MicroRNA Profiling of Hepatocellular Carcinomas in B6C3F1 Mice Treated with Ginkgo biloba Extract by Gavage for 2 Years

1. MicroRNA
Profiling
of
Hepatocellular
Carcinomas
in
B6C3F1
Mice
Treated
with
Ginkgo
biloba
Extract
by
gavage
1Cellular
&
Molecular
Pathology
Branch,
NaPonal
Toxicology
Program,
NaPonal
InsPtute
of
Environmental
Health
Sciences
(NIEHS),
Research
Triangle
Park,
NC,
United
States,
2Taisho
PharmaceuPcal
Co.
Ltd.,
Saitama,
Japan,
3Experimental
Pathology
Laboratories,
Research
Triangle
Park,
NC,
United
States,
4BiostaPsPcs
Branch,
NIEHS,
Research
Triangle
Park,
NC,
United
States,
5Molecular
Genomics
Core
Laboratory,
NIEHS,
Research
Triangle
Park,
NC,
United
States.
Abstract
Ginkgo
biloba
Yamashita
H1,2,
Pandiri
AR1,3,
Bhusari
S1,
Shockley
KR4,
Peddada
SD4,
Gerrish
KE5,
Rider
CV1,
Hoenerhoff
MJ1,
Sills
RC1.
leaf
extract
(GBE)
has
been
used
for
centuries
in
tradiPonal
Chinese
medicine
and
today
is
used
as
an
herbal
supplement
touted
for
improving
neural
funcPon
and
for
its
anPoxidant
and
anPcancer
effects.
Exposure
of
B6C3F1
mice
to
GBE
in
the
2-­‐year
NaPonal
Toxicology
Program
(NTP)
bioassay
resulted
in
a
dose-­‐dependent
increase
in
hepatocellular
carcinomas
(HCC).
We
have
previously
reported
increased
and
alteraPons
in
Wnt/Ctnnb1
signaling
in
GBE-­‐induced
HCC
compared
to
spontaneous
HCC
in
vehicle
controls.
MicroRNAs
(miRNAs)
are
small
non-­‐
coding
RNAs
that
are
ogen
dysregulated
in
various
diseases
including
cancer.
To
idenPfy
key
miRNAs
that
modulate
GBE-­‐induced
hepatocarcinogenesis,
we
examined
global
miRNA
expression
using
Affymetrix
GeneChip®
miRNA
3.0
arrays
and
two
pairwise
analyses
(n=5/group)
comparing
GBE-­‐induced
HCCs
and
spontaneous
HCCs
with
vehicle
control
age-­‐matched
normal
livers
from
B6C3F1
mice.
Using
a
false
discovery
rate
threshold
of
5%,
we
observed
16
and
3
unique
differenPally
expressed
miRNAs
in
GBE-­‐induced
HCC
and
spontaneous
HCC,
respecPvely.
Ingenuity
Pathway
Analysis
of
the
miRNA
and
mRNA
array
data
from
these
tumors
demonstrated
altered
molecular
pathways
associated
with
hepatocarcinogenesis,
cell
cycle
progression,
cell
migraPon
and
cell
proliferaPon.
AddiPonally,
miRs-­‐31,
145,
329
and
433-­‐3p,
which
were
uniquely
expressed
in
GBE-­‐induced
HCC,
are
known
or
predicted
to
regulate
Wnt/Ctnnb1
signaling.
In
the
miRNA
expression
analysis
in
livers
from
the
90-­‐day
GBE
mouse
study,
miRs-­‐411,
300,
127
and
134
were
upregulated
more
than
double
in
GBE-­‐treated
group
compared
to
vehicle
control
group,
indicaPng
that
these
miRNAs
could
serve
as
potenPal
biomarkers
for
GBE
exposure
or
hepatocellular
carcinogenesis.
It
has
become
increasingly
apparent
that
epigenePc
mechanisms
are
at
play
in
the
mechanisms
of
carcinogenesis.
