Acta Med. Okayama, 2018
Vol. 72, No. 2, pp. 165‑174
CopyrightⒸ 2018 by Okayama University Medical School.

Original Article

http://escholarship.lib.okayama-u.ac.jp/amo/

Clinical and Functional Significance of Intracellular and Extracellular
microRNA-25-3p in Osteosarcoma
Aki Yoshidaa, Tomohiro Fujiwaraa＊, Koji Uotania, Takuya Moritaa,
Masahiro Kiyonoa, Suguru Yokooa, Joe Haseia, Eiji Nakataa,
Toshiyuki Kunisadaa,b, and Toshifumi Ozakia
Departments of aOrthopaedic Surgery, bMedical Materials for Musculoskeletal Reconstruction,
Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan

Although there is considerable evidence indicating that the dysregulation of microRNAs (miRNAs) in malignant tumors plays a role in tumor development, the overall function of miRNAs and their clinicopathologicalsignificance are not well understood. In this retrospective analysis of 45 biopsy specimens from osteosarcoma(OS) patients, we investigated the functional and clinical significance of miR-25-3p in OS, which we previously
identified as a highly expressed miRNA in OS patients’ serum. We observed that miR-25-3p dysregulation inhuman OS tissues was negatively correlated with the clinical prognosis, whereas the expression level of its target
gene, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3), was positively correlated with the clinical prognosis.
Endogenous miR-25-3p upregulation promoted tumor growth, invasion, and drug resistance, which was consistent with DKK3 silencing in OS cells. In addition, secretory miR-25-3p was embedded in tumor-derived
exosomes, where it promoted capillary formation and the invasion of vascular endothelial cells. Overall, ourresults show that miR-25-3p has intracellular and extracellular oncogenic functions as well as clinicopathologicalrelevance in OS, indicating its potential as a novel diagnostic and therapeutic tool for the clinical managementof this disease.
Key words: microRNA, circulating microRNA, osteosarcoma, prognosisOsteosarcoma (OS) is the most common primarymalignant bone tumor, and occurs mainly inchildren and adolescents [1]. Historically, the clinicalprognosis of OS improved significantly in the 1970s,
with the survival rate reaching 60-70% after the adventof high-dose, multi-agent chemotherapy. However, survival measures have failed to demonstrate any further
improvements in the prognosis of OS for more than
30 years, despite advances in surgical techniques andchanges in chemotherapy regimens [2 , 3]. To date,there are few useful biomarkers for monitoring tumor
Received October 31, 2017 ; accepted November 27, 2017.
Corresponding author. Phone : +81-86-235-7273; Fax : +81-86-223-9727
E-mail : tomomedvn@gmail.com (T. Fujiwara)＊

burden and response to treatment in OS. Salvage chemotherapy is of limited efficacy, even after identifying
relapse [2]. The development of diagnostic and therapeutic strategies based on a thorough understanding of
the molecular basis of metastasis is thus required to
improve patient survival.
MicroRNAs (miRNAs), a class of small noncoding
RNAs, are involved in the post-translational regulationof gene expression via their ability to inhibit the stability
and translation of messenger RNA (mRNA) [4]. miRNAs
play important roles in a variety of physiologicaland pathological processes by modulating the expresConflict of Interest Disclosures: No potential conflict of interest relevantto this article was reported.

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Yoshida et al.

sion of their multiple target genes [4 , 5]. To date,miRNA dysregulation has been documented in malignant tumors, demonstrating that miRNAs can have
either an oncogenic or tumor suppressive function[6 , 7]. In addition, miRNAs were recently demonstrated to be secreted into the circulation in a cell-freeform [8 , 9].
Despite the presence of RNase activity in humanblood, circulating miRNAs are detectable in extracellular vesicles such as exosomes, which are secreted fromtumor cells and play a biological and pathological role inintercellular communication within the tumor microenvironment and promote cell motility and invasion incancer [10]. The demonstration of circulating miRNAlevels in the blood or urine of patients may present a
novel approach to predicting clinical behavior and therapeutic responses [9 , 11]. We previously identified thesecretion of the miRNA miR-25-3p in OS cells, andmiR-25-3p was also detected in the serum of OS patients[12]. Serum miR-25-3p at the pretreatment status miR25-3p levels of the OS patients were significantly correlated with patient prognosis [12].
However, the clinicopathological significance andintracellular and extracellular functions of miR-25-3p
dysregulation in OS have been unclear, and few reports
have documented the functional and clinical value ofmiR-25-3p in the management of malignant tumors. In
the present study, we investigated the functional andclinicopathological significance of intracellular andextracellular miR-25-3p dysregulation in OS toward anunderstanding of the overall function of miR-25-3p intumor progression.

