and puromycin (see Supplementary Figure 3A).
In this study 293T cells were used for lentiviral infection and purification. Twenty-four
hours before transfection, 293T cellsin logarithmic growth phase were digested with trypsin and
the cell density was adjusted to about 5 x 106 cells / 15 ml in medium containing 10% serum.
The cells were re-suspended in 10 cm cell culture dishes and cultured to 70-80% confluence.A
prepared DNA solution (20 µg of GV358 vector, 15 µg of pHelper 1.0 vector and 10 µg of
pHelper 2.0 vector) was added to a sterile centrifuge tube and mixed with the corresponding
volume of the transfection reagent to adjust the total volume to 1 ml.
293T cells were harvested 48 hours after transfection by centrifugation at 4000g for 10 min
at 4° C, after which supernatant was collected and filtered through a 0.45 µm filter. Supernatant
was subsequently transferred to 40 ml ultracentrifuge tube and centrifuged at 25000rpm for2hat
4 ℃. After ultra-centrifugation, the supernatant was discarded and pellet was re-suspended in
virus preservation solution.
To assess the titer of the lentivirus, 293T adherent cells were plated on 96-well plates, at a
density of 4 x 104 cells/well. Various concentrations of purified virus were preparedaccording to
the expected titer of the virus. After 24 h, 100 µl of complete medium was added and after 4 days,
fluorescence was observed. Due to the expression of the GFP tag, fluorescent cells indicated the
infection of the cells with the lentivirus. Subsequently, virus titer was determined according to
the following equation:
Virus titer = fluorescence cell count / virus stock solution = 2 / (1E-6) = 2E + 6 (TU / µl) =
2E + 9 (TU / ml)
A PDCD4 full-length clone was purchased from Shanghai Ji Kai Gene Chemical
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Technology Co., Ltd. as a template to amplify the PDCD4 coding frame sequence. The PCR
products were identified by 1% agarose gel electrophoresis followed by gel digestion.
Sequencing was performed to confirm the amplification of the cloned plasmid. Following
confirmation, restriction enzyme digestion was performed and fragments were ligated to the
lentivirus expression vector GV358. Subsequently, the ligation product was transformed into
competent E. coli. The plasmid was transfected into 293T cells and the light emission of GFP
was used to assess transfection. Finally, RT-PCR was used to identify the expression of
PDCD4.Two lentiviruses were obtained using this method, lentiviral LV-PDCD4 (17318-1)
overexpressing PDCD4 and blank control lentivirus LV-NC (CON238). All of the above
processes were carried out by Shanghai Ji Kai Gene Chemical Technology Co., Ltd.
Lentiviral infection of SNU-899 cells
SNU-899 cells were cultured to 80% confluence. Following tripsin digestion, complete
medium containing 3-5 × 104 / mL cell suspension was inoculated with 2ml of lentivirus in 6-
well culture plate. After 16 h incubation, virus-containing medium was replaced with
conventional culture medium to continue culture. Fluorescent microscopy was used to evaluate
the efficiency of infection. Only wells observed at 70% infection or higher were used for
downstream experiments. Seventy-two hours after initial infection, a subset of cells were
screened for cell viability via 48 hour incubation in medium containing puromycin at a final
concentration of 2µg / ml. Puromycin-containing medium was subsequently replaced and cells
were cultured for 2-3 weeks at which timecells remained healthy and proliferating and expressed
afluorescent signal, indicating stable expression of the lentivirus.
RNA isolation and RT-PCR
Extraction of total RNA from cells was performed viaTRIzol reagent, according to the
following instructions. Cells were trypsinized and aspirated for centrifugation. Cell pellets were
incubated with 1 ml of Trizol. Cells were transferred into 1.5 ml sterile tubes and incubated in
200 µl of chloroform for 5 min at room temperature. Following centrifugation at 12000 rpm for
15 min, the upper aqueous (RNA) phase was transferred to a clean 1.5mL tube and an equal
volume of pre-cooled isopropanol was gently mixed and incubated for 10 min at room temp.
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Subsequent 12000 rpm centrifugation was performed for 5 min at 4 ℃ and supernatant was
removed to reveal RNA pellet. Pellet was washed in 1 ml of pre-cooled 75% ethanol two times.
A final 7500 rpm centrifugation was performed for 5 min and supernatant was discarded. Pellet
was dried for 10-15 min at room temp and precipitate was re-dissolved in 50 µl of DEPC-treated
water and stored at -70 ° C. RNA concentration was measured by UV spectrophotometer at the
A260 / A280 ratio.
A total of 20 µlof purified RNA (containing 1 µg of total RNA)was used for reverse
transcription synthesis of cDNA by Promega RT mixtureaccording to the manufacturer’s
instructions. Thermocycling parameters were carried out as follows:50℃ for 60 min, 70 ℃ for
10 min, and ice cooling.
Real-time (RT) PCR amplification of PDCD4 and internal control gene GAPDH was
performed in this study using the following primer sequences synthesized by Bioengineering
(Shanghai) Co., Ltd. Promega.
PDCD4: upstream primer: 5 'G T G A C G C C T T A G A A G T G G A 3'
Downstream primer: 5 'C T G C A C C A C C T TTTTT G G T 3'
GAPDH: upstream primer: 5 'A T G A C A T C A A G A A G G T G G T G 3'
Downstream primer: 5 'C A T A C C A G G A AA T G A G C T T G 3'
PromegaGoTaq reagents were used in conjunction with the above primers and 2uL of
cDNA for fluorescent, quantitative PCR according to the following parameters: 95 ° C for 30 s,
95 ° C for 5 s, and 60 ° C for 30 sfor 40 cycles using Applied Biosystems Fluorescence
Quantification PCR 7500 instrument.
Total protein extraction and Western Blot
Total protein was extracted from isolated cells by mechanical disruption. After 4 , ℃
12000 rpm centrifugation, the supernatant (containing total protein) was transferred into a clean
centrifuge tube. The appropriate amount of BCA working solution was prepared according to the
number of samples required. Samples were assessed in triplicates following 5% CO2 incubator at
37 ° C for 30 min. Absorbance was read at 562nm on a microplate reader and a standard protein
curve was used to quantify experimental samples.
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200 µl of the measured protein samples was addedto 5 µl of protein loading bufferin 50 µl,
boiling water for 10 min to denature the protein. Polyacrylamide gel (4-8%) was prepared and
40µg of the sample was loaded. Protein was separated by electrophoretic system at 100V in
running buffer until bromophenol blue was observed near the bottom of the separation plastic.
Transfer of the gel plate was performed on PVDF membrane by sandwich transfer at 100 V for
90 min in transfer buffer. PVDF membrane was incubated in 15 ml of blocking solution (TBST
solution containing 5% skimmed milk powder) and sealed at room temperature for 120 min. The
primary antibodies against PDCD4 (1: 1000), GAPDH (1: 2000), E-cadherin (1:1000), and N-
cadherin (1:1000), STAT-3 (1:1000), and ß-catenin (1:1000) were incubated on membrane
overnight at 4 ° C. The following day secondary antibody (1:5000) was added at room temp for
120 min. Finally, membrane was incubated in ECL fluorescent substrate A and B solution for 7
min in the dark. Membrane was subsequently scanned by Image pro-plus image analysis
software. Protein density of the bands was expressed relative to the optical density of the
reference (GAPDH) band.
MTT proliferation assay
The MTT colorimetric method was used to detect cell survival and growth. Transfected
cells in the logarithmic growth phase were digested and counted. Equal amounts of cells were
seeded on 96-well plates in 200 µl basal medium RPMI1640. Groups were inoculated with MTT
(10mg/ml) solution at 37 ° C for 5 h in a 5% CO2 incubator. Next, MTT medium was aspirated
and 200µl of DMSO was added to mix the formed crystals. Wells were evaluated at 1, 2, 3, 4, 5,
6, and 7 days (five wells per time point). The absorbance (OD) of each well was measured by
enzyme-linked immunoassay at a wavelength of 490 nm. The OD values indicate cell
proliferation capacity. Values were plotted against time point to produce a growth curve.
Colony formation assay
Tripsinizedcells(~500) seeded onto 6-well plates with 2ml complete medium and were
allowed to proliferate for more than sixpassages (~10-14 days). The cells were then fixed with
6% glutaraldehyde and 0.5% crystal violet for 30 min. CTL S5 Versa analyzerwas used for clone
quantitation. Each group was measured in triplicates and the colony formation rate was
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calculated according to the following equation: number of clones formed / number of inoculated
cells x 100%.
Flow cytometry and cell cycle analysis
Tripsinizedcells in the logarithmic growth phase were harvested and centrifuged to remove
non-cellular fluid (2000 rpm for 5 min). Cell precipitates were incubated in 1 ml of ethanol (70%)
overnight to fix the cells.Ethanol-immobilized cells were centrifuged at 2000 rpm for 5 min,
discarded with ethanol and aspirated with a pipette to remove residual ethanol. Following a PBS
wash, 200 µl of PI working fluid was mixed with cells for 30 min in a dark room for subsequent
flow cytommetry (FACS).