MicroRNAs
(miRNAs)
have
been
idenPfied
as
a
new
layer
of
gene
regulatory
mechanisms
(Lujambio
and
Lowe,
2012).
The
importance
of
miRNAs
in
cancer
is
highlighted
by
the
observaPon
that
half
of
the
known
aberrant
expressions
of
miRNAs
are
located
in
cancer
associated
genomic
regions
(Wiklund
et
al.,
2010).
On
the
relaPonship
between
miRNAs
and
hepatocellular
carcinoma
(HCC)
in
humans,
several
studies
have
detected
the
aberrant
expression
of
specific
miRNAs
in
malignant
HCC,
compared
to
normal
hepatocyte
(Masaki,
2009).
There
is
widespread
and
unregulated
use
of
GBE
as
a
dietary
supplement
by
the
American
public,
and
thus
is
a
significant
public
health
concern.
NTP’s
Ginkgo
biloba
leaf
extract
(GBE)
bioassay
has
indicated
that
chronic
GBE
exposure
to
B6C3F1
mouse
resulted
in
a
dose
dependent
increase
in
hepatocarcinogenicity.
Recent
transcriptomic
studies
on
GBE-­‐induced
HCC
indicated
dysregulated
cancer
gene
expression.
In
AddiPon,
increased
Ctnnb1
mutaPons
and
alteraPons
in
Wnt/Ctnnb1
signaling
were
demonstrated
in
GBE-­‐induced
HCC
compared
to
spontaneous
HCC
(Hoenerhoff
et
al.,
2013).
Determining
the
mechanisms
of
GBE-­‐induced
hepatocarcinogenicity
in
rodents
may
aid
in
assessing
the
health
risks
of
human
exposure.
We
hypothesize
that
genePc
and
epigenePc
pathways
dysregulated
in
GBE-­‐induced
mouse
HCC
may
reflect
key
pathways
altered
in
human
HCC.
The
objecPve
of
this
study
is
to
characterize
the
pamern
of
dysregulated
miRNAs
occurring
in
spontaneous
and
GBE-­‐induced
HCC
and
compare
it
to
the
corresponding
mRNA
alteraPons
in
HCC.
Tissue
collec*on
and
miRNA
extrac*on
for
miRNA
array:
Ctnnb1
mutaPons
Frozen
samples
from
GBE-­‐induced
HCCs,
spontaneous
HCCs
and
vehicle
control
age-­‐matched
normal
livers
from
B6C3F1
mice
from
the
2-­‐year
NTP
bioassay
were
used
for
miRNA
array
analysis
(n=5/group).
miRNA
extracPon
was
performed
using
mirVana
miRNA
IsolaPon
Kit
(Life
technologies,
Carlsbad,
CA)
and
RNA
integrity
was
measured
with
Bioanalyzer
(Agilent
Technologies,
Santa
Clara,
CA).
miRNA
array
hybridiza*on
&
data
analysis:
miRNA
expression
analysis
was
conducted
using
Affymetrix
GeneChip®
miRNA
3.0
Array
(Affymetrix,
Santa
Clara,
CA)
following
manufacturer’s
direcPons.
miRNA
expression
data
were
normalized
across
all
samples
using
the
robust
mulParray
analysis
(RMA)
(Guo
et
al.,
2010).
RMA-­‐normalized
data
were
used
for
idenPfying
differenPally
expressed
miRNAs
using
two
pairwise
analyses
comparing
GBE-­‐
induced
HCCs
and
spontaneous
HCCs
with
vehicle
control
age-­‐matched
normal
livers
from
B6C3F1
mice.
Using
Ingenuity
Pathway
Analysis
(IPA),
we
have
analyzed
the
differenPally
expressed
miRNAs
together
with
the
corresponding
transcriptomic
data
that
we
have
previously
obtained
from
these
samples
(Hoenerhoff
et
al.,
2013).
miRNA
array
data
valida*on:
QuanPtaPve
RT-­‐PCR
(QRT-­‐PCR)
was
used
to
validate
miRNA
array
results.
