| Abstract |
The
Ras
p21
proto-‐oncogenes
(H-‐Ras,
K-‐Ras
and
N-‐Ras)
are
a
part
of
intracellular
signalling
pathways
that
play
an
important
role
in
cell
growth,
regulation
and
malignant
transformation.
In
this
study
we
investigated
the
presence
of
mutations
at
codon
12
of
H-‐,
K-‐
and
N-‐Ras
oncogenes
in
a
series
of
32
benign
and
malignant
thyroid
tumours
matched
with
normal
adjacent
tissue,
using
a
PCR-‐RFLP
technique.
Additionally,
we
measured
the
expression
levels
of
the
three
Ras
genes
using
a
quantitative
Real-‐Time
Polymerase
Chain
Reaction
(qRT-‐PCR)
assay
with
SYBR®
Green
I
chemistry.
Beta-‐actin
served
as
internal
control.
No
H-‐Ras
mutations
were
detected
in
our
series
of
thyroid
samples.
Additionally,
N-‐Ras
was
mutated
in
only
1/32
(3.1%)
malignant
specimen.
On
the
contrary,
K-‐Ras
mutations
were
detected
in
10/32
(31.3%)
tumor
samples.
Expression
analysis
showed
that
K-‐Ras
was
deregulated
in
50%
of
thyroid
tumors,
exhibiting
overexpression
in
9/32
(28.1%)
and
downregulation
in
7/32
(21.9%)
samples,
respectively.
A
similar
pattern
was
observed
for
N-‐Ras
expression,
since
in
9/32
(28.1%)
samples
its
mRNA
levels
were
elevated
and
in
10/32
(31.3%)
were
reduced.
Interestingly,
in
11/32
(34.4%)
sample
pairs
N-‐Ras
was
not
expressed
in
neither
the
neoplastic
nor
the
normal
specimen.
On
the
contrary,
H-‐Ras
mRNA
levels
were
downregulated
in
12/32
(37.5%)
and
elevated
in
5/32
(15.6%)
samples,
respectively.
From
the
above
results
we
deduce
that
only
K-‐Ras
mutations
are
a
frequent
event
in
thyroid
malignancies
and
that
all
three
Ras
oncogenes
deviate
from
the
normal
expression
pattern,
suggesting
an
important
role
for
this
gene
family
in
thyroid
tumorigenesis.
B-‐Raf,
a
member
of
the
Raf
serine/threonine
kinase
family,
is
an
intermediate
molecule
in
the
mitogen-‐activated
protein
(MAP)
kinase
pathway,
which
relays
extracellular
signals
from
the
cell
membrane
to
the
nucleus
via
a
cascade
of
phosphorylation
events,
ultimately
promoting
cancer
development.
This
pathway
is
usually
activated
in
human
neoplasias.
The
purpose
of
this
study
was
to
investigate
the
role
of
B-‐Raf
in
thyroid
pathology.
We
scanned
for
the
presence
of
mutations
at
codon
600
(VèE)
of
the
B-‐Raf
gene,
using
a
PCRRFLP
assay.
In
tumors
with
no
mutation
(32
benign
and
malignant
thyroid
tumors)
and
in
their
adjacent
normal
tissue,
we
measured
the
expression
levels
of
B-‐Raf
gene,
using
a
quantitative
Real-‐Time
PCR
(qPCR)
assay.
B-‐Raf
expression
in
V600E-‐negative
tumors
deviated
from
the
normal
pattern,
since
it
was
overexpressed
in
42%
of
benign
samples
and
downregulated
in
54%
of
malignant
specimens.
Hashimoto’s
thyroiditis
also
seemed
to
play
an
important
role,
since
benign
specimens
with
Hashimoto’s
thyroiditis
had
a
2.2-‐fold
higher
B-‐Raf
expression
than
samples
V
without
thyroiditis
(1.71±0.63
vs.
0.78±0.13).
Statistical
analysis
revealed
that
B-‐Raf
deregulation
postponed
disease
onset
by
more
than
10
years
in
both
benign
and
malignant
thyroid.
(benign:
55.6±3.9
vs.
45.3±3.3,
p=0.049;
malignant:
52.2±3.5
vs.
33.0±7.9,
p=0.020).
From
the
above
results
we
deduce
that
in
the
absence
of
mutation
activation,
B-‐Raf
overexpression
or
downregulation
is
a
protective
event,
since
it
delays
the
development
of
both
malignant
and
benign
thyroid
tumors.
|
Μελέτη της έκφρασης των ογκογονιδίων RAS σε σχέση με τα γονίδια RAF σε καλοήθης όγκους του θυρεοειδή αδένα
