Programmed cell death 4 and microRNA 21 inverse expression is maintained in cells and exosomes from...

Programmed Cell Death 4 and MicroRNA 21 Inverse

Expression Is Maintained in Cells and Exosomes From

Ovarian Serous Carcinoma Effusions

Rocco Cappellesso, MD1; Andrea Tinazzi, Technician1; Thomas Giurici, Technician1;

Francesca Simonato, BD1; Vincenza Guzzardo, Technician1; Laura Ventura, PhD2; Marika Crescenzi, BD3;

Silvia Chiarelli, MD1; and Ambrogio Fassina, MD1

BACKGROUND: Ovarian serous carcinoma (OSC) is a fatal gynecologic malignancy usually presenting with bilateral local-

ization and malignant peritoneal effusion. Programmed cell death 4 (PDCD4) is a tumor suppressor gene whose expression

is directly controlled by microRNA-21 (miR-21). Exosomes are small cell-derived vesicles that participate in intercellular com-

munication, delivering their cargo of molecules to specific cells. Exosomes are involved in several physiological and patho-

logical processes including oncogenesis, immunomodulation, angiogenesis, and metastasis. The current study analyzed the

expression of PDCD4 and miR-21 in resected OSC specimens and in cells and exosomes from OSC peritoneal effusions.

METHODS: PDCD4 was immunohistochemically examined in 14 normal ovaries, 14 serous cystadenoma (CA), and 14 OSC

cases. Quantitative reverse transcriptase-polymerase chain reaction analysis of PDCD4 and miR-21 expression was per-

formed in CA and OSC cases and in cells and exosomes obtained from 10 OSC and 10 nonneoplastic peritoneal effusions.

miR-21 was also evaluated by in situ hybridization. RESULTS: Immunohistochemistry demonstrated a gradual PDCD4 loss

from normal ovaries to CA and OSC specimens. Quantitative reverse transcriptase-polymerase chain reaction displayed

higher PDCD4 messenger RNA levels in CA specimens compared with OSC cases and highlighted miR-21 overexpression

in OSC specimens. In situ hybridization detected miR-21 only in OSC cells. This PDCD4 and miR-21 inverse expression was

also noted in cells and exosomes from OSC peritoneal effusions compared with nonneoplastic effusions. CONCLUSIONS:

PDCD4 and miR-21 are involved in OSC oncogenesis. The transfer of miR-21 by exosomes could promote oncogenic trans-

formation in target cells distant from the primary tumor without direct colonization by cancer cells and could be used as

a diagnostic tool. Cancer (Cancer Cytopathol) 2014;122:685-93. VC 2014 American Cancer Society.

KEY WORDS: ovarian carcinoma; microRNA-21 (miR-21); programmed cell death 4 (PDCD4); exosomes; malignant

effusions.

INTRODUCTION

Ovarian cancer is the leading cause of gynecologic tumor-related death in the Western world.1,2 Malignant sur-

face epithelial-stromal tumors account for approximately 90% of ovarian cancer cases, among which borderline

and invasive ovarian serous carcinomas (OSC) represent the large majority.2 The majority of patients with OSC

are initially asymptomatic and present with advanced stage disease (International Federation of Gynecology and

Obstetrics stages III-IV), often characterized by bilateral localization and malignant peritoneal effusion.2-6

Despite advances in treatment, survival is still low. Understanding the molecular alterations underlying OSC

could provide the basis for novel diagnostic or therapeutic strategies.3-6

Corresponding author: Ambrogio Fassina, MD, Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Via Aris-

tide Gabelli, 61, 35121-Padua, Italy; Fax: (011) 39 049 827 3782; [email protected]

1Surgical Pathology and Cytopathology Unit, Department of Medicine, University of Padua, Padua, Italy; 2Department of Statistical Sciences, Univer-

sity of Padua, Padua, Italy; 3Radiotherapy Unit, Istituto Oncologico Veneto-IRCCS, Padua, Italy

Received: April 16, 2014; Revised: April 30, 2014; Accepted: May 5, 2014

Published online May 28, 2014 in Wiley Online Library (wileyonlinelibrary.com)