Flow cytometry for detection of cell apoptosis
Early apoptosis can be detected by Annexin V. 7-AAD can be used to distinguish between
surviving early cells and necrotic or late apoptotic cells. Annexin V and 7-AAD collectively can
be used to profile cells in different apoptotic states using flow cytometry.In a scatter plot of
bivariate flow cytometry, the lower left quadrant shows live cells, the lower quadrant is early
apoptotic cells, the upper right quadrant is apoptotic cells, and the upper left quadrant shows
necrotic cells. To achieve this, 3 × 105
cells were seeded on 6-well plates and cultured for 24
hours. Cells were tripsinized and transferred to 1.5ml tubes for centrifugation (2000 rpm for 3
min). Precipitate was re-suspended in 200µl of binding buffer with 5 µl of Annexin V-PE and 10
ul of 7-AAD for 10 minutes at room temperature. Finally, cells were centrifuged and washed
with binding buffer repeatedly, after which the precipitate of cells was used for flow cytometry.
Transwell invasion assay
The lower level of a Transwell chamber was filled with extracellular matrix-mimicking
Matrigel matrix glue. Cells which enter the lower chamber must first secrete matrix
metalloproteinases (MMPs) for matrix degradation, through the polycarbonate film. The number
of cells entering the lower chamber can thus reflect the invasion capacity of tumor cells. Here,
matrix rubber was diluted with the serum-free medium RPMI 1640. The diluted matrix gel was
added to thebottom chamber at 50 µl / well and incubated at 37 ℃, 5% CO2 for 30 min.A single
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cell suspension (concentration 5.0x 105 / ml) of cells was seeded in a 6-well cell culture plate and
cultured for 24 hours. Cells were subsequently seeded at 1 x 105 cells / well in the upper
chamber. Matrix-coated bottom chambers were subsequently filled with RPMI + 10% FBS
medium and chambers were incubated for 24 hours at 37 ℃, 5% CO2.Transwell membranes
were next removed and allowed to dry before fixation in 70% ethanol. Membrane was then
immersed in 0.1% crystal violet for 30min, after which cells could be quantified by inverted
microscope (200 X). Five visual fields were selected from each room at random for quantitation.
The average was recorded as the number of cells migrating through the membrane.
Mouse xenograft procedures and tissue processing
The experimental animals used in this experiment were 4 week old SPF grade BALB / C
nude mice (about 20g,SHANGHAI SLAC LABORATORY ANIMAL CO. LTD). Ten nude
mice were randomly divided into two groups. Stable shRNA-PDCD4 and shRNA-NC expressing
Hep-2 cells were screened and confirmed. A single cell suspension was used for cell
quantification and an additional aliquot (10µl) was used to assess the viability of the cells with 1
drop of trypan blue dye. Only cell suspensions containing live cell ratios of > 95% were used for
in vivo experiments.
BALB / C nude mice were acclimated for one week. Mice were disinfected in the right
armpit skin with 75% medical grade alcohol.Previously prepared cell suspensions (0.2ml,5
x105)were slowly injected subcutaneously at the disinfection site.Mice were observed daily for
tumor occurrence and growthfor 30days. This included the time of occurrence and the
measurement of tumor size every 3 days using a vernier caliper to determine the maximum
diameter (a) and minimum diameter (b) of the tumor. Tumor volume was subsequently
calculated as, V = a × b2 / 2, and values were plotted on a 30 day growth curve.After the final
measurement, animals were removed from the sterile laminar flow chamber, euthanized by
cervical dislocation and tumor specimens were removed. Images were taken of the tumors and
tissues were subsequently weighed, blocked, and separated into 2/3 sections for cryopreservation
and 1/3 for wax embedding.
miR-21TaqMan Real Time RT-PCR
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RNA was extracted from frozen tissues according to the DNA-free kit instructions.
Briefly, 1X rDNase I buffer and 1 µl of rDNase were incorporated into total RNA for 30 min.
Next, 1X of DNase Inaction Reagent was added for 2 minutes. Following 10000 rpm
centrifugation for 90 seconds, RNA containing supernatant was removed and RNA (5µl, 10ng)
was incubated with TaqMan MicroRNA Assay KitforRNAmiRNA stem-loop (stem-loop) primer
reverse transcription. Reagents were incorporated according to manufacturer’s instructions. The
following primers were added:
Human miR-21 primer sequence
The upstream primer is: 5'-TAGCTTATCAGACTGATG-3 '
The downstream primer is: 5'-TGGTGTCGTGGAGTCG-3 '
U6 primer sequence
The upstream primers are: 5'-CTCGCTTCGGCAGCACA-3 '
The downstream primers are: 5'-AACGCTTCACGAATTTGCGT- 3 '
Thermal cycler conditions were set to 16 ℃ 30min, 42 ℃ 30min, 85 ℃ 5min, and indefinite
4 ℃.
Quantitation of miR-21 expression was subsequently performed using U6 as an internal
reference gene. PCR amplification was performed on ABI7000 thermal cycler (Applied
Biosystems) using TaqMan MicroRNA Assay Kit and the above listed gene primers. Thermal
cycler conditions were set to50 2 mins, 95 10 mins, 95 15s, and 60 1 min for 40 cycles. ℃ ℃ ℃ ℃
Amplification curve was checked using the 7500 system SDS software and the Ct values were
automatically analyzed. Measurements for each sample were made in triplicates and the
experiment was additionally repeated three times. Using U6 as the internal reference, the Ct
value of Hep-2 / shRNA-NC group and Hep-2 / shRNA-PDCD4 group was calculated as:
∆ Ct value = miR-21 Ct value -U6 Ct value.
Statistics
All data were presented as Mean ± SD. All data processing was performed using SPSS
22.0 statistical software. Statistical analysis of immunohistochemistry and clinicopathological
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data was performed using a chi-square test. The mean value of multiple groups was analyzed by
single factor analysis of variance, where P <0.05 indicated statistical significance. Comparison of
the means of two groups was performed by t-test, where statistical significance was determined
as P <0.05.
Results
Expression of PDCD4, E-cadherin and N-cadherin in laryngeal squamous cell carcinoma
PDCD4 protein expression was mainly located in the nucleus and cytoplasm though
PDCD4 expression in adjacent normal tissues was significantly higher than in laryngeal
squamous cell carcinoma (Figure 1). PDCD4 protein expression was differentially expressed in
different carcinoma grades, clinical stages, and states of cervical lymph node metastasis (Table 1,
P <0.05). Specifically, the positive rate of PDCD4 expression in laryngeal squamous cell
carcinoma was significantly lower in the low differentiation stages than that in the high/mid
differentiation group. On the other hand, the positive rate of PDCD4 in advanced squamous cell
carcinoma clinical stages was significantly lower than that in early clinical stage group. PDCD4
protein was also significantly lower in cervical lymph node metastasis group compared to the
non-metastatic tissues.
E-cadherin is mainly located in cytoplasm-facing side of the cell membrane. The
expression of E-cadherin protein in normal epithelium was significantly higher than that in
laryngeal squamous cell carcinoma (see Table 1 and Figure 1). The expression of E-cadherin
was also differential according to carcinoma grade, clinical stage, and cervical lymph node
metastasis (Table 1, P <0.05). The positive expression rate of E-cadherin in low differentiated
laryngeal squamous cell carcinoma was significantly lower than that in the mid/high
differentiated group. On the other hand, the expression of E-cadherin was decreased as clinical
stage of the tissue became advanced, similar to PDCD4. The positive rate of protein expression
in cervical lymph node metastatic carcinoma was also significantly lower than that in non -
metastasis group.
The expression of N-cadherin protein is mainly located in the cytoplasm. In the
paracancerous normal epithelium, N-cad was almost completely undetected whereas the
expression in laryngeal squamous cell carcinoma was about 15% (see Table 1, Figure 1). The
expression of N-cadherin protein in different laryngeal squamous cell carcinoma grades, clinical
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stages, cervical lymph node metastasis was significantly different between groups (P <0.05). The
positive rate of N-cadherin in the low differentiated group was significantly higher than that in
mid/high group. Conversely, N-cadherin was significantly increased parallel to advanced clinical
stage. Similarly, the positive rate of expression was increased by metastasis. No statistically
significant changes in PDCD4, E-cadherin, or N-cadherin were detectable across age and gender
categories or glottis or superior/inferior positioning of the carcinoma.
Relationship of PDCD4 and E-cadherin and N-cadherin in laryngeal squamous cell carcinoma
There was a positive correlation between PDCD4 and E-cadherin protein expression in
laryngeal squamous cell carcinoma (Table 2, P <0.05). Unlike E-cadherin, PDCD4 and N-
cadherin protein expression was negatively correlated (Table 2, P <0.05).We had previously
hypothesized that PDCD4 expression and EMT-related proteins had some intrinsic relationship.
Here we see that PDCD4 is associated (negatively and positively) with two criticalEMT proteins
in laryngeal squamous cell carcinoma.
Endogenous expression of PDCD4 mRNA in Hep-2, SNU-899, human immortalized epidermal
(Hcat) cells
Hep-2, SNU-899, and HacatRNAwerereverse transcribed to generate cDNA which was
subjected to fluorescence quantitative PCR using GAPDH as the internal reference gene. The
expression of endogenous PDCD4 mRNA in each of these cell lines was determined by ∆∆CT
method. The expression of PDCD4 in Hacatcells was on par with the endogenous level observed
in the reference gene. The expression in the two carcinoma cell lines was lower by comparison
(F = 54.03, P <0.01), with Hep-2 cells expressing slightly more PDCD4 than SNU-899 cells
(Supplementary Figure 1). Due to these findings, the Hep-2 cell line seemed most appropriate
for PDCD4 silencing and SNU-899 for PDCD4 overexpression.