QRT-­‐PCR
was
performed
using
TaqMan®
MicroRNA
Assay
(Life
technologies,
Carlsbad,
CA)
on
ABI
PRISM
7900HT
Sequence
DetecPon
System
(Applied
Biosystems,
Foster
City,
CA).
snoRNA202
was
used
as
the
endogenous
control
for
normalizaPon
of
miRNA
levels.
miRNA
expression
analysis
in
livers
from
90-­‐day
GBE
mouse
study:
miRNA
was
isolated
and
extracted
from
two
20
μm
secPons
of
formalin-­‐fixed,
paraffin-­‐embedded
(FFPE)
livers
from
control
mice
and
from
mice
treated
with
2000
mg/kg
GBE
for
90
days
(n=6/group)
with
RecoverAll™
Total
Nucleic
Acid
IsolaPon
Kit
for
FFPE
(Life
technologies,
Carlsbad,
CA).
QRT-­‐PCR
was
performed
as
described
above.
In
the
NTP
2-­‐year
mouse
GBE
bioassay,
there
was
a
dose
dependent
increase
in
HCC
(NTP
TR
578,
Table
1).
Using
the
Affymetrix
GeneChip®
miRNA
3.0
Array
plaoorm,
when
compared
to
normal
livers,
there
were
3
and
16
unique
differenPally
expressed
mouse
miRNAs
in
spontaneous
HCC
and
GBE-­‐induced
HCC,
respecPvely,
at
FDR
≤
0.05
(Figure
1
and
2,
Table
2).
Analyzing
miRNA
and
the
corresponding
mRNA
array
data
in
IPA,
we
have
found
several
differenPally
altered
molecular
pathways
associated
with
HCC
development
in
both
GBE-­‐
induced
HCCs
and
spontaneous
HCCs
(Tables
3,
4,
and
5).
Therefore,
these
data
show
that
GBE-­‐induced
HCCs
are
disPnguishable
from
spontaneous
HCC
in
terms
of
their
miRNA
expression
profile.
We
have
previously
reported
increased
Ctnnb1
mutaPons
and
alteraPons
in
Wnt/Ctnnb1
signaling
in
GBE-­‐induced
HCC
compared
to
spontaneous
HCC
in
vehicle
controls.
In
addiPon,
in
GBE-­‐induced
HCC,
there
was
cytoplasmic
accumulaPon
of
CTNNB1
and
loss
of
normal
CDH1
membrane
immunoreacPvity,
with
accumulaPon
of
the
protein
in
the
cytoplasm
that
suggests
disrupPon
of
CTNNB1/CDH1
complexes
within
adherens
juncPons,
which
is
associated
with
a
more
malignant
phenotype
(Hoenerhoff
et
al.,
2013).
In
this
study,
we
found
4
miRNAs
that
were
uniquely
expressed
in
GBE-­‐
induced
HCC
and
known
or
predicted
to
regulate
Wnt/Ctnnb1
signaling
(Table
5).
One
of
them,
miR-­‐31
was
strongly
downregulated
(83-­‐fold
by
QRT-­‐PCR)
in
GBE-­‐induced
HCC
with
no
change
in
spontaneous
HCC
compared
to
normal
livers
and
predicted
to
regulate
Cdk1,
which
was
upregurated
in
GBE
induced
HCC.
CDK1
plays
a
key
role
in
cell
cycle
regulaPon
and
increases
Src
kinase
acPvity
(Roskoski,
2005).
PhosphorylaPon
by
Src
kinase
disrupts
binding
of
CTNNB1/CDH1
and
results
in
loss
of
the
complexes
from
the
cell
surface
(Nelson
and
Nusse,
2004).
Therefore,
miR-­‐31
seems
to
indirectly
modulate
Wnt/Ctnnb1
signaling
in
GBE-­‐induced
HCC.
However,
further
studies
are
needed
to
evaluate
the
effect
of
miR-­‐31
on
Wnt/Ctnnb1
signaling.