Materials and Methods
Human tissues. The study protocol was approvedby the Institutional Review Board of Okayama
University Hospital (No. 1509-037). All patients provided written, informed consent authorizing the collection and use of their samples for research purposes.
Tumor samples from OS patients were collected
between 2000 and 2015 at the Okayama University
Hospital.
Cell lines and cell culture.
The human OS celllines 143B and U2OS were purchased from theAmerican Type Culture Collection (Manassas, VA,
USA). Human mesenchymal stem cells (hMSCs) werepurchased from Lonza (Walkersville, MD, USA).

Acta Med. Okayama Vol. 72, No. 2

Human umbilical vein endothelial cells (HUVECs) werepurchased from PromoCell (Heidelberg, Germany).
143B and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies,
Grand Island, NY, USA). All media were supplemented with 10% fetal bovine serum (FBS; Life
Technologies), 50 units/ml penicillin, and 50 μg/ml
streptomycin (Life Technologies). Cells were maintained under 5% CO2 in a humidified incubator at 37°C.
Exosome isolation. Exosomes were purified fromthe culture medium supernatant as described previously[14]. The supernatant was ultracentrifuged using an
Optima TL-100 ultracentrifuge (Beckman Coulter,
Fullerton, CA, USA) at 110,000 g for 70 min to pellet
the exosomes. The pellet was washed in phosphatebuffered saline (PBS) to eliminate contaminating proteins, and centrifuged again at 110,000 g for 70 min.
The PBS was removed and the exosomes were re-suspended in 100 µl of PBS.
RNA extraction and RT-PCR.
Total RNA(including miRNA) was extracted using an miRNeasy
mini Kit (Qiagen, Valencia, CA, USA) according to the
manufacturer’s instructions. RNA samples were reverse
transcribed using the TaqMan MicroRNA Reverse
Transcription Kit (Applied Biosystems, Foster, CA,
USA). For mRNA detection, total RNA was reverse
transcribed using a PrimeScript RT Reagent Kit (Takara,
Tokyo, Japan) according to the manufacturer’s protocol.
Quantitative polymerase chain reaction (PCR) was performed on an Agilent Mx3000P QPCR System (Agilent
Technologies, Santa Clara, CA, USA) using TaqMan
2 × Universal PCR Master mix and each primer. Dataobtained from RT-PCR were analyzed using the 2−∆∆Ct
method. The miRNA expression levels were normalizedusing the miRNA cel-miR-39 for exosomes and thesmall non-coding RNA RNU6B for tumor cells, andthe mRNA expression levels were normalized using
GAPDH.
Western blots. Total protein was extracted fromthe cells using Mammalian Protein Extraction Buffer(GE Healthcare, Chicago, IL, USA). Fifteen μg of eachprotein extract was separated on Mini-PROTEAN®
TGX gels (Bio-Rad Laboratories, Hercules, CA, USA)and transferred onto Immun-Blot® PVDF membranes(Bio-Rad). The membranes were probed with the following primary antibodies: anti-CD9 mouse monoclonal antibody (Abcam, Cambridge, UK), anti-CD81
mouse monoclonal antibody (Santa Cruz Biotechnology,