DOI: 10.1002/cncy.21442, wileyonlinelibrary.com

Cancer Cytopathology September 2014 685

Original Article

Programmed cell death 4 (PDCD4) is a recently dis-

covered tumor suppressor gene involved in apoptosis that

affects cell transformation, oncogenesis, and tumor inva-

sion.7-10 Function and subcellular localization of PDCD4

are controlled by several mechanisms, in particular by the

oncogenic microRNA-21 (miR-21). Indeed, miR-21

directly targets the 30-untranslated region of PDCD4,

downregulating its expression.11,12 Moreover, low

PDCD4 protein levels have been found to be inversely

correlated with miR-21 expression and to correlate with

prognosis in different tumors, such as thyroid, colon,

esophageal, and ovarian carcinomas.8,11-15

Exosomes are small (30-120 nanometers [nm] in

size) membrane-bounded vesicles of cellular derivation

detected in several bodily fluids, such as plasma, urine,

saliva, breast milk, and effusions.16-18 Exosomes have

been demonstrated to participate in intercellular commu-

nication, delivering their cargo of molecules to specific

target cells.16-18 The transfer of molecules via exosomes

may modulate the activity of cellular mechanisms and

pathways in recipient cells. In particular, exosomes have

been involved in oncogenesis, immunomodulation,

angiogenesis, and metastasis.16-18 Exosome content (pro-

teins and RNA) is related to the cells of derivation.

Indeed, exosomes are produced by a variety of cells,

including lymphocytes and epithelial, endothelial, dendri-

tic, and cancer cells.16-18

The objective of the current study was to assess miR-

21/PDCD4 involvement in resected OSC specimens and

in cells and exosomes from OSC peritoneal effusions.

MATERIALS AND METHODS

Materials

From the archives of the Surgical Pathology and Cytopa-

thology Unit of the University of Padua for the period

between 2009 and 2013, we retrieved resected specimens

of 14 normal ovaries from cases of uterine prolapse, 14

serous cystadenoma (CA), and 14 OSC (clinical and path-

ological data for these cases are reported in Table 1). All

cases were reviewed and the diagnoses confirmed in all

instances by 2 pathologists (S.C. and R.C.) according to

the World Health Organization classification.19

Peritoneal effusions were submitted for routine diag-

nostic purposes to the study unit and immediately proc-

essed. Fresh peritoneal effusions were collected between

2012 and 2013 from 10 patients diagnosed with OSC

before the administration of chemotherapy and surgical

treatment. Clinical and pathological data are detailed in

Table 1. All effusions were proven to be malignant by

cytology and OSCs were subsequently histologically con-

firmed in all instances. As a negative control, 10 nonneo-

plastic (cytologically negative) peritoneal effusions were

also included.

This study was approved by the Institutional Ethical

Review Board of Padua University and the institute’s ethi-

cal regulations on research conducted on human tissues

were followed.

Isolation of Cells and Exosomes

On their arrival at the laboratory, all effusion samples

were divided in half: one half was used for the usual diag-

nostic process and the other for the study. The latter half

was centrifuged for 6 minutes at 6000 g to separate the

cells from the supernatant fluid. The cell pellet was readily

stored at 280�C until further use, whereas the superna-

tant fluid was centrifuged again at 2000 g for 30 minutes

to remove cellular debris and contaminants and 1 mL of

the clarified fluid was transferred to a fresh container for

isolation of the exosomes without disturbing the pellet.

Then, 500 lL of Total Exosome Isolation Reagent (Invi-

trogen, Carlsbad, Calif), a polymer-based mixture that

separates exosomes from solution by tying up water mole-

cules thereby allowing their collection by low-speed

TABLE 1. Clinicopathological Characteristics of the Considered Series

No. of

Cases

Mean Age 6 SD,

Years

Histologic

Grade, No.

FIGO

Stage, No.

Bilaterality,

No. (%)

Malignant Effu-sion,

No. (%)

Surgical specimens Normal ovaries 14 52.866.2 — — — —

CA 14 55.664.7 — — — —

OSC 14 68.065.4 2 (3) 3 (11) II (4) III (10) 11 (78.6%) 9 (64.3%)

Peritoneal effusions NEG 10 64.265.8 — — — —

OSC 10 70.066.1 2 (1) 3 (9) II (2) III (8) 8 (80.0%) 10 (100%)

Abbreviations: CA, cystadenoma; FIGO, International Federation of Gynecology and Obstetrics; NEG, nonneoplastic; OSC, ovarian serous carcinomas; SD,

standard deviation.