Stable PDCD4 gene silencing in Hep-2 cells
Hep-2 cells were transfected with lipofectamine (TM) 3000 liposomes, and after 2 weeks of
puromycin screening, the transfected cells were observed under UV and fluorescence
microscopy (Figure 2A-B). Fluorescent Hep-2 cells indicate successful transfection of the
plasmid, which was designed to carry the GFP fluorescent protein tag as a marker. Transfection
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of the shRNA-PDCD4 and shRNA-NC plasmid groups was above 90%, indicating successful
transfection efficiency. Positive clones were screened by addition of Puromycin 48 h-12 days
after transfection. Clones containing GFP were picked up and used for RNA processing.
Fluorescence quantitative PCR was used to detect PDCD4 mRNA levels among the four
shRNA-PDVD4 (“RNAi” for short) interfering groups (Figure 2C). The most significant
PDCD4-silencing RNAi was shRNA8653 (RNAi3), which was used for all subsequent PDCD4-
silencing experiments.
Stable overexpression of PDCD4gene in SNU-899 cells
SNU-899 cells were infected with lentiviral vector LV-PDCD4 (17318-1) and LV-NC
(CON238) containing PDCD4 fragments respectively. After 72 hours of infection, cell infection
efficiency was determined by fluorescent microscopy (Figure 2E-H) to be 70% or higher,
indicating successful transfection. Cells were also screened by puromycin and determined to
have healthy, viable cells.
The relative expression of PDCD4 mRNA in SNU-899 LV-NC and LV-PDCD4 groups
were assessed by quantitative PCR as 1.014 ± 0.117 and 7.382 ± 0.153, respectively. The
expression level of LV-PDCD4 group was significantly higher than that of the negative control
group (see Figure 2I, t = 33.11, P <0.001, n = 3), indicating successful overexpression of the
PDCD4 gene in SNU-899 cells.
PDCD4 protein following sh-RNA transfection of Hep-2 cells and letiviral transfection of SNU-
899 cells
Experimental and control group cells were cultured and harvested for protein detection of
PDCD4. The total protein concentration was measured and the appropriate concentration of
protein was used for Western blot analysis. Optical density of detectable bands was quantified for
statistical comparison and the results are shown in Figure 2D. The expression of PDCD4 in the
Hep-2 / PDCD4-shRNA8653 group was 0.449 ± 0.022, significantly lower than in the control
group (t = 4.918,P﹤0.01,n=3) and echoing the earlier observations of PDCD4 mRNA
knockdown. Conversely, in SNU-899 cells the expression of PDCD4 protein was 0.43 ± 0.12 in
the LV-NC group and 0.96 ± 0.17 in the overexpression group (Figure 2J, t = 4.41, P <0.05,n=3),
indicating increased expression of PDCD4 reminiscent of mRNA patterns observed earlier.
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Taken together, the observations of PDCD4 mRNA and protein in Hep-2 and SNU-899 cells
suggest broad and stable down and up-regulation of the gene and product, respectively.
Colony formation/growth assessment of transfected cells
Transfected Hep-2cells were cultured for 12-14 days. The colony formation rate (%) of the
Hep-2/ shRNA-PDCD4 group was 12.467 ± 1.361, which was significantly higher than that of
Hep-2/ ShRNA-NC group at 8.067 ± 0.945. The results showed that the clonal formation ability
of PDCD4 cells was significantly increased by PDCD4 knockdown (Figure 3B, t = -4.598, P
<0.05, n =3), indicative of increased growth. In SNU-899 cells, the colony formation rate (%) of
the LV-NC / SNU-899 group was 11.26 ± 1.16, which was significantly higher than that of LV -
PDCD4 / SNU-899 group at 6.06 ± 0.34. The results showed that the clonal formation ability of
PDCD4-overexpressing cells was significantly decreased compared with controls (Figure 3E, t =
7.45,P﹤0.01,n=3). Collectively, the results suggest a negative correlation between PDCD4 and
clonal formation/growth ability.
MTT proliferation assay of transfected cells
MTT assay was used to detect the cell proliferation. The OD value of 490 nm was measured
at 24 hours after inoculation of the cells with MTT reagents. The OD values of the inoculated
cells was measured daily for 7 days. Values were plotted in time-course for both the
experimental and control groups. The results of Hep-2 cells showed that cell proliferation of
PDCD4-silenced cells was significantly higher than control cells at every measured time point
(Figure 3C, P﹤0.05,n=3). In SNU-899 cells, the overexpression of PDCD4 yielded
significantly lower proliferation than controls across all measured time points (Figure 3F,P﹤
0.01,n=3). Collectively the results support the notion that PDCD4 expression is negatively
correlated with cell proliferation. Further, this inverse relationship echoes the early finding that
PDCD4 is negatively associated with cancer cell colony formation and growth ability.
Cell cycle distribution of transfected cells as detected by flow cytometry
Propidium iodide was used along with flow cytometry to evaluate the intracellular DNA
content of control and experimental cells. In this manner, the percentage of cells in G1/G0 phase
could be distinguished from cells in G2/M (synthesizing) phases of the cell cycle. In Hep-2 cells,
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the percentage of cells in G1 phase in the shRNA-NC group was 61.78±3.98% while the
shRNA-PDCD4 group had 46.74 ± 3.04%, indicating a significant reduction (Figure 4B, t =
5.37, P <0.01, n = 3). Thepercentage of G2 cells was 38.22 ± 3.74% for the shRNA-NC group
and 53.76 ± 3.98% in the shRNA-PDCD4 group (Figure 4B, t = 4.93, P <0.01, n = 3), indicating
that PDCD4 silencing increased the proportion of cells in G2 significantly. Overall, the results
agreed that stable down - regulation of PDCD4 promoted cells from G1 into the G2 phase,
indicative of enhanced intracellular DNA of the cells.
The percentage of G1 phase cells in the stage of SNU-899 cells was 48.97 ± 2.32% in the
LC-NC group and 57.37 ± 3.58% in LV-PDCD4 group (Figure 4D, t = 3.41, P < 0.05,n=3),
indicating a significant increase after PDCD4 knockdown. The percentage of SNU-899 cells in
G2 phase was 44.87 ± 2.78% in the LV-NC group and 17.75 ± 1.08% in the LV-PDCD4 group
(Figure 4D, t = 15.75, P <0.001,n=3), indicating that PDCD4 overexpression inhibited the
progression of cells from G1 to G2 phase.
Apoptosis of transfected cells as detected by flow cytometry
Annexin V and flow cytometrywere used to determine the apoptosis of transfected cells.In a
scatter plot of bivariate flow cytometry, the lower left quadrant indicates live cells, the lower
quadrant are early apoptotic cells, the upper right quadrant are apoptotic cells, and the upper left
quadrant show necrotic cells. The results demonstrated that amount of apoptotic shRNA-PDCD4
cells were significantly lower than the apoptotic cells in theshRNA-NC group, 3.71 ± 0.98% and
13.01 ± 1.24% respectively (Figure 5B, t = 10.19, P <0.01, n = 3). This indicated that the stable
silencing of PDCD4 decreased the proportion of apoptotic cells.
The results of SNU-899 cells indicated that the number of apoptotic cells in the LV-NC
group was 2.37 ± 0.12% and that the proportion of apoptotic cells in the LV-PDCD4 group was
14.97 ± 3.5%. This PDCD4-mediated increased was statistically significant (Figure 5D, t = 6.23,
P <0.01, n = 3) and corroborate the Hep-2 findings indicating a positive correlation between
PDCD4 expression and apoptosis.
Invasion capacity of transfected cells
A Transwell invasion assay was carried out using a Matrigel-coated lower chamber to
mimic the extra-cellular matrix. The number of cells that were detectable on the lower-facing
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membrane (Figure 6A, C) indicated successful cell “invasion” of the Matrigel. In Hep-2 cells,
PDCD4 knockdown group was41 ± 8 while the number of cells in the control group was 20 ±
7. This indicated a statistically significant increase in the number of invasive cells in response
to PDCD4 knockdown (Figure 6B, t = 3.42, P <0.05, n = 3). In SNU-899 cells, the number of
invasive cells in the LV-PDCD4 group was significantly lower compared to 17 ± 4 in the LV-
NC control group. This demonstrated a PDCD4-induced decrease in the invasive ability of
SNU-899 cancer cells (Figure 6D, t = 3.81, P <0.05, n = 3). These findings agree with Hep-2
cell assays which depict PDCD4 as a suppressor of cancer cell invasiveness. Moreover, they
support earlier experiments suggesting a negative correlation between PDCD4 expression and
cancer cell migration (Supplementary Figure 4).