April 2018

Santa Cruz, CA, USA), anti-β-actin mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA),and anti-DKK3 rabbit polyclonal antibody (Abcam).
The secondary antibody was IRDye® 800CW anti-rabbit
IgG or IRDye® 680RD anti-mouse IgG (LI-COR,
Lincoln, NE, USA).
Transfection with synthetic miRNAs, LNAs, andsiRNAs.
Synthetic hsa-miRs (pre-miR-hsa-miR-25
and negative control; Life Technologies) and locked
nucleic acids (LNAs) (LNA-antimiR-25-3p and scramble control; Gene Design, Ibaraki, Japan) were transfected at a final concentration of 30 nM each with theuse of the transfection agent DharmaFECT 1 (GEHealthcare). Synthetic siRNAs (Life Technologies) weretransfected into the cells at a final concentration of
10 nM each using DharmaFECT 1 (GE Healthcare).
Prediction of miRNA target genes.
The miRNA
analysis web (http://www.microrna.org/) was used to
identify candidate miRNAs. This web-based tool is
integrated with three of the most commonly used target
gene prediction algorithms: miRanda, MiRtarget2 andTargetScan.
Cell proliferation and cytotoxicity assays. Cell
proliferation rates and cell viability were used as indicators of the relative sensitivity of the cells to doxorubicin,
cisplatin, methotrexate, and docetaxel. Measurements
were determined using the WST-1 Proliferation Assay(Sigma-Aldrich) per the manufacturer’s instructions.
Cells were seeded in 96-well plates after 24-h transfection, and treated with varying doses of doxorubicin(Sigma-Aldrich), cisplatin (Enzo Life Sciences,
Farmingdale, NY, USA), methotrexate (SigmaAldrich), or docetaxel (Sigma-Aldrich) for 72 h.
Absorbance was measured at 450 nm with a reference
wavelength at 630 nm by a microplate reader (BioRad). The relative number of viable cells is expressed as
the percent of viable cells.
Cell migration and invasion assay. Cell invasion
and migration were examined with 24-well BD BioCoat
invasion chambers with and without Matrigel matrix(BD Biosciences, San Jose, CA, USA). Cells of thelines 143B, U2OS and HUVECs were trypsinized andseeded in chambers 48 h after transfection. The cells
were subsequently suspended in medium without FBS
and added to the upper chamber. Medium with 10%
FBS was added to the lower chamber. After a 24-h
incubation, the cells on the upper side were removed,
and the filters were fixed in methanol and stained with

Intra/extracellular miR-25-3p in OS

167

Hemacolor® solution 3 (Merck, Darmstadt, Germany).
The number of cells was counted in 6 separated highpower fields.
Luciferase assay.
First, 143B cells were cotransfected with the pGL3-DKK3-3’ untranslated region(UTR) or with the pGL3-DKK3-3’UTR Mut plasmid,
the phRL-SV40 control vector (Promega, Fitchburg,
WI, USA), and 30 nM miR-25-3p mimic or AccuTargetTM
miRNA mimic negative control #1 (Bioneer, Alameda,
CA, USA) using Lipofectamine® 3000 (Invitrogen,
Waltham, MA, USA). After transfection for 48 h, we
determined the renilla and firefly luciferase activities
using a Dual Luciferase Reporter Gene Assay kit(Promega) according to the manufacturer’s instructions. Luciferase activity was detected by a FlexStation
3 microplate reader (Molecular Devices, Sunnyvale,
CA, USA). The firefly luciferase activity of each sample
was normalized using the renilla luciferase activity.
Immunohistochemistry.
Slides were heated for
antigen retrieval in 10 mM sodium citrate (pH 6.0),
followed by incubation with polyclonal goat anti-human DKK3 (Abcam) or isotype-matched control antibodies overnight at 4°C. Immunodetection was performed using Histofine anti-goat IgG (Nichirei, Tokyo,
Japan) and the DAB substrate kit (Nichirei) according
to the manufacturer’s instructions. Sections were counterstained with hematoxylin to create contrast. The
immunohistochemical (IHC) score was defined as the
proportion of tumor cells with stained cell membrane:
negative, 0-49%; focal, 50-79%; diffuse, 80-100%.
Tube formation assay.
HUVECs were plated on
top of Matrigel and treated with exosomes (50 ng perwell) derived from OS cells 48 h after transfection. Tube
formation was quantified by counting the branch point
number in three wells after 24 h. The average number of
each group was calculated and normalized to the average number of the control group.
Statistical analysis.
Experiments were repeatedat least 3 times. Statistical analyses were performed
using SPSS ver. 23 software (SPSS, Chicago, IL, USA).
Student’s two-tailed t-test was used for comparisons ofpairs of groups. For comparisons of more than 2groups, we performed a one-way analysis of variance(ANOVA). The differences in miR-25-3p expression
among different clinicopathological data were analyzed
by the chi-squared test. The Kaplan-Meier method andthe log-rank test were used to compare the survival ofpatients. P-values < 0.05 were considered significant.

Yoshida et al.