Original Article

686 Cancer Cytopathology September 2014

centrifugation, were added and mixed by vortexing,

according to the manufacturer’s instructions. After incu-

bation at room temperature for 30 minutes, the sample/

reagent mixture was centrifuged at 10,000 g for 10

minutes at room temperature and the supernatant fluid

was discarded. Finally, the exosome pellet was resus-

pended in 200 lL of 1X phosphate-buffered saline buffer

and stored at 280�C until further use.

Immunohistochemistry

Immunohistochemistry was performed on 4-lm to 5-lm

thick formalin-fixed and paraffin-embedded (FFPE) sec-

tions from each tissue sample. Staining was performed

automatically (BondmaX; Menarini, Florence, Italy) as

described elsewhere,20,21 using the Bond Polymer Refine

Detection kit (Leica Microsystems, Wetzlar, Germany),

with anti-PDCD4 antibody (polyclonal; working dilu-

tion, 1:100 for 30 minutes, citrate buffer) (Atlas Antibod-

ies, Stockholm, Sweden). Sections were then slightly

counterstained with hematoxylin. Appropriate positive

and negative controls were run concurrently. PDCD4

nuclear expression was jointly scored by 2 pathologists

(A.F. and R.C.) who were unaware of any clinical infor-

mation. PDCD4 nuclear staining was scored semiquanti-

tatively on a 4-tiered scale on the basis of the percentage of

positive cells, with 0 indicating no stain, 1 indicating 1%

to 30% staining, 2 indicating 31% to 70% staining, and 3

indicating 71% to 100% staining. Cytoplasmic expres-

sion was evaluated on a 4-tired scale based on the staining

intensity, in which 0 indicated absent staining, 1 indicated

weak staining, 2 indicated moderate staining, and 3 indi-

cated strong staining. According to scoring system of

Mudduluru et al, nuclear and cytoplasmic scores were

summed to obtain a final value: 1 to 2 indicated weak

expression, 3 to 4 indicated moderate expression, and 5 to

6 indicated intense expression.8

In Situ Hybridization

Reactions were performed on 4-lm to 5-lm thick FFPE

sections from 5 CA and 5 OSC randomly selected cases

using the GenPoint Catalyzed Signal Amplification Sys-

tem (DakoCytomation, Glostrup, Denmark) according

to the manufacturer’s protocol and applying the 50–

biotin-labeled miR-21 miRCURY LNA microRNA

detection probe (Exiqon, Vedbaek, Denmark) or the

scrambled negative control probe (U6; Exiqon) at a final

concentration of 200 nM, as described elsewhere.22 The

slides were finally counterstained with hematoxylin. Reac-

tions were jointly assessed by 2 pathologists (A.F. and

R.C.) and were considered positive when granular cyto-

plasmic staining was present.

RNA Extraction

CA and OSC samples were enriched in the neoplastic

component by manual microdissection to ensure a higher

neoplastic component. Briefly, 5 consecutive, unstained,

10-lm thick, FFPE sections of each specimen were

scraped in a 1.5-mL tube using the hematoxylin and

eosin-stained slide as a guide. Total RNA was extracted

using the RecoverAll Total Nucleic Acid Isolation Kit

(Ambion, Austin, Tex) as described elsewhere.23,24 Cell

pellets from peritoneal effusions were resuspended in 500

lL of TRIzol reagent (Invitrogen) for total RNA isolation,

according to the manufacturer’s instructions.25 RNA

recovery from the exosome samples was performed with

the Total Exosome RNA and Protein Isolation Kit (Invi-

trogen), following the manufacturer’s protocol. Each sam-

ple was mixed with 200 lL of 23 denaturing solution,

vortexed, and incubated on ice for 5 minutes. Then, 400

lL of acid-phenol:chloroform were added. The mixture

was briefly vortexed and centrifuged at 10,000 g for 5

minutes at room temperature to separate the aqueous and

organic phases. The aqueous phase was transferred to a

fresh tube and a 1.25-volume of 100% ethanol was added.

