Defining the Regulatory Role of Programmed Cell Death 4 in … · 2018. 8. 29. · Draft Defining...

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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

https://mc06.manuscriptcentral.com/bcb-pubs

Biochemistry and Cell Biology

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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


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


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


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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


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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