The regulatory role of PDCD4 on the expression of EMT-related proteins
To address the pathways mediating the functional nature of PDCD4 on carcinoma cells,
the relationship between PDCD4 and two critical EMT factors was more closely examined. E-
cadherin and N-cadherin play important roles in conferring the epithelial to mesenchymal
transition of cells during tumorigenesis. Based on previous observations, it was hypothesized that
PDCD4 may have a regulatory influence on these factors. The relative expression of E-cadherin
and N-cadherin protein were observed in PDCD4 manipulated Hep-2 and SNU-899 cells. Frist,
in Hep-2 cells, the expression of E-cadherin was 0.801 ± 0.190 in the control group and 0.287 ±
0.090 in the experimental group. This demonstrated a significant inhibition of E-cadherin
following PDCD4 silencing (Figure 7B, t = 4.23, P <0.01, n = 3). On the contrary, the relative
expression of N-cadherin in the shRNA-NC group was 0.223 ± 0.092, compared to 0.687 ±
0.132 in the experimental group. This indicated that in contrast to E-cadherin, N-cadherin is
significantly increased in the wake of PDCD4 silencing (Figure 7B, t = 4.99,P<0.01, n = 3).
The relative expression of E-cadherin protein in SNU-899 cells revealed that PDCD4
overexpression displayed 0.78 ± 0.13, compared to the 0.31 ± 0.07 observed by the LV-NC
control group. This PDCD4-related spike in E-cadherin protein was significant (Figure 7D, t =
5.51, P <0.01, n = 3). In contrast, the relative expression of N-cadherin protein in the LV-PDCD
group and LV-NC group (0.54 ± 0.11) was significantly lower than in the control group (0.89 ±
0.09), indicating a significant PDCD4-related increase in the context of N-cadherin (Figure 7D, t
= 4.27, P <0.05, n = 3). Across both cell lines, E-cadherin and N-cadherin display inverse
expression patterns in relation to PDCD4.
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Assessment of PDCD4-silencing on tumor growth in vivo
Nude mice were selected for Hep-2, shRNA-PDCD4 and shRNA-NC-expressing xenograft
for the assessment of the role of PDCD4 on tumor growth in a live system. Healthy mice were
acclimated and xenografted with the plasmid-expressing Hep-2 cell suspension at random (n=5
per group). Thirty days after Hep-2 xenograft, tumor appearance in the PDCD4-knockdown
group was visibly larger (Figure 8A) than controls. The experimental PDCD4 group also
displayed a higher incidence of tumor-related skin ulcerations (data not shown). Following
removal and measurement of all tumors, it was clear that tumor weights were significantly higher
in the PDCD-4 silenced group compared to controls (Figure 8B, t=5.51, P <0.05). Additionally,
calipers were used to evaluate the dimensions of the tumor. With these data, a mean volume was
calculated throughout the entire course of the experimental treatment. These volumes are plotted
in a time-course analysis in Figure 8C. Statistically significant increases in tumor volume were
detected between the PDCD4-knockdown relative to the control group between days 10 and 30.
Collectively, the average tumor volume and final weights corroborate the idea that PDCD4
inhibition enhances the tumorigenic ability of nude mice.
Immunohistochemcial evaluation of PDCD4 and EMT protein in vivo
Immunohistochemical staining showed that PDCD4 was mainly located in the cytoplasm
and nucleus while E-cadherin was mainly located on the cytoplasm-facing side of the cell
membrane and partially in the cytoplasm. N-cadherin, on the other hand, appeared to be
exclusively located in the cytoplasm. PDCD4- knockdown observably diminished the expression
of PDCD4 and E-cadherin protein in the tumors (Figure 9 E-F), compared to control tumors. In
contrast, PDCD4-knockdown enhanced the expression of N-cadherin (Figure 9 C-D). These
results mirror cellular trends observed in vitro (Figure 7) and re-iterate the bi-lateral relationship
of PDCD4 on EMT proteins.
Expression of p-STAT3 and β-Catenin in Hepatocellular Carcinoma Cell Line with Stable
Silencing of PDCD4
To investigate the mechanistic drivers of PDCD4 regulation on EMT, we performed a
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proteomic analysis of phosphorylated STAT3 and beta catenin proteins via Western Blot. The
expression of p-STAT3 protein in the shRNA-NC expressing Hep-2 cells was 0.418 ± 0.013,
while the expression in the shRNA-PDCD4 group was significantly increased to1.240 ± 0.233
(Figure 10B, t = 6.10, P <0.01, n = 3). A similar trend was observed in regard to the expression
of ß-catenin protein, where the control cells expressed significantly lower protein than the
shRNA-PDCD4 group (Figure 10B, t = 25.95, P <0.01, n = 3).
Expression of miR-21 in Hepatocellular Carcinoma Cell Line with Stable Silencing of PDCD4
A secondary conduit for PDCD4 regulation of EMT was explored via quantitative PCR.
MiR-21 is a well-known carcinogenic micro RNA which has been previously been implicated in
the regulation of the EMT phenotype. Here, the expression of miR-21 was significantly
enhanced by PDCD4 knockdown (Figure 11, t = 9.96, P <0.01, n = 3), indicating an inverse
relationship between PDCD4 and the miR.
Discussion
With an estimated impact of 159,000 new cases/year and 90,000 deaths/year globally,
laryngeal squamous cell carcinoma remains a global concern. Although the surgical methods and
chemo/radiotherapies have improved in the past 30 years, the overall survival rate of laryngeal
cancer patients has not improved significantly. Therefore, it is important to study the
developmental mechanism of laryngeal carcinoma, and subsequently new treatment strategies.
To accomplish that aim, the mechanisms of tumor etiology should be more closely examined.
Due to the correlation of laryngeal cancer prognosis and the size and invasiveness/metastasis of
the growth, emphasis has been aimed at factors which play a role in configuring cell proliferation
and motility. The evidence for PDCD4 as a correlate in various cancers, along with its
demonstrated role in the EMT axis (a cellular transformation endowing cell invasion/motility)
has made it a promising target for investigation.
Clinical data suggest that high expression of PDCD4 protein is associated with improved
prognosis in patients with lung, colon and ovarian cancer ( Wei et al. 2009;Horiuchi et al. 2012;
Vikhreva et al. 2014), suggesting that PDCD4 may be an important tumor suppressor in the
progression of diverse cancer cells. Wang J et al.(Wang et al. 2012)previously reported that
PDCD4 protein is significantly lowered in laryngeal squamous cell carcinomas. Moreover, they
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reported that PDCD4 exhibited a significant correlation with tumor differentiation and cervical
lymph node metastasis, hinting at a probable role of PDCD4 in tumorigenesis. In this study 80
cases of laryngeal carcinoma were examined for PDCD4 protein and the results confirm the
findings of Wang et al. Additionally, our findings detail the distribution of PDCD4 protein across
tumor grades, where PDCD4 was progressively lowered from normal, to grade II/II, and III-IV
(Table 1), supporting that PDCD4 protein is closely related to the occurrence of laryngeal
squamous cell carcinoma and indicating that PDCD4 may be a complimentary tool for tumor
assessment. These findings are also complicit with a preliminary study done by our group,
detailing the loss of PDCD4 mRNA in laryngeal carcinoma.
The transformation of epithelial cells (EMT) requires robust changes including the
disappearance of cell polarity and intercellular adherence structures, which form the basis of
structural de-differentiation and migration. Cell adhesion molecules such as cadherins are thus
key elements in EMT. Through β-catenin E-cadherin forms connections with actin filaments to
stabilize epithelial cell-to-cell contacts. There is much evidence that EMT plays an important
role in the transformation of the normal epithelium to atypical hyperplasia in the later stages of
cancer progression(Gravdal et al. 2007). Cappellesso R and other studies have shown that low
expression of E-cadherin and high expression of Slug were closely related with the recurrence of
laryngeal cancer and shorter disease-free survival (DFS)(Cappellesso et al. 2015). Additionally,
in a comparison of 76 laryngeal squamous cell carcinomas, Song PP and others found the
decreased expression of E-cadherin and the abnormally high expression of N-cadherin and β-
catenin in the tissues(Song et al. 2016). While both cadherins were correlated with β-catenin
expression, expression of the two proteins was independent of one another. This is in line with
the known dynamics of E and N-cadherin during EMT, whereby E-cadherin loss is closely tied to
the loss of epithelial cell features and N-cadherin rise with the acquisition of mesenchymal
properties. In fact, in a study by Hazan et al 1997, the investigators demonstrated that EMT in
breast cancer cells is a direct result of N-cadherin up-regulation, independent of E-cadherin
loss(Hazan et al. 1997). Neiman et al 1999 went on to explain that up-regulation of N-cadherin in
breast cancer cells promoted motility and invasion, independent of E-cadherin
expression(Nieman et al. 1999).
Here, our findings support the dynamic expression of E and N-cadherin in laryngeal
squamous cell carcinoma tissues. We found a significant decrease in the expression of E-cadherin
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by advancement of tumor grade and metastatic state (Table 1). Parallel to PDCD4, we also report
a positive correlation between E-cadherin and differentiation state of the cancer cells. In the case
of N-cadherin, significant expression differences were noted across the same three clinical
characteristics, with the exception that N-cadherin increased with cancer progression. Our
findings support the work of previous investigations implicating E- and N-cadherin in the
transformation of tumorigenic cells. Additionally, our study characterized a positive correlation
between PDCD4 and E-cadherin and an inverse correlation between PDCD4 and N-cadherin
(Table 2). Collectively, the data support not only the role of EMT proteins in the clinical etiology
of laryngeal carcinoma, but also frame the presumptive role of PDCD4 in laryngeal cancer
progression in the scope of EMT regulation.