The mixture was then pipetted onto a filter cartridge in a

collection tube and spun at 10,000 g for 15 seconds. The

flow-through was discarded and the filter cartridge was

washed once with 700 lL of Wash Solution 1 and twice

with 500 lL of Wash Solution 2/3. After discarding the

flow-through from the last wash, the filter cartridge was

transferred into a fresh collection tube and 50 lL of pre-

heated (95�C) nuclease-free water was twice applied to

the center of the filter and briefly centrifuged to collect

RNA. All RNA extractions were assessed for the amount

and purity of RNA with Nanodrop technology (Thermo

Scientific, Wilmington, Del) and stored at 280�C until

further use. To avoid any potential variation between

assays, analyses were then performed on all of the extracts

simultaneously.

Quantitative Reverse Transcriptase-Polymerase Chain Reaction

PDCD4 reverse transcription was performed using 100

ng of total RNA, M-MLV Reverse Transcriptase

PDCD4 and miR-21 Expression in OSC and Exosomes/Cappellesso et al


(Invitrogen), and 250 mM of random primers (Invitro-

gen). PDCD4 primers (forward 50-TGGAAAGCGTA

AAGATAGTGTGTG-30; reverse 50-TTCTTTCAGCA

GCATATCAATCTC-30) were designed using the Probe-

Finder software (roche-appliedscience.com) and the

respective probe was selected among the Universal Probe-

Library (Roche Diagnostics, Mannheim, Germany).

Experiments were performed according to the standard

protocol provided by the manufacturer, including a

housekeeping gene control (b-2 microglobulin) to adjust

for unequal RNA amounts, as explained elsewhere.22

Mature miR-21 (primer sequence 50-GATACCAAAAT

GTCAGACAGCC-30) was retrotranscribed from 100 ng

of total RNA with the SuperScript VILO cDNA Synthesis

Kit (Invitrogen) and quantified using the NCode miRNA

quantitative reverse transcriptase-polymerase chain reac-

tion (qRT-PCR) method (Invitrogen), according to the

manufacturer’s instructions, as previously stated.25,26

Normalization was done with the small nuclear RNA

U6B (RNU6B; Invitrogen). All the reactions were run in

triplicate, including no-template controls, on the Light-

Cycler 480 Real-Time PCR System (Roche Diagnostics).

Statistical Analysis

Statistical significance was determined for immunohisto-

chemistry results using the Kruskal-Wallis test. Differen-

tial expression of PDCD4 and miR-21 was tested on the

logarithmic scale using a 2-sided Student t test, after

checking both the assumption of normality (Shapiro-

Wilk test) and the assumption of homogeneity of variance

(F-test). In situ hybridization results were not statistically

analyzed. A P value< .05 was considered to be statistically

significant. All statistical analyses were performed using R

software (R Development Core Team, version 2.9; R

Foundation for Statistical Computing, Vienna, Austria).

RESULTS

Surgical Specimens

In normal ovaries, PDCD4 immunostaining demon-

strated a moderate cytoplasmic intensity and a strong

nuclear positivity in virtually all surface cells (Fig. 1), with

an intense final score in all instances (Fig. 2).8 CA speci-

mens displayed a moderate-to-strong cytoplasmic immu-

noreaction with a scattered nuclear positivity (Fig. 1).

Overall, PDCD4 immunolabeling scoring resulted an

intense score in 21%, a moderate score in 71%, and a

Figure 1. Representative images from the considered series

are shown. Programmed cell death 4 (PDCD4) immunostain-

ing in (A) normal ovary demonstrated moderate cytoplasmic

intensity and nuclear positivity in>71% of surface cells. (B) In

serous cystadenoma, nuclear immunolabeling decreased (<

70% of cells) whereas cytoplasmic intensity was moderate to

strong. (C) In ovarian serous carcinoma, nuclear positivity

was totally absent with weak-to-moderate cytoplasmic stain-

ing noted (original magnification, A and B: 3 400; C: 3 200).

Original Article


weak score in 8% of CA specimens (Fig. 2).8 In OSC

specimens, nuclear immunoreaction was found to disap-

pear completely whereas cytoplasmic immunostaining

ranged in intensity from weak to moderate (Fig. 1).

Approximately one-half of OSC cases displayed a weak

final expression whereas the remaining specimens were

found to have a moderate score (Fig. 2).8 Statistical analy-

sis of immunohistochemistry results highlighted a sub-

stantial decrease in PDCD4 expression in both OSC and

CA specimens compared with normal ovary (all

P< .001). Such findings were further supported by qRT-

PCR analysis of PDCD4 expression in CA and OSC

specimens. Indeed, PDCD4 messenger RNA (mRNA)

levels were found to be significantly higher in CA speci-

mens compared with OSC specimens (P< .05) (Fig. 2).