Acta Med. Okayama Vol. 72, No. 2

Results
The dysregulated miR-25-3p expression levels in OS
tissue specimens were negatively correlated with thepatient prognosis.
To investigate the clinical significance of miR-25-3p expression levels in OS tissue specimens, we performed a Kaplan-Meier analysis on the
RT-PCR results of 45 fresh frozen human biopsy specimens. High miR-25-3p expression in the pretreatment
OS tissues was significantly correlated with poor overall
survival (p = 0.004, log-rank test; Fig. 1A). High miR25-3p levels were also correlated with poor metastasis-free survival (p = 0.005, log-rank test; Fig. 1B). The
clinicopathological features of the patients in relation to
the expression of miR-25-3p are summarized in Table 1.
The statistical analysis revealed a significant correlation
between high-miR-25-3p expression and the presence ofmetastasis (p = 0.014) and the oncologic outcome(p = 0.003). These data were consistent with thoseobtained previously for circulating miR-25-3p [12],indicating that both cellular and circulating miR-25-3p
expression levels are significantly correlated with theprognosis of OS patients.
miR-25-3p regulates lethal phenotypes of OS cells.
To investigate the role of miR-25-3p in OS cells, we performed a functional analysis by altering miR-25-3p
expression. Because miR-25-3p was confirmed to be
expressed at a higher level in OS cells compared to normal cells [12] (Fig. 2A), we performed functional analysis using locked nucleic acids (LNAs). LNA-antimiR25-3p suppressed the miR-25-3p expression in OS cells
A 100

Low miR-25-3p

Overall survival (%)

80
60
40

High miR-25-3p

20
P = 0.004

0
0

30

60
90
Months120

150(Fig. 2B). The proliferation assays revealed that miR25-3p silencing suppressed the tumor growth ability in
OS cells (Fig. 2C). In addition, cellular invasiveness was
also reduced by LNA-antimiR-25-3p (Fig. 2D).
Interestingly, miR-25-3p silencing improved the multidrug resistance in OS cells (Fig. 2E). These data
revealed that miR-25-3p is a potential oncogenic miRNAin OS and contributes to the development of OS.
DKK3, a direct target of miR-25-3p, regulates cellular proliferation and invasion in OS.
We screened
the mRNA targets of miR-25-3p by using the in silico
prediction algorithms miRanda, MiRtarget2 andTargetScan to determine the potential downstream
pathway regulated by miR-25-3p. Among the 122 downstream targets of miR-25-3p revealed by both algorithms,
DKK3 was identified as a miR-25-3p target (Fig. 3A).
DKK3 plays an important role in the Wnt-β catenin signaling pathway, which is inactive in OS [13]. DKK3
expression was inversely correlated with miR-25-3p in
OS cells, confirming increased DKK3 levels by miR25-3p silencing (Fig. 3B).
To validate whether DKK3 is regulated by miR-25-3p,
we cloned the 3’UTR fragment containing the putative
miR-25-3p binding site downstream of a luciferase coding sequence, and the luciferase reporter and miR25-3p oligos were cotransfected into U2OS cells. The
luciferase activity levels were approximately 72% lower
in the cells cotransfected with miR-25-3p compared tothe cells cotransfected with miR-NC oligos (Fig. 3C). We
evaluated the functional consequences of silencing
DKK3 expression by using siRNA (Fig. 3D). Silencing
B 100
Metastasis-free survival (%)

168

Low miR-25-3p

8060High miR-25-3p

40
20

P = 0.005

0
0

30

60
90
Months120

150

Fig. 1
Clinical relevance of miR-25-3p expression in OS. A, Kaplan-Meier survival curves for overall survival according to miR-25-3p
expression (log-rank test, p = 0.004); B, Kaplan-Meier survival curves for metastasis-free survival according to miR-25-3p expression (logrank test, p = 0.005).