To investigate the hypothesized regulatory role of PDCD4 on the EMT-related cadherins,
we established a loss and gain of function model in two laryngeal carcinoma cell lines, Hep-2
and SNU-899, which naturally under-express PDCD4 mRNA (Supplementary Figure 1).
Though the two lines under-expressed the PDCD4 transcript (compared to an immortalized
human epithelial cell line), there was a significantly higher PDCD4 expression in the Hep-2 cells
compared to SNU-899. As such, it was determined that Hep-2 cells would be the more suitable
candidate for PDCD4 knockdown and SNU-899 for PDCD4 overexpression. Using stable
manipulation of PDCD4 in these lines, we probed the regulatory role of PDCD4 on E-cadherin
and N-cadherin in a more controlled and precise manner. Additionally, this approach allowed us
to more clearly define the developmental role of PDCD4 in various aspects of laryngeal cancer
progression, including growth and apoptosis, cell cycle distribution and proliferation, and
migration and invasiveness of the cells.
We report that shRNA can be successfully used for the stable and efficient knockdown of
PDCD4 mRNA and protein in Hep-2 cells (Figure 2). Moreover, lentiviral infection of SNU-899
cells with a PDCD4 overexpression vector successfully increased PDCD4 mRNA and protein
(Figure 2). A previous investigation utilizing shRNA for PDCD4 knockdown in colon cancer
HT29 cells found that tumor cell proliferation was enhanced via the activation of the NF-κB
signaling pathway(Guo et al. 2011). Yet another study of PDCD4 inhibition performed in
nasopharyngeal carcinoma cells demonstrated the enhanced growth and proliferation of the
cells(Zhen et al. 2013). Here our findings re-capitulate the proliferation-enhancing function of
PDCD4 knockdown in our squamous cancer cells (Figure 3C). In addition, our study
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strengthens the relationship of PDCD4 on cancer cell proliferation through our observations in
PDCD4 over-expressing SNU-899 cells, where proliferation was significantly diminished below
baseline levels (Figure 3F). In line with results from our MTT proliferation assay, clonal
formation rates conform to similar patterns. Overall, we conclude that PDCD4 plays a significant
biological role in growth and proliferation of laryngeal squamous cancer cells.
Another suspected role of PDCD4 in cancer etiology revolves around its modulation of
cell survival via c-Myc-controlled cell cycle regulation and the BCL-2-controlled apoptosis
pathway(Zhen et al. 2013). Using flow cytometry for cell cycle detection, we found that PDCD4
inhibition increased the proportion of cancer cells in G2 phase and decreased those in G1 phase,
parameters indicative of increased cell proliferation (Figure 4B). Further, in a direct assessment
of apoptotic state, PDCD4 knockdown drastically reduced the number of apoptotic cells (Figure
5B). In contrast, SNU-899 cells overexpressing the gene demonstrated inverse patterns-increased
G1 to G2 ratios, and increased apoptosis (Figure 5D). Our results are consistent with those in
ovarian, gastric, and nasopharyngeal carcinoma( Wei et al. 2009;Wang et al. 2010;Zhen et al.
2013), demonstrating that PDCD4-related inhibition of tumor growth and survival is
substantiated in laryngeal squamous cell carcinoma.
As previously mentioned, the occurrence of PDCD4 in laryngeal squamous cell
carcinomas is significantly correlated with metastatic state. Metastasis is generally associated
with poor prognosis and advanced cancer state. While many elements are involved in metastasis,
one of the fundamental factors is the ability of cancer cells to invade neighboring cells and
tissues (recall that this process is partially mediated by EMT). In a previous study of colon
cancer GEO cells, down-regulation of PDCD4 gene expression demonstrated associated
increased cell invasion and migration capacity. Further, when these cells were inoculated into the
colon wall, investigators observed that the probability of liver metastasis was significantly
increased(Wang et al. 2013). On the other hand, one study observed that overexpression of
PDCD4 in breast cancer cells inhibited cell invasion and migration(Santhanam et al. 2010). Here
we used Transwell assays to detect the invasion and migration of transfected Hep-2 and SNU-
899. The migration and invasiveness of Hep-2 cells was significantly enhanced by PDCD4
knockdown (Supplementary Figure 4B, Figure 6B). Additionally, these functions were
restricted by PDCD4 overexpression, collectively supporting the observations made in previous
literature. The results of PDCD4-regulated migration and invasiveness may underlie the basis for
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the clinical correlations observed between PDCD4 expression and metastatic state of laryngeal
squamous cell carcinomas.
Due to the significant alterations in biological function which were observed by PDCD4
manipulation in vitro, we set out verify the effect of PDCD4 expression on the progression of
laryngeal cancer in a live model. Using nude mice lacking the T-cell-mediated rejection reflex,
we could 1) exploit naturally-occurring tumorigenesis to examine the effectiveness of shRNA-
PDCD4 transfected Hep-2 cell xenograft and 2) test the hypothesis that laryngeal carcinoma is
regulated by PDCD4 inhibition in a live model of laryngeal squamous cell carcinoma. As
hypothesized, we found that mice xenografted with PDCD4-inhibited cells displayed larger
tumors indicative of advanced tumor progression. This was demonstrated by increased tumor
weight and volume (Figure 7), as well as by pathological loss of tumor cell adhesion, advanced
interstitial fibrous tissue hyperplasia, and the early appearance of lymphocyte infiltration
(Supplementary Figure 5). These findings support previous correlative evidence linking loss of
PDCD4 with laryngeal cancer tumorigenicity(Wang et al. 2012;Li et al. 2016; Shuang et al.
2017). While PDCD4 knockdown in vitro has yielded valuable information regarding the tumor
suppressing functions of PDCD4 in laryngeal carcinoma cell lines, our animal model is the first
evidence of a stable PDCD4 knockdown approach in vivo. Moreover, it is the first study to
demonstrate a direct and causative role for PDCD4 loss in a live system of laryngeal squamous
cell carcinoma.
To probe the molecular mechanisms by which PDCD4 may regulate tumorigenesis in
laryngeal carcinoma, we first centered on the critical EMT components E- and N-cadherin,
which had previously demonstrated strong and bilateral associations with PDCD (Table 2).
Turning back to our successful model of PDCD4 manipulation in laryngeal carcinoma cells, we
demonstrated that the expression of E-cadherin was downregulated in the shRNA-PDCD4 Hep2-
cells but up-regulated in tandem with PDCD4 overexpression in SNU-899 cells (Figure 7 B, D).
As inpatient tumors, the opposite was true of N-cadherin expression, where N-cad was up-
regulated in response to PDCD4 knockdown and down-regulated by PDCD4 overexpression.
These findings confirm our group’sand other investigators’ descriptions of the dynamic
regulatory relationship of PDCD4 and EMT associated proteins and provide a direct and causal
demonstration of PDCD4’s control over the cadherins in laryngeal carcinoma cells. These results
indicate that PDCD4 governs EMT at least partially by recruiting E-cadherin and/or suppressing
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N-cadherin, though a more complete and specific mechanism remains to be unraveled in future
studies.
A first step in expanding our understanding of the mechanisms endowing PDCD4
regulation in laryngeal carcinoma was our investigation of β-catenin based on the premise that it
can form a complex with E-cadherin in the maintenance of cytoskeletal integrity of epithelial
cells. When E-cadherin is downregulated, the complex formation ability with β-catenin
decreased, prompting translocation of cytoplasmic β-catenin to the nucleus. There, nuclear β-
catenin activatestranscription of downstream targets, including various cancer-regulating genes
including Cyclin D1, C-myc, C-Jun, VEGF, etc. ( MacDonald et al. 2009;Astudillo et al. 2014).
The expression of β-catenin was significantly up-regulated in PDCD4 inhibited cells (Figure
10B). Across various cancer models, PDCD4 knockdown has demonstrated accumulation of
active β-catenin in the nucleus(Wang et al. 2008). In laryngeal carcinoma studies, however,
cytoplasmic β-catenin is specifically overexpressed while membranous β-catenin appears to be
diminished(Andrews et al. 1997; Lopez-Gonzalez et al. 2004). In a report of simultaneous
cytoplasmic and membranous β-catenin, a reported decrease in both types was associated with
advanced laryngeal carcinoma tumor grade(Greco et al. 2016). Due to the assessment of total
protein in this study, it is not possible to know whether our observations reflect the cytoplasmic
or nuclear β-catenin overexpression which has been previously published. The associations of β-
catenin and N-cadherin should also be taken into consideration in future studies, as the two have
previously demonstrated associations in laryngeal carcinoma(Song et al. 2016).