Conversely, miR-21 was found to be overexpressed in

OSC compared with CA specimens (P< .05) (Fig. 2).8

This finding was also confirmed by in situ hybridization

analysis performed in a limited number of CA and OSC

cases that demonstrated considerable granular cytoplas-

mic staining in OSC cells only (Fig. 3).

Peritoneal Effusions

Inverse expression of PDCD4 and miR-21 also were con-

firmed in cells and exosomes from OSC peritoneal effu-

sions compared with nonneoplastic effusions. Indeed,

qRT-PCR results demonstrated higher PDCD4 mRNA

levels in the cells and exosomes of nontumoral compared

with OSC effusions (both P< .01) (Table 2) (Fig. 4).

Conversely, miR-21 was found to be significantly down-

regulated in both cells (P< .05) and exosomes (P< .01)

from nonneoplastic controls in comparison with OSC

Figure 2. Immunohistochemical staining scores for programmed cell death 4 (PDCD4) are shown using the scoring system of

Mudduluru et al.8 (A) PDCD4 immunostaining was found to be significantly weaker in ovarian serous carcinoma (OSC) specimens

than in cystadenoma (CA) specimens and normal ovaries (both P< .001). Box plots show the relative expression of (B) PDCD4

and (C) microRNA-21 (miR-21) in CA and OSC specimens. PDCD4 expression levels were found to be higher in CA than in OSC

specimens (all P< .05), whereas for miR-21 amounts it was the opposite (P<.05). The y-axis represents expression levels of

PDCD4 or miR-21 (logarithmic scale). * indicates P< .05.



effusions (Table 2) (Fig. 4). Thus, exosomes in OSC peri-

toneal effusions presented with high miR-21 content sim-

ilar to that of the presumed cells of origin (ie, OSC cells).

DISCUSSION

Ovarian cancer is the leading cause of gynecologic tumor-

related deaths in the Western world and OSC represents

the most common histologic subtype.1,2 Early symptoms

are usually absent, minor, and overlooked, and the large

majority of patients present with advanced disease.2-6

This is the main reason for the poor prognosis of OSC

and there is a pressing need for the development of novel

diagnostic and therapeutic strategies.3-6

The current study investigated the expression of the

recently discovered tumor suppressor gene PDCD4 and

its regulator miR-21 among a series of normal ovary, CA,

and OSC surgical specimens and in cells and exosomes

from OSC peritoneal effusions. PDCD4 is a tumor sup-

pressor gene located at chromosome 10q24 that is associ-

ated with apoptosis-regulating activator protein 1 (AP-1)-

dependent transcriptional activity, cyclin-dependent

kinase 4 (CDK4), p27, p21, and eukaryotic translation

initiation factor 4A (eIF4A) in response to several

inducers.7-10 PDCD4 loss or downregulation has been

correlated with tumor progression and poor prognosis in

different tumors, such as thyroid, colon, esophageal, and

ovarian cancers.8,11-15 In ovarian cancer cell lines,

PDCD4 overexpression has been demonstrated to inhibit

the malignant behavior enhancing apoptosis and chemo-

sensitivity.27-30 The results of the current study in resected

samples are in keeping with data from the literature.

Indeed, a substantial decrease in PDCD4 levels was

observed in OSC specimens compared with normal con-

trols and CA specimens at both the protein and mRNA

level, as reported by other authors.14,15

Previous studies have demonstrated that the func-

tion and subcellular localization of PDCD4 are controlled

by several mechanisms, mainly miR-21.11-13 Indeed,

miR-21 is an oncogenic microRNA that recognizes and

directly binds the 30-untranslated region of PDCD4,

thereby controlling its expression. Nam et al found over-

expression of miR-21 in OSC specimens.31 Moreover,

miR-21 downregulation has been reported to promote

apoptosis and chemosensitivity, while conversely inhibi-

ting migration and invasion in ovarian cancer cell

lines.29,31,32 Accordingly, the results of our qRT-PCR

demonstrated higher expression of miR-21 in OSC com-

pared with CA cases, findings that were further confirmed

by in situ hybridization. Overall, such findings endorse

the involvement of PDCD4/miR-21 in the oncogenesis

of OSC.