In
order
to
determine
if
there
are
any
miRNAs
that
could
potenPally
serve
as
a
biomarker
for
GBE
exposure
and/or
early
biomarkers
of
hepatocellular
carcinogenesis,
we
have
also
analyzed
the
miRNA
expression
in
livers
from
the
90-­‐day
GBE
mouse
study.
The
expression
of
miRs-­‐411,
300,
127
and
134
more
than
doubled
in
GBE-­‐treated
group
compared
to
vehicle
control
group
(Figure
3).
In
the
90-­‐day
GBE
study,
although
hepatocellular
hypertrophy
and
focal
necrosis
were
found
in
the
livers,
there
were
no
preneoplasPc
hepaPc
foci
(NTP
TR
578,
Table
1).
Since
these
miRNAs
were
uniquely
expressed
in
livers
from
90-­‐day
exposures
and
in
HCCs
from
GBE
exposure
or
arising
spontaneously,
they
could
serve
as
potenPal
biomarkers
for
GBE
exposure
or
hepatocellular
carcinogenesis.
These
results
suggest
that
these
miRNAs
might
be
useful
as
biomarkers
of
exposure
and
apical
endpoints.
However,
further
validaPons
in
prospecPve
studies
are
necessary
in
order
to
validate
these
findings.
In
addiPon,
although
this
QRT-­‐PCR
analysis
was
performed
using
miRNA
extracted
from
secPons
of
FFPE
livers,
all
the
miRNAs
analyzed
were
amplified
with
relaPve
ease,
indicaPng
that
archival
FFPE
Pssues
can
be
leveraged
for
miRNA-­‐based
biomarker
idenPficaPon.
Introduc.on
Materials
and
Methods
Result
Results
and
Discussion
Table
1
Table
1.
Incidences
of
select
hepaPc
lesions
in
B6C3F1
mice
treated
with
Ginkgo
biloba
leaf
extract
(GBE)
by
gavage
in
subchronic
(90-­‐day)
and
chronic
(2-­‐year)
NaPonal
Toxicology
Program
studies
(NTP
TR
578).
Figure
1
Figure
1.
Normal
liver
Spontaneous
HCC
(A)
Principal
component
analysis
(PCA)
of
global
miRNA
expression
profiles
demonstrated
nearly
disPnct
clustering
of
normal
liver
(blue),
spontaneous
HCC
(green)
and
GBE-­‐induced
HCC
(red)
samples.
(B)
Using
a
false
discovery
rate
threshold
of
5%,
3
and
16
unique
mouse
miRNAs
were
differenPally
expressed
in
spontaneous
HCC
and
GBE-­‐induced
HCC,
respecPvely.
The
number
in
the
parenthesis
indicates
the
number
of
differenPally
expressed
miRNAs
from
other
species
besides
mice.
Conclusions
• GBE-­‐induced
mouse
HCCs
are
markedly
different
from
spontaneous
HCCs
in
terms
of
their
global
miRNA
expression
profile.
• The
miRNA
and
mRNA
array
data
from
these
tumors
demonstrated
altered
molecular
pathways
associated
with
hepatocellular
carcinogenesis.
• miRs-­‐31,
145,
329
and
433
that
were
uniquely
expressed
in
GBE-­‐induced
HCC
are
known
or
predicted
to
regulate
Wnt/Ctnnb1
signaling.
• miRs-­‐411,
300,
127
and
134
were
upregulated
in
the
livers
from
GBE-­‐
treated
group
compared
to
vehicle
control
group
from
90-­‐day
GBE
mouse
study
and
these
miRNAs
could
serve
as
potenPal
biomarkers
for
GBE
exposure
or
hepatocellular
carcinogenesis.
References
Acknowledgements
We
would
like
to
thank
DNTP
and
DIR,
NIEHS
for
funding
this
project.