STAT3 is an important factor downstream of the Wnt / β-catenin pathway and studies
have shown that activation of β-catenin in hepatocellular carcinoma can lead to the activation of
downstream STAT3(Wang et al. 2011). In turn, the activation of STAT3 has been linked with
reductions in apoptosis and increases in growth, migration and invasion of breast cancer
cells(Liao et al. 2017). STAT3 is proposed to affect the development of tumors through the
regulation of downstream gene targets(Rebouissou et al. 2009), many of which overlap with the
targets of β-catenin (survivin, cyclinD1 etc.). STAT3, like PDCD4 and cadherins, has
demonstrated independent clinical correlations with tumor grade and metastatic state(Masuda et
al. 2002). It is suspected to play a role in EMT-associated processes which underlie de-
differentiation and metastasis, increasing as tumors progress(Tao et al. 2009). In fact, inhibition
of STAT3 in Hep-2 cells was shown to inhibit the proliferation of the cells and increase
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apoptosis(Liu et al. 2008). In this study, we uncovered STAT3 as a novel regulatory domain of
PDCD4 in Hep-2 cells, one in which PDCD4-knockdown produced a phosphorylated
(activated)-STAT3 overexpression. These results are in line with the aforementioned literature.
Future studies are required to validate the suspected recruitment of Wnt/ β-catenin signaling by
PDCD4-mediated STAT3. Some important questions to consider include whether functional β-
catenin signaling events, such as nuclear translocation and gene regulation, are detected under
the manipulation of PDCD4 and STAT3 activation. Equally important is the gathering of
evidence that more directly implicates wnt/β-catenin signal activation in PDCD4-EMT
regulation, such as treatment of cells with Wnt inhibitors, etc.
To resolve a possible missing link between PDCD4 and STAT3, we looked toward a
known activator of STAT3 which has been implicated in EMT and has been acknowledged as a
biomarker across a range of epithelial and squamous cell cancers(Danielsson et al. 2012;
Fukushima et al. 2015). The microRNA-21 promotes EMT by activating STAT3 in LIF-induced
tumor cells(Yue et al. 2015) and inhibition of the miR21/STAT3 axis restricted EMT in a head
and neck squamous cell carcinoma model(Sun et al. 2015). Here, we report that PDCD4
knockdown yielded a significant increase in the expression of miR-21 (Figure 11), supporting
previous results demonstrating PDCD4-dependent increases in activated STAT3. Collectively,
the results of our mechanism study begin to elucidate EMT networks downstream of PDCD4,
consequently increasing our understanding of the molecular mechanisms by which PDCD4 acts
as a regulator in laryngeal carcinoma etiology.
In this study, we confirm PDCD4 as a correlate of various clinicopathalogic factors in
human laryngeal carcinoma, adding for the first time an analysis of PDCD4-EMT related
cadherins in patient samples. Next, we successfully established stable PDCD4-knockdown and
overexpression in laryngeal carcinoma cell lines to describe the biological functions of PDCD4,
including the suppression of cancer cell growth and proliferation, promotion of apoptosis, and
inhibition of cell migration and invasiveness. Moreover, we established a successful animal
model of PDCD4 knockdown to demonstrate the anti-tumorigenic role of PDCD4 in vivo for the
first time in a model of laryngeal squamous cell carcinoma. Finally, we expand our current
understanding of the regulatory role of PDCD4 on EMT in laryngeal cancer etiology, positioning
EMT-related β-catenin and the miR-21/STAT3 axis as downstream mediators of PDCD4 function.
Our findings not only strengthen the case for PDCD4 as an anti-tumorigenic but elevate PDCD4
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from a prognostic and correlate to a definitive regulator of EMT and tumorigenesis in laryngeal
carcinoma. While the picture is far from complete, this work begins to fill the gap of PDCD4’s
mechanistic network. In lieu of the lacking advancements of effective and novel strategies,
PDCD4 is increasingly poised as a therapeutic recourse for future laryngeal carcinoma studies
and EMT-targeting approaches at large.
Acknowledgements We thank our colleagues from Department of Otolaryngology of The First Affiliated Hospital of
Fujian Medical University, Central Laboratory of the First Affiliated Hospital of Fujian Medical
University and the Clinical Laboratory of the First Affiliated Hospital of Fujian Medical
University for their support.
Funding sources This work was supported by the Foundation for Young and Middle-aged Backbone Talents of
the Health and Family Planning Commission of Fujian Province (2016-ZQN-43).
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Figure Legends
Figure 1. PDCD4, E-cadherin, N-cadherin expression in laryngeal carcinoma and adjacent
tissues
H&E Staining of Normal tissue (A) and carcinoma tissues (B: Mid/High grade, C:low/mid
grade). PDCD4 immunoreactivitywasstronger in adjacent normal tissue compared to
laryngealcarcinoma tissue.(D) Strong signals in adjacent normal tissue, (E) weak signals in
carcinoma tissue, and (F) strong signals in carcinoma tissue.E-cadherin immunoreactivity was
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stronger in adjacent normal tissue than inlaryngeal carcinoma. (G) Strong signals in adjacent
normal tissue, (H) weak signals in carcinoma tissue, and (I) strong signals in carcinoma tissue.
N-cadherin immunoreactivity was non-existent in adjacent normal tissue but strong in laryngreal
carcinoma. (J) Negative signals in adjacent normal tissue, (K) weak signals in carcinoma tissue
but strong in mesenchymal cells, and (L) strong signals in carcinoma tissue. Representative
images displayed at 400X magnification. Black lines denote the demarcation between normal
“N” and tumorigenic “T” tissues.
Figure 2. Efficient PDCD4 knockdown by sh-RNA in Hep-2 cells and PDCD4
overexpression by lentiviral infection of SNU-899 cells
(A) Brightfield imaging of PDCD4-shRNA transfected Hep-2cells and (B) expression of green
fluorescence in Hep-2 cells after transfection with PDCD4-shRNA; representative images
displayed at 100 X magnification. (C) The relative expression of PDCD4mRNA in Hep-2
cellstransfected with different PDCD4 sh-RNA sequences according to RT- PCR. ANOVA
(F=4.478, P﹤0.05). (D) PDCD4 protein expression detected by Western Blot in shRNA-NC and
PDCD4 sh-RNA8653 transfected Hep-2cells. GAPDH was used as a loading control.
Quantitative assessment of PDCD4 protein expression in shRNA-NC and PDCD4 sh-RNA8653
transfected Hep-2 cells (t = 4.918, **p﹤0.01).(E,G) Brightfield imaging of lentiviral infected
SNU-899 cells (E,LV-NC;G,LV-PDCD4), representative images displayed at 200X magnification.
(F,H) SNU-899 cells displaying green fluorescence after lentiviral infection (F,LV-NC;H,LV-
PDCD4). (I) The relative expression of PDCD4 mRNA in different lentiviral transfection SNU-
899 groups as assessed by RT-PCR. (t = 33.11,***p<0.001). (J) PDCD4 protein expression
detected by Western Blot in LV-NC and LV-PDCD4 infected SNU-899 cells. GAPDH was used
as a loading control. Quantitative assessment of PDCD4 protein expression in LV-NC and LV-
PDCD4 infected SNU-899 cells (t = 4.41,*P <0.05). All quantitative experiments were
performed with n=3 biological lysates and replicated three times, independently. The average of
all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
Figure 3. Effect of PDCD4 gene manipulation on colony formation and cellular growth
(A-B) Result of colony-formation assay of Hep-2 cells transfected with PDCD4-shRNA (t =
4.598, *P﹤0.05). (C) Growth curves of cells in the experimental and control Hep-2 cells as
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assessed by MTT assay(*P﹤0.05). (D-E) Result of colony-assay of SNU-899 cells with
PDCD4 overexpression (t = 7.45, **P﹤0.01). (F) Growth curves of cells inexperimental and
control SNU-899 cells as assessed by MTT assay( **P﹤ 0.01). OD=optical density; all
quantitative experiments were performed with n=3 replicated three times, independently. The
average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
Figure 4. Cell cycle distribution of PDCD4-silenced and PDCD4-overexpressed cancer cells
(A) Cell cycle distribution of shRNA-NC Hep-2 Cells and shRNA-PDCD4 Hep-2 cells profiled
by flow cytometry. (B) Percent of Hep-2 cells designated in different cell cycle
phases,G1phaseofshRNA-NC Hep-2 Cells are significant reduced in shRNA-PDCD4 Hep-2 cells
(t=5.37, **P﹤0.01), but G2 phase cells increasedsignificantly(t=4.93, **P﹤0.01). (C) Cell
cycle distribution of LV-NC and LV-PDCD4 SNU-899 cells profiled by flow cytometry. (D)
Percent of SNU-899 cells designated in different cell cycle phases,G1 phase of LV-NCSNU-899
cellsincreasedsignificantly in LV-PDCD4 SNU-899 cells (t=3.41, *p<0.05), but G2 phase cells
significant reduced(t = 15.75, *** P <0.001). All quantitative experiments were performed with
n=3 replicated three times, independently. The average of all three runs was used for statistical
analysis. All data reflect statistical mean ±SD.
Figure 5. Apoptotic profiles of PDCD4-silenced and PDCD4 overexpressed cancer cells.
(A) Representative flowcytometry profile of shRNA-NC and shRNA-PDCD4-treated Hep-2
cells. (B) Percentage of Annexin V-positive Hep-2 cells in apoptotic state in control versus
experimental PDCD4 silenced cells (t = 10.19, **P﹤0.01). (C) Representative flowcytometry
profile of LV-NC and LV-PDCD4-treated SNU-899 cells. (D) Percentage of Annexin V-positive
SNU-899 cells in apoptotic state in control versus experimental PDCD4 overexpressing cells (t =
6.23, **P﹤0.01). All quantitative experiments were performed with n=3 replicated three times,
independently. The average of all three runs was used for statistical analysis. All data reflect
statistical mean ±SD.