April 2018the DKK3 expression promoted the migration and invasion of the OS cells, but did not affect the drug resistance (Fig. 3E , F). Collectively, these results suggestthat DKK3, a direct target of miR-25-3p, could act toprevent tumor growth and metastasis.
The DKK3 expression levels in the OS tissues werepositively correlated with the patient prognosis.
To
determine the clinicopathological significance of DKK3,
we performed a statistical analysis of the DKK3 expression in OS tissues. Immunohistochemical staining
revealed DKK3 to be diffusely positive in 55%, focally
positive in 6%, and negative in 39% of the diagnostic
incisional biopsy specimens, with DKK3 localized tothe cytoplasm in the positive cases (Fig. 4A). The prognosis of the patients whose biopsy specimens were negative for DKK3 was poor, with 57% of these patients
dying of the disease. Our Kaplan-Meier analysis of the
RT-PCR results revealed that the DKK3 expression levels
were significantly correlated with the patients’ progno-

Intra/extracellular miR-25-3p in OS

169

ses. Low DKK3 expression was significantly correlatedwith poor overall survival (p = 0.022, log-rank test) andpoor disease-free survival (p = 0.017, log-rank test)(Fig. 4B, C). These data show the clinicopathologicalsignificance of DKK3 to be inversely correlated with thatof miR-25-3p, corresponding to the demonstrated functional relationship between miR-25-3p and its target.
Secretory miR-25-3p is embedded in OS-derived
exosomes and mediates angiogenesis. We previously
observed that miR-25-3p was released into the extracellular space in OS cells [12]. Here, to examine the delivery of secretory miR-25-3p into extracellular fluid, we
purified tumor-derived exosomes and analyzed themiR-25-3p expression in exosome fractions. Scanningelectron microscopy (SEM) revealed that the exosomes
had a round shape with 40-100-nm-sized membrane
vesicles (Fig. 5A). A nanoparticle tracking analysis of
the purified exosomes revealed a fraction containing
nanoparticles with a peak at 101 nm (Fig. 5B).
Our western blot analysis conTable 1
Patient characteristics
firmed the expression of common
exosomal surface markers, CD9
miR-25-3p expression
No. ofand CD81, in the exosome fracFactor
p-valuepatients
Low
High
tions (Fig. 5C). The RT-PCR
revealed higher miR-25-3p expresAge at diagnosis (yrs):
0.229
sion levels in the tumor-derived
0-10
3
3
0
11-20
16
8
8
exosome fractions compared to the
≥ 21
26
17
9
exosome fractions from the hMSCs
Gender:0.258(Fig. 5D). These data suggest that
Male
20
14
6
secretory miR-25-3p in extracellular
Female25
14
11
fluid may acquire stability via
Location:
0.316
Femur
22
15
7
encapsulation by OS-derived exoTibia
642
somes. In addition, the capillary
Humerus3
2
1
formation of HUVECs was signifiOthers
14
7
7
cantly enhanced by the addition ofHistological subtype:
0.398
OS-derived exosomes (Fig.5E).
Conventional
34
22
12
Other
11
6
5
Silencing of the miR-25-3p in
Response to chemotherapy:
0.287
HUVECs decreased the capillary
< 90%
19
12
7
formation; however, the addition
≥ 90%
11
5
6
of OS-derived exosomes restored
Local recurrence:
0.538
the capillary formation in silenced
Yes
7
4
3
No
38
24
14
HUVECs (Fig. 5F), suggesting that
Distant metastasis:
0.014
the transport of OS-derived exoYes
21
912
somes containing miR-25-3p to
No
24
19
5
endothelial cells may disrupt their
Oncologic outcome:0.003
microenvironment and promote OS
CDF/NED
26
21
5
DOD
19
7
12
development.
Finally, increased miR-25-3p
CDF, continuous disease-free; NED, no evidence of disease; DOD, dead of disease.

Acta Med. Okayama Vol. 72, No. 2

10
0

SC

M

B
43

1

S

2O

U

＊＊

0.0

C R
N mi
A- nti p
LN A-a 5-3
2
LN 143B

LNAantimiR-25-3p

E

143B

1.0

＊

LNA-NC

0.5

0.5
U2OS

Invasion index

D

0.0

C R
N mi
A- nti pN
L A-a 5-3
2
LN -

C R
N mi
A- nti pN
L A-a 5-3
2
LN -

143B

U2OS

Relative cell viability

＊

＊＊＊

1.0

1.5
1.0
0.5
0.0
10-4 10-2 100 102 104
Methotrexate (µM)
1.5
1.0
0.5
0.0 -510

10-3 10-1 101
Cisplatin (µg/ml)