It is interesting to note that the same inverse expres-

sion of PDCD4 and miR-21 was maintained also in cells

and exosomes from OSC peritoneal effusions compared

with nonneoplastic effusions. These characteristics reveal

that the frequent bilaterality and multiple peritoneal local-

ization of OSC could be based on double mechanisms.

The first and more obvious mechanism is direct adhesion

and proliferation of neoplastic cells to other sites of the

peritoneal cavity, a point that does not need further com-

ments. The latter and intriguing process is unrelated to

the direct migration of OSC cells and depends on

Figure 3. Representative images from the considered series

are shown. In situ hybridization for microRNA-21 displayed

(A) scant and haphazard cytoplasmic staining in neoplastic

cells of serous cystadenoma and (B) strong and diffuse gran-

ular cytoplasmic staining in ovarian serous carcinoma (origi-

nal magnification 3 400).

Original Article


exosomes. Indeed, exosomes could promote oncogenic

transformation in target cells distant from the primary

tumor. These 30-nm to 120-nm sized vesicles play a role

in intercellular communication by transferring proteins

and RNA from donor to recipient cells.16-18 Exosomes are

implicated in several tumor mechanisms, such as onco-

genesis, immunomodulation, angiogenesis, and metasta-

sis.16-18 In particular, many of these functions are related

to the transfer of miRNAs in recipient cells, in which they

modulate tumor transformation, proliferation, and pro-

gression.16-18,33,34 miRNAs are selectively incorporated

into exosomes and delivered to specific cells, although it

remains unclear whether the sorting mechanism is related

to the association with the RNA-induced silencing com-

plex (RISC) components or the target mRNAs.16,35,36

Moreover, the detection of miR-21 in exosomes also

could be used as a diagnostic tool in peritoneal effusions.

Recently, Taylor et al demonstrated that miR-21 levels

were higher in exosomes isolated from the sera of patients

with OSC than those from women with benign disease

and precancerous lesions.37 In addition, miR-21 was

included in a panel of miRNAs from tumor-derived exo-

somes whose expression analysis was effective for diagnos-

ing OSC.37 Currently, exosome analysis is performed

TABLE 2. qRT-PCR Results in Cells and ExosomesObtained From OSC and NEG Peritoneal Effusions

PDCD4 expression miR-21 expression

Cells Exosomes Cells Exosomes

Peritoneal

effusions

NEG High High Low Low

OSC Low Low High High

Abbreviations: miR-21, microRNA 21, NEG, nonneoplastic; OSC, ovarian

serous carcinomas; PDCD4, programmed cell death 4; qRT-PCR, quantita-

tive reverse transcriptase-polymerase chain reaction.

Figure 4. Box plots are shown demonstrating the relative expression of programmed cell death 4 (PDCD4) and microRNA-21

(miR-21) in (A and B) cells and (C and D) exosomes obtained from ovarian serous carcinoma (OSC) specimens and nonneoplastic

(NEG) peritoneal effusions. PDCD4 expression was found to be significantly downregulated in both OSC cells and exosomes

compared with negative controls (P< .01 for both). On the contrary, miR-21 was found to be consistently overexpressed (P< .05

in cells and P< .01 in exosomes). The y-axis represents expression levels of PDCD4 or miR-21 (logarithmic scale). ** indicates

P<.01; *, P< .05.



only for research purposes. However, the availability of

fast, simple, and inexpensive isolation methods could

soon increase its application as an ancillary technique to

effusion cytology (and it most likely will replace light

microscopy evaluation in the future). Currently, exosome

analysis is 5 to 6 times more costly than routine cytology,

thereby limiting its diffusion.

The results of the current study demonstrate that the

loss of PDCD4 with a concurrent increase in miR-21

expression occurs in OSC, confirming their involvement

in the oncogenesis of the neoplasm. The presence of miR-

21 in OSC exosomes suggests the involvement of this

mechanism in the oncogenic transformation of target cells

distant from the primary tumor. This could be an alterna-

tive to direct colonization by cancer cells underlying the

multiple peritoneal localizations commonly observed in

OSC. In addition, the presence of high miR-21 expression

levels in the cells and exosomes of OSC peritoneal effu-

sions could be used as a diagnostic tool.

FUNDING SUPPORT

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

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