Figure 6.Invasion assay of PDCD4-silenced and PDCD4-overexpressing cancer cells.
(A) Representative images of Transwell invasion assay between control and experimental Hep-
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2cells. (B) Number of transfected Hep-2 cells determined to pass through Transwell membrane
(t=3.42, *P <0.05). (C) Representative images of Transwell invasion assay between control and
experimental SNU-899 cells. (D) Number of LV-infected SNU-899 cells determined to pass
through Transwell membrane (t=3.81, *P <0.05).All quantitative experiments were performed
with n=3 replicated three times, independently. The average of all three runs was used for
statistical analysis. All data reflect statistical mean ±SD.
Figure 7. Expression of E-cadherin and N-cadherin in PDCD4-silenced and PDCD4-
overexpressing cancer cells
(A) Western Blot of EMT-associated proteins from shRNA-NC and shRNA-PDCD4 transected
Hep2 cells. (B) Quantitative assessment of E-cadherin (t = 4.23, **P﹤0.01) and N-cadherin (t =
4.99, **P﹤0.01) protein in shRNA-PDCD4 and shRNA-NC groups, where GAPDH was used as
a loading control. (C) Western Blot of EMT-associated proteins from LV-NC and LV-PDCD4
infected SNU-899 cells. (D) Quantitative assessment of E-cadherin (t = 5.51**P﹤0.01) and N-
cadherin (t = 4.27, *P <0.05) protein in LV-NC and LV-PDCD4 groups, where GAPDH was used
as a loading control. All quantitative experiments were performed with n=3 biological lysates
and replicated three times, independently. The average of all three runs was used for statistical
analysis. All data reflect statistical mean ±SD.
Figure 8. Tumor weight and volume in sh-RNA PDCD4 xenografted mice
(A)Qualitative comparison of tumor size between experimental and control,xenografted nude
mice. (B) Quantitative tumor weight assessment between the experimental and control group
revealed significant differences (P<0.05). (C)The tumor volume curve of mice xenograted with
PDCD4-silenced Hep-2 cells compared to controls over time(*P<0.05).Quantitative experiments
were performed with n=5. Data reflect statistical mean ±SD.
Figure 9.Immunohistochemicalevaluation of EMT proteins in xenografted mice
(A)Representative immunostaining of PDCD4 protein expression in shRNA-NC groupand (B)
PDCD4 expression in shRNA-PDCD4 group.(C) N-cadherin expression in shRNA-NC groupand
(D) N-cadherin expression in shRNA-PDCD4 group. (E)E-cadherin expression in shRNA-NC
groupand (F) E-cadherin expression in the shRNA-PDCD4 group. Representative images were
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captured at 400X magnification. Scale bars= 20µm.
Figure 10. Expression of p-STAT3 and β-catenin in Hep-2 cells after PDCD4 knockdown
(A) Representative Western Blot of shRNA-NC and shRNA-PDCD4 treated Hep-2 cells
incubated with p-STAT3 and β-catenin primary antibodies; GAPDH was used as a loading
control in each run. (B) Quantitative protein expression of P-STAT3 (t = 6.10,**P﹤0.01) and β-
catenin protein (t= 25.95,**P﹤0.01) in experimental versus control Hep-2 cells, where GAPDH
was used as the normalizing protein. All quantitative experiments were performed with n=3
biological lysates and replicated three times, independently. The average of all three runs was
used for statistical analysis. All data reflect statistical mean ±SD.
Figure 11. Expression of miR-21 in Hep-2 cells after PDCD4 silencing
Quantitative PCR was used to examine the expression of miR-21 in the experimental and control
transfected Hep-2 cells (t=9.96,** P<0.01).All quantitative experiments were performed with
n=3 biological lysates and replicated three times, independently. The average of all three runs
was used for statistical analysis. All data reflect statistical mean ±SD.
Supplementary Methods
Transwell migration assay
Transwell cell migration used a 12.0µm micropore film-separated dual chamber to assess
the migration capacity of cells. Cells are placed in the upper chamber and the bottom chamber is
filled with fetal bovine serum or specific chemokines. By counting the number of cells in the
lower-facing side of the membrane, the ability of tumor cells to migrate was assessed. Here,
cells were seeded in 5cm Petri dishes at 1 × 106 cells until adherent to 70-80% confluence. After
tripsin digestion, 5 × 105
/ ml single cell suspension were seeded into 24-well Transwell
chambers at a concentration of 1 × 105 cells (200 µl) per well. After 24 hour incubation, the
bottom-facing side of the membrane was washed and fixed with methanol for 30 min. Next, film
was washed and incubated with 0.1% crystal violet for 30min. The cells of the upper and lower
layer s of the membrane were observed under a microscope and photographed byinverted
microscope (200 X). Five visual fields were selected from each room at random for quantitation.
The average was recorded as the number of cells migrating through the membrane.
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H&E staining
Tumor tissues slated for wax embedding were fixed with 10% neutral formalin solution for
24 hours, dehydrated in gradient alcohol, xylene, and were dipped in wax for conventional
paraffin embedding. Slices were prepared at a thickness of 5µm, for HE staining with
hematoxylin and eosin dyes.
Supplementary Figure Legends
bcb-2017-0293.R3suppla. PDCD4 mRNA expression in laryngeal carcinoma cell lines Hep-
2, SNU-899 and immortalized human epithelial cell line Hacat
Compared with Hacat, the expression level of PDCD4 in two laryngeal squamous cell carcinoma
cells was lower, and the difference was statistically significant (F = 54.03, P <0.01, single factor
ANOVA). All quantitative experiments were performed with n=3 biological lysates and
replicated three times, independently. The average of all three runs was used for statistical
analysis. All data reflect statistical mean ±SD.
bcb-2017-0293.R3supplb. GV248 structure diagram and PDCD4 sh-RNA sequences
(A) The gene structure of the GV248 vector plasmid. (B)Four short hairpin RNAs (shRNA) and
one negative control sequence were designed according to the PDCD4 mRNA sequence of SEQ
ID NO: NM_014456 published on NCBI.
bcb-2017-0293.R3supplc. GV358 structure diagram and lentiviral infection in 293T cells
(A) The GV358 lentiviral vector contains the basic components of HIV 5'LTR and 3'LTR as well
as other auxiliary components.Vector plasmid (GV358) Element sequence: Ubi-MCS-3FLAG-
SV40-EGFP-IRES-puromycin. Cloning site: AgeI / AgeI. Labeling / resistance: 3FLAG (tag),
EGFP, puromycin(B) Gene structure of Helper1 Helper1.0 and Helper 2.0 plasmids (C) 293T
cells display green fluorescence after lentivirus infection (original magnification:200x).
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bcb-2017-0293.R3suppld.Transwell migration assay of PDCD4-knockdown Hep-2 and
PDCD4-overexpressing SNU-899 cells
(A)Representative images of Transwell migration assay between control and experimental Hep-2
cells. (B) Number of transfected Hep-2 cells determined to pass through Transwellmembrane (t =
4.64,**P﹤0.01). (C) Representative images of Transwell migration assay between control and
experimental SNU-899 cells. (D) Number of LV-infected SNU-899 cells determined to pass
through Transwell membrane (t=3.60,*P <0.05).All quantitative experiments were performed
with n=3 replicated three times, independently. The average of all three runs was used for
statistical analysis. All data reflect statistical mean ±SD.
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Table 1. Correlation between clinicopathlogic feature and expression of PDCD4,E-cadherin and
N-cadherin in laryngeal carcinoma
Characteristic Total PDCD4 E-cadherin N-cadherin
pos neg P pos neg P pos neg P
Ages(y)
≥60 37 12 25 0.499 13 24 0.487 5 32 0.730
<60 43 11 32 12 31 7 36
Gender
Male 75 21 54 0.566 22 53 0.152 10 65 0.106
Female 5 2 3 3 2 2 3
Subsite
Glottic 47 10 37 0.078 11 36 0.071 7 40 0.975
Superior/inferior glottic 33 13 20 14 19 5 28
Histopathological Grade
High/middle 57 21 36 0.012 22 35 0.026 3 54 0.000
low 23 2 21 3 20 9 14
Clinical stage
Ⅰ-Ⅰ 40 17 23 0.007 18 32 0.011 2 38 0.012
Ⅰ-Ⅰ 40 6 34 7 43 10 30
Lymph node metastasis
Positive 31 4 27 0.017 5 26 0.02 9 22 0.005
Negative 49 19 30 20 29 3 46
pos positive ,neg negtive
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Table2.Correlation between PDCD4 and E-cadherin/N-cadherin expression in laryngeal
carcinoma
PDCD4 X2 P value
positive negative total
E-cadherin positive 22 3 25 62.32 P<0.01
negative 1 54 55
total 23 57 80
N-cadherin positive 0 12 12 5.696 0.017
negative 23 45 68
total 23 57 80
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Figure 1. PDCD4, E-cadherin, N-cadherin expression in laryngeal carcinoma and adjacent tissues H&E Staining of Normal tissue (A) and carcinoma tissues (B: Mid/High grade, C:low/mid grade). PDCD4 immunoreactivity was stronger in adjacent normal tissue compared to laryngeal carcinoma tissue.(D)
Strong signals in adjacent normal tissue, (E) weak signals in carcinoma tissue, and (F) strong signals in carcinoma tissue. E-cadherin immunoreactivity was stronger in adjacent normal tissue than in laryngeal carcinoma. (G) Strong signals in adjacent normal tissue, (H) weak signals in carcinoma tissue, and (I)
strong signals in carcinoma tissue. N-cadherin immunoreactivity was non-existent in adjacent normal tissue but strong in laryngreal carcinoma. (J) Negative signals in adjacent normal tissue, (K) weak signals in
carcinoma tissue but strong in mesenchymal cells, and (L) strong signals in carcinoma tissue. Representative images displayed at 400X magnification. Black lines denote the demarcation between normal
“N” and tumorigenic “T” tissues.