We
would
also
like
to
thank
the
NIEHS
Microarray
core
and
the
Histology
core
Laboratory
in
the
Cellular
and
Molecular
Pathology
Branch
for
their
technical
assistance
on
this
project.
Table
2
Table
2.
DifferenPally
expressed
miRNAs
and
their
target
genes
in
spontaneous
and
GBE-­‐induced
HCC
compared
to
age-­‐matched
normal
livers.
Table
3
ê:
Known
to
decrease
the
diseases
or
funcPon
and
is
down-­‐regulated
in
the
dataset.
é:
Known
to
increase
the
diseases
or
funcPon
and
is
up-­‐regulated
in
the
dataset.
êé:
Literature
indicates
this
gene
is
involved
in
the
diseases
or
funcPon
but
does
not
indicate
whether
it
increases
or
decreases
it.
And
the
gene
is
down-­‐
or
up-­‐regulated
in
the
dataset.
#1
Target
genes
that
showed
relaPonship
with
the
respecPve
miRNA
in
IPA
analysis
(experimentally
observed
or
predicted)
and
down-­‐
or
up-­‐regulated
in
GBE-­‐induced
HCC
microarray
analysis.
Table
3.
RepresentaPve
altered
miRNA-­‐mRNA
interacPons
and
molecular
pathways.
Ingenuity
Pathway
Analysis
of
target
mRNAs
of
differenPally
altered
miRNAs
in
spontaneous
and
GBE-­‐induced
HCC
demonstrated
dysregulated
molecular
pathways
associated
with
hepatocarcinogenesis,
cell
cycle
progression,
cell
migraPon
and
cell
proliferaPon.
Table
4.
DifferenPally
altered
miRNAs
from
other
species
that
are
staPsPcally
significant
(but
not
in
mice)
and
that
play
a
role
in
human
hepatocellular
carcinogenesis.
These
miRNA
sequences
are
typically
conserved
across
species
and
are
likely
to
be
relevant
even
in
mouse
hepatocellular
carcinogenesis.
Table
5
Table
5.
miRs-­‐329,
31,
145
and
433-­‐3p,
which
were
uniquely
expressed
in
GBE-­‐induced
HCC,
are
known
or
predicted
to
regulate
Wnt/Ctnnb1
signaling.
Figure
2
Figure
2.
QuanPtaPve
RT-­‐PCR
validaPon
of
miRNA
array
expression
changes
observed
in
spontaneous
HCC
and
GBE-­‐induced
HCC
normalized
to
normal
livers.
Figure
3.
QuanPtaPve
RT-­‐PCR
of
miRNAs
in
livers
from
B6C3F1
mice
treated
with
2000
mg/kg
GBE
for
90
days.
The
miRNA
expression
in
GBE
livers
was
normalized
to
normal
livers.
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(B)
a
10
male
and
female
B6C3F1
mice
were
exposed
to
0,
125,
250,
500,
1,000,
and
2,000
mg/kg
GBE
by
gavage,
once
daily,
5
days
per
week
for
90
days.
b
Severity
grade
based
on
0–4
grading
scale
(0
=
no
significant
lesion,
1
=
minimal,
2
=
mild,
3
=
moderate,
4
=
severe).
c
50
male
and
female
B6C3F1
mice
were
exposed
to
0,
200,
600,
and
2,000mg/kg
GBE
by
gavage,
once
daily,
5
days
per
week
for
two
years.
d
StaPsPcal
analysis
not
available
for
metastaPc
lesions.
Significantly
different
from
controls
*p
<
.05,
**p
<
.01
by
the
poly-­‐3
test.
#1
Target
genes
that
showed
relaPonship
with
the
respecPve
miRNA
in
IPA
analysis
(experimentally
observed
or
predicted)
and
down-­‐
or
up-­‐regulated
in
GBE-­‐induced
HCC
microarray
analysis.
#2
No
informaPon
on
the
target
genes
was
found
in
IPA
knowledge
base
on
these
miRNAs.
Figure
3
Table
4
(A)
GBE-­‐induced
HCC