0.5

0.0

C
N iR
A- ntim p
N
L -a 5-3
A 2
LN 143B

C iR
N
A- ntim p
LN -a 5-3
A 2
LN U2OS

＊＊＊

＊＊＊

1.0

Relative cell viability

20

＊＊＊

1.5

Relative cell viability

30

C

1.5

Relative cell viability

B＊

Relative cell viability

40

Relative expression of miR-25

A

Yoshida et al.
Relative expression of miR-25

170

1.5

C
N iR
A- ntim p
N
L -a 5-3
A 2
LN U2OS

LNA-NC
LNA-antimiR-25-3p

1.0
0.5
0.0 -210

100
102
Doxorubicin

104
(µM)

0.0
10-4 10-2 100 102
Docetaxel

104
(µM)

1.0
0.5

Fig. 2
miR-25-3p promotes OS cell proliferation, migration, and drug resistance. A, miR-25-3p expression levels in OS cells andhuman mesenchymal stem cells (hMSCs); B, miR-25-3p expression levels in OS cells after transfection with LNA scramble control(LNA-NC) or LNA-antimiR-25-3p; C, Relative proliferation rates of OS cells transfected with LNA-NC or LNA-antimiR-25-3p; D: Invasion
analysis of OS cells treated with LNA-NC or LNA-antimiR-25-3p. Scale bar, 100 μm; E, Drug sensitivity according to miR-25-3p expression in the presence of methotrexate, doxorubicin, cisplatin, or docetaxel. Columns, mean of at least three independent experiments.
Bars, SEM. ＊p < 0.05, ＊＊p < 0.01, ＊＊＊p < 0.001.

expression promoted the invasion and migration of
HUVECs, whereas miR-25-3p silencing reduced these
HUVEC phenotypes (Fig. 5G). These data suggest thatextracellular miRNAs embedded in secretory exosome
vesicles could be delivered to the tumor microenvironment and act as physiologically functional molecules topromote tumor progression.

Discussion
Accumulating evidence suggests that dysregulated
miRNAs are involved in the oncogenesis, maintenance,and development of human malignant tumors [6 , 7]. In
addition, cell-free circulating miRNAs have been iden-tified in patients with malignant tumors and may function in communication with the tumor microenvironment, indicating that they could be used as novel
biomarkers in tumor diagnosis and monitoring [14].
However, few reports have addressed the clinicopathological significance and functional relevance of single
dysregulated specific miRNAs in tumor development.
Ma and colleagues were the first to report an aspectof the functional significance of miRNAs by showingthat the overexpression of a specific miRNA, miR-10b,
contributed to the development of breast cancer metastasis [15]. In addition, oncogenic miR-10b secreted bybreast cancer cells has been shown to suppress the protein level of its target genes, such as HOXD10 and KLF4,

10-2 100 102 104
Cisplatin (µg/ml)

0.5
0.0
10-4 10-2 100 102 104
Docetaxel (µM)

3
2

＊

1
0

3

1.0

＊

C

101
(µM)

4

+
+

KK

10-3
10-1
Doxorubicin

Invasion

N

0.0
10-5

Migration

+
+
-

D

0.5

F

+
+

si

0.0
10-4

1.5

1.0

143B

0.0
miR-25-3p +miR-NCDKK3 wild +DKK3 mut -3

0.0
10-4 10-2 100 102 104
Methotrexate (µM)
1.5

siNC
siDKK3C iR
N
A- ntim p
LN A-a 5-3
2
LN U2OS

si

0.5

1.5

C iR
N
A- ntim p
LN A-a 5-3
2
LN -

C

37-

1.0

0.5

0.0

5050-

1.5

1.0

0.5

KK

Relative cell viability

E

Relative cell viability

β-actin75-

siNC siDKK3

0.5

D

DKK3

siNC siDKK3

Relative cell viability

kD

U2OS

Relative cell viability

D

143B

1.0

1.0

N

DKK3 3’ UTR mut: 5’-AGGCUGUGGGUAGAUCGAACGC -3’

＊

1.5

si

3’-AGUCUGGCUCUGUU--CACGUUAC-5’

171

＊

si

hsa-miR-25-3p:＊＊

Invasion index

5’-AGGCUGUGGGUAGAUGUGCAAUA-3’

143B
siDKK3 siNC

DKK3 3’ UTR:

C

2.0

U2OS
siDKK3 siNC

B

Relative DKK3 expression

A

Relative luciferase activity

Intra/extracellular miR-25-3p in OS

April 2018

143B

U2OS

Fig. 3
Dickkopf-3 (DKK3) is a direct target of miR-25-3p. A, Alignments of DKK3-3’UTR, hsa-miR-25-3p, and DKK3-3’UTR mut,in which the complementary sites for the seed region of miR-25-3p are indicated; B, Relative DKK3 expression in OS cells treated with
LNA-NC or LNA-antimiR-25-3p; C, Luciferase activities of 143B cells co-transfected with miR-25-3p oligonucleotides and wild-type (wild)or mutant (mut) reporter vectors of DKK3; D, Western blot analysis of DKK3 expression after negative control siRNA (siNC) or siDKK3transfection into 143B and U2OS cells, respectively; E, Drug sensitivity according to DKK3 expression in the presence of methotrexate,
doxorubicin, cisplatin, or docetaxel; F, Invasion assays performed using 143B and U2OS cells at 24 h post-transfection with siNC or
siDKK3. Scale bar, 100 μm. Columns, mean of at least three independent experiments. Bars, SEM. ＊p < 0.05, ＊＊p < 0.01.

in recipient cells, which can lead to a favorable environment for breast cancer development and progression [16]. However, miR-10b has rarely been reported
to be present in the circulation and to have potential asa breast cancer biomarker. miR-21, an oncomiR, stimulates the invasion, intravasation, and metastasis ofcolorectal cancer [17]. Serum miR-21 is a promisingbiomarker for the early detection and prognosis of colorectal cancer [18], but the extracellular function of
miR-21 in colorectal cancer is not fully understood.
Our present findings demonstrated that miR-25-3p had
functional and clinical significance in the intracellularand extracellular regions of OS cells and that it has a rolein the promotion of tumor progression and develop-

ment, suggesting that miR-25-3p could have potential asa diagnostic biomarker and a therapeutic target in OS
patients.
We also observed that miR-25-3p affected not only
the proliferative and invasive abilities of OS cells, butalso their drug resistance. These phenotypes are consistent with the lethal phenotypes of tumor-initiating cells(TICs), which are designated cancer stem cells (CSCs).
The hallmarks of TICs are not only their self-renewal
ability, but also their resistance to conventional chemotherapeutic agents and high metastatic potential[19 , 20]. Recent studies have shown that miRNAs areassociated with TIC phenotypes via their ability tosimultaneously regulate their various target genes

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Acta Med. Okayama Vol. 72, No. 2

DKK3

H.E.

A

100

Focal / strong(6%)
High DKK3806040
Low DKK320

p = 0.022

0
0

30

60

Diﬀuse / weak
(22%)

C 100

Metastasis-free survival (%)

Overall survival (%)

B

Negative(39%)

90 120 150 180
Months

Diﬀuse / strong
(33%)

80
60
40

High DKK3

20

Low DKK3
p = 0.017

0
0

30

60
90
Months120

150

Fig. 4
Clinicopathological signiﬁcance of DKK3 expression. A, Immunohistochemical staining of DKK3 and hematoxylin and eosin
staining of OS biopsy specimens. Scale bar, 50 μm; B, Kaplan-Meier survival curves for overall survival according to DKK3 expression(log-rank test, p = 0.022); C, Kaplan-Meier survival curves for metastasis-free survival according to DKK3 expression (log-rank test,
p = 0.017).

[21 , 22]. miR-34a, a p53 target, is reported to be a key
negative regulator of prostate CSCs with a CD44-positive
phenotype, which are responsible for the clonogenic
expansion, tumor regeneration, and metastasis of
prostate cancer [21]. In addition, let-7 is a master regulator of the CD44+/CD24−/low cell population, a breast
CSC fraction with a markedly high TIC [23].
Among these TIC phenotypes, the most important
clinical phenotype is drug resistance. In the present
study, we did not investigate the relevance of TICs inosteosarcoma; however, miR-25-3p, which can regulate drug resistance, could constitute a new therapeutictarget for this cancer, and drugs targeting this miRNA
could be used in combination with conventional therapeutic drugs. Since LNAs are currently undergoingphase II clinical trials for the treatment of hepatitis [24],it is to be expected that similar strategies could be used
to develop miRNA therapeutics for the treatment ofmalignant diseases.
Several studies have demonstrated the contributionof exosomal miRNAs to cancer metastasis [25 , 26].
miR-181c promotes the destruction of the blood-brain