156x154mm (300 x 300 DPI)
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Figure 2. Efficient PDCD4 knockdown by sh-RNA in Hep-2 cells and PDCD4 overexpression by lentiviral infection of SNU-899 cells
(A) Brightfield imaging of PDCD4-shRNA transfected Hep-2cells and (B) expression of green fluorescence in
Hep-2 cells after transfection with PDCD4-shRNA; representative images displayed at 100 X magnification. (C) The relative expression of PDCD4mRNA in Hep-2 cells transfected with different PDCD4 sh-RNA
sequences according to RT- PCR. ANOVA (F=4.478, P﹤0.05). (D) PDCD4 protein expression detected by
Western Blot in shRNA-NC and PDCD4 sh-RNA8653 transfected Hep-2 cells. GAPDH was used as a loading control. Quantitative assessment of PDCD4 protein expression in shRNA-NC and PDCD4 sh-RNA8653
transfected Hep-2 cells (t = 4.918, **p﹤0.01).(E,G) Brightfield imaging of lentiviral infected SNU-899 cells
(E,LV-NC;G,LV-PDCD4), representative images displayed at 200X magnification. (F,H) SNU-899 cells displaying green fluorescence after lentiviral infection (F,LV-NC;H,LV-PDCD4). (I) The relative expression of
PDCD4 mRNA in different lentiviral transfection SNU-899 groups as assessed by RT-PCR. (t = 33.11,***p<0.001). (J) PDCD4 protein expression detected by Western Blot in LV-NC and LV-PDCD4
infected SNU-899 cells. GAPDH was used as a loading control. Quantitative assessment of PDCD4 protein expression in LV-NC and LV-PDCD4 infected SNU-899 cells (t = 4.41,*P <0.05). All quantitative experiments were performed with n=3 biological lysates and replicated three times, independently. The average of all
three runs was used for statistical analysis. All data reflect statistical mean ±SD.
173x85mm (300 x 300 DPI)
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Figure 3. Effect of PDCD4 gene manipulation on colony formation and cellular growth (A-B) Result of colony-formation assay of Hep-2 cells transfected with PDCD4-shRNA (t = 4.598, *P﹤0.05).
(C) Growth curves of cells in the experimental and control Hep-2 cells as assessed by MTT assay(*P﹤
0.05). (D-E) Result of colony-assay of SNU-899 cells with PDCD4 overexpression (t = 7.45, **P﹤0.01). (F)
Growth curves of cells in experimental and control SNU-899 cells as assessed by MTT assay(**P﹤0.01).
OD=optical density; all quantitative experiments were performed with n=3 replicated three times, independently. The average of all three runs was used for statistical analysis. All data reflect statistical mean
±SD.
151x93mm (300 x 300 DPI)
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Figure 4. Cell cycle distribution of PDCD4-silenced and PDCD4-overexpressed cancer cells (A) Cell cycle distribution of shRNA-NC Hep-2 Cells and shRNA-PDCD4 Hep-2 cells profiled by flow
cytometry. (B) Percent of Hep-2 cells designated in different cell cycle phases,G1 phase of shRNA-NC Hep-2 Cells are significant reduced in shRNA-PDCD4 Hep-2 cells (t=5.37, **P﹤0.01), but G2 phase cells increased
significantly(t=4.93, **P﹤0.01). (C) Cell cycle distribution of LV-NC and LV-PDCD4 SNU-899 cells profiled
by flow cytometry. (D) Percent of SNU-899 cells designated in different cell cycle phases,G1 phase of LV-NC SNU-899 cells increased significantly in LV-PDCD4 SNU-899 cells (t=3.41, *p<0.05), but G2 phase cells significant reduced(t = 15.75, *** P <0.001). All quantitative experiments were performed with n=3
replicated three times, independently. The average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
361x198mm (300 x 300 DPI)
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Figure 5. Apoptotic profiles of PDCD4- � �silenced and PDCD4 overexpressed cancer cells. (A) Representative flowcytometry profile of shRNA-NC and shRNA-PDCD4-treated Hep-2 cells. (B) Percentage of Annexin V-
positive Hep-2 cells in apoptotic state in control versus experimental PDCD4 silenced cells (t = 10.19, **P﹤
0.01). (C) Representative flowcytometry profile of LV-NC and LV-PDCD4-treated SNU-899 cells. (D)
Percentage of Annexin V-positive SNU-899 cells in apoptotic state in control versus experimental PDCD4 overexpressing cells (t = 6.23, **P﹤0.01). All quantitative experiments were performed with n=3 biological
lysates and replicated three times, independently. The average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD
268x167mm (300 x 300 DPI)
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Figure 6.Invasion assay of PDCD4-silenced and PDCD4-overexpressing cancer cells. (A) Representative images of Transwell invasion assay between control and experimental Hep-2cells. (B)
Number of transfected Hep-2 cells determined to pass through Transwell membrane (t=3.42, *P <0.05). (C)
Representative images of Transwell invasion assay between control and experimental SNU-899 cells. (D) Number of LV-infected SNU-899 cells determined to pass through Transwell membrane (t=3.81, *P
<0.05).All quantitative experiments were performed with n=3 replicated three times, independently. The average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
223x116mm (300 x 300 DPI)
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Figure 7. Expression of E-cadherin and N-cadherin in PDCD4-silenced and PDCD4-overexpressing cancer cells
(A) Western Blot of EMT-associated proteins from shRNA-NC and shRNA-PDCD4 transected Hep2 cells. (B) Quantitative assessment of E-cadherin (t = 4.23, **P﹤0.01) and N-cadherin (t = 4.99, **P﹤0.01) protein
in shRNA-PDCD4 and shRNA-NC groups, where GAPDH was used as a loading control. (C) Western Blot of EMT-associated proteins from LV-NC and LV-PDCD4 infected SNU-899 cells. (D) Quantitative assessment of E-cadherin (t = 5.51**P﹤0.01) and N-cadherin (t = 4.27, *P <0.05) protein in LV-NC and LV-PDCD4
groups, where GAPDH was used as a loading control. All quantitative experiments were performed with n=3 biological lysates and replicated three times, independently. The average of all three runs was used for
statistical analysis. All data reflect statistical mean ±SD.
213x129mm (300 x 300 DPI)
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Figure 8. Tumor weight and volume in sh-RNA PDCD4 xenografted mice (A)Qualitative comparison of tumor size between experimental and control,xenografted nude mice. (B) Quantitative tumor weight assessment between the experimental and control group revealed significant
differences (P<0.05). (C)The tumor volume curve of mice xenograted with PDCD4-silenced Hep-2 cells compared to controls over time(*P<0.05).Quantitative experiments were performed with n=5. Data reflect
statistical mean ±SD.
257x166mm (300 x 300 DPI)
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Figure 9.Immunohistochemicalevaluation of EMT proteins in xenografted mice (A)Representative immunostaining of PDCD4 protein expression in shRNA-NC groupand (B) PDCD4
expression in shRNA-PDCD4 group.(C) N-cadherin expression in shRNA-NC groupand (D) N-cadherin
expression in shRNA-PDCD4 group. (E)E-cadherin expression in shRNA-NC groupand (F) E-cadherin expression in the shRNA-PDCD4 group. Representative images were captured at 400X magnification. Scale
bars= 20µm.
104x103mm (300 x 300 DPI)
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Figure 10. Expression of p-STAT3 and β-catenin in Hep-2 cells after PDCD4 knockdown (A) Representative Western Blot of shRNA-NC and shRNA-PDCD4 treated Hep-2 cells incubated with p-
STAT3 and β-catenin primary antibodies; GAPDH was used as a loading control in each run. (B) Quantitative protein expression of P-STAT3 (t = 6.10,**P﹤0.01) and β-catenin protein (t= 25.95,**P﹤0.01) in
experimental versus control Hep-2 cells, where GAPDH was used as the normalizing protein. All quantitative experiments were performed with n=3 biological lysates and replicated three times, independently. The
average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
467x146mm (300 x 300 DPI)
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Figure 11. Expression of miR-21 in Hep-2 cells after PDCD4 silencing Quantitative PCR was used to examine the expression of miR-21 in the experimental and control transfected Hep-2 cells (t=9.96,** P<0.01).All quantitative experiments were performed with n=3 biological lysates
and replicated three times, independently. The average of all three runs was used for statistical analysis. All data reflect statistical mean ±SD.
283x174mm (300 x 300 DPI)
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