barrier (BBB) by mediating the abnormal localization ofactin via its ability to downregulate its target gene,
PDPK1 [26]. miR-210, which is released by metastatic
breast cancer cells, is transported to endothelial cells via
cancer-derived exosomes and suppresses the expressionof specific target genes, resulting in enhanced angiogenesis [27]. In the present study, miR-25-3p was
secreted by OS cells and induced the formation andmigration of vascular endothelial cells. Although theeffects of miR-25-3p have not yet been demonstrated invivo, miR-25-3p, which is also upregulated in OS cells,
altered the surrounding tumor microenvironment andstimulated tumor progression. The molecular mechanism linking OS-secreted exosomal miR-25-3p to themicroenvironment of the lungs, a common site of distant metastasis, remains unclear and requires further
exploration. A broad understanding of all of the functions of oncomiRs in tumor development will provide
insights into how they could be used to develop newnovel diagnostic and therapeutic approaches to malignant diseases.
Overall, our present findings demonstrate the clini-Intra/extracellular miR-25-3p in OS

143B

U2OS

D

CD9
CD81

Size (nm)

60
40
20
0

LNA-NC

20
0

MSC

＊＊＊
＊＊＊

60
40
20
0
Ex

o

ex

143B U2OS

＊＊＊

(-)

o

40

14

LNAantimiR-25-3p＊

Invasion index

miR-25-3p

60

LNA-NC

2.0

LNAantimiR-25-3p

1.5

＊＊＊

1.0
0.5
0
m
iR
-N
C
m
iR
-2
53p
LN
ANC
LN
Aa
-2 nt
5- im
3p iR

Invasion Migration

miR-NC

OS

14

3B

o
Ex

G

80

80
Total Branch points＊(-)

Exosome(+)

80oExosome
(-)

F

＊＊＊

ex

Total Branch points

100

＊＊

＊＊

U2

E

100

o
(
3B -)
ex
U2
o
OS
ex
o

C

101 nm

10
0
20
0
30
0
40
0
50
0
60
0
70
0
80
0
90
10 0
00

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
0

173

Ex

B
Concentration(particles / ml)A

miR-25-3p expression

April 2018

Fig. 5
Exosome-embedded secretory miR-25-3p aﬀects function of normal vascular endothelial cells. A, SEM image of puriﬁed exosomes. Scale bar, 100 nm; B, Size distribution of exosomes assessed using the NanoSight® nanoparticle tracking system. The size range
of isolated exosomes was approx. 40-200 nm, with a peak at 101 nm; C, Western blot of CD9 and CD81 in exosomes derived from 143B
and U2OS cells; D, RT-PCR analysis of miR-25-3p expression in secreted exosomes derived from MSC and OS cells; E, HUVECs tube
forming assay with or without exosomes derived from OS cells. Scale bar, 50 μm; F, Tube formation analysis with alteration of miR25-3p expression and tumor-derived exosomes; G, Invasion analysis of HUVECs treated with mimic or LNA. Scale bar, 100 μm. Columns,
mean of at least three independent experiments performed in duplicate. Bars, SEM. ＊p < 0.05, ＊＊p < 0.01, ＊＊＊p < 0.001.

copathological and functional significance of intracellular and extracellular miR-25-3p in OS. Intracellular
miR-25-3p contributed to cellular proliferation, invasion, and drug resistance, which are lethal OS phenotypes. Extracellular miR-25-3p, which was enriched in
OS-derived exosomes, was transported to endothelial
cells and stimulated angiogenesis. The levels of circulating and intracellular miR-25-3p were significantly
correlated with a poor prognosis in the present series ofOS patients. By contrast, the expression of the Wnt
signaling inhibitor DKK3, a miR-25-3p target, was correlated with a good prognosis in the OS patients. Thesefindings contribute to our understanding of the intracellular and extracellular functions of dysregulated
miRNAs in tumor biology and suggest that these functions could be analyzed in liquid biopsies and developed
to provide novel therapeutics for OS patients.

174

Yoshida et al.

Acta Med. Okayama Vol. 72, No. 2

Acknowledgments. We thank the member of the Central ResearchLaboratory of the Okayama University Medical School for the technicalsupport for the isolation and validation of exosomes and histological analysis. The authors are supported by the Japan Orthopaedics and Traumatology
Research Foundation (Grant no. 311), the Uehara Memorial Foundation,
and JSPS KAKENHI Grants nos. 16K20054 and 16H05449.

16.

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