Vol.
174,
February
No. 14,
BIOCHEMICAL
3, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1991
THE
mRNA
LEVELS
THYROGLOBULIN
AND
NEOPLASTIC Kazuyasu The Third
Received
THYROID
Toyoshi
Department
of
Medical
Endo
RECEPTOR,
PEROXIDASE
HUMAN THYROID
Ohta,
Yamanashi
THYROTROPIN
OF
TISSUES
Medicine,
School,
IN
and Toshimasa
Internal
1148-1153
Tamaho
Onaya
University
409-38,
of
Japan
December 17, 1990
SUMMARY Expression levels of thyrotropin receptor (TSH-R), thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA in normal and neoplastic human thyroid tissues (6 adenomas and I carcinomas) were investigated by Northern-blot and slotblot analyses. We found that TSH-R mRNA levels were significantly lower in carcinoma tissues than in normal tissues. The levels of Tg mRNA were also significantly lowered in adenoma and carcinoma tissues as compared to normal tissues. In contrast, no significant difference was observed in the expression levels of TPO mRNA between these tissues. TSH-R mRNA levels were wellFurthermore, correlated with Tg mRNA levels in neoplastic tissues. These results suggest that mRNAs of TSH-R and Tg are expressed in relation to their degree of differentiation. 0 1991Academic P?xSS,Inc. Since cells
the
is
degree
important
characterization proteins, is out
(l), TSH-R,
of for
differentiation
diagnostic the subject
Tg and TPO,
in
of theoretical and practical For this reason, a number on the
TSH-binding
4) and on the
glycosylated
Tg from
thyroid
of
activity
characterization
These studies (5). binding activity in
of
tumor
neoplastic
interest. studies
have
tissues
been
carried
of thyroid
tumor
of Tg from
malignant
tissues
existence of and poorly
lz51-TSH
demonstrated the the tumor tissues the
thyroid
and prognostic of thyroid specific
tissues
tissues.
However,
it can occasionally be problematic to decide whether a particular thyroid nodule is hyperplastic or neoplastic, and in the case of neoplasm, to differentiate follicular difficulties biology
of
adenoma from carcinoma. stem from major gaps thyroid neoplasm.
in
We think these our knowledge
of the
(Z-
Vol.
174,
No.
3, 1991
BIOCHEMICAL
Furthermore, markers of
thyroid
for
we have the
assessment
AND
BIOPHYSICAL
no information of
the
degree
RESEARCH
about of
the
COMMUNICATIONS
reliable
differentiation
tumors.
In this report, we investigated the expression TSH-R, Tg and TPO mRNAs in neoplastic human thyroid and compared with those in normal tissues.
MATERIALS
AND
levels
of
tissues
METHODS
Material: Neoplastic human thyroid tissues (follicular adenoma (n=6), carcinoma (papillary carcinoma (n=3), follicular carcinoma (n=4) ), their adjacent normal tissues (n=8) and thyroid tissues from patients with Graves' disease were obtained from surgery and immediately stored at -8OOC. a cloned line of functional rat thyroid cells, FRTL5 cells, was cultured in the presence of 10 mU/ml of thyrotropin (TSH) as reported previously (6). Probes for detecting TSH-R, TPO and Ts mRNAS: cDNA probes for human TSH-R and TPO mRNAs were synthesized by polymerase chain reaction (PCR) methods (7) using synthetic oligonucleotides as primers. Template cDNAs for preparing these probes were synthesized from purified mRNA from thyroid glands of a patients with Graves' disease by avian reverse transcriptase (8). The size of PCR products and its purity were checked by agarose electrophoresis, and nucleotide sequence of human TSH-R cDNA (1096-2468th base (9,lO)) and human TPO cDNA (2351-2928th base (11)) were confirmed by asymmetrical PCR method (12) using 32P-labeled primers. For detecting Tg mRNA, we used rat Tg cDNA as probe, which we cloned from cDNA libraly constructed from rat thyroid glands (13). As the control we detected p-actin mRNA in the tissues using chicken p-actin cDNA (2 kb Pst I/PST I fragment of pA1 (14)). For hybridization, these cDNA probes were radiolabeled with [a-32P]dCTP by multiprimed labeling system (Amersham, England). Northern-blot and slot-blot analyses: Total RNAs were extracted from the tissues with guanidine isothiocyanate followed by centrifugation through CsCl, and poly (A)+ RNA was further purified on oligo-(dT) cellulose chromatography gel analysis, mRNA (2 pg) was denatured (8) . For Northern by glyoxisal at 50°C, then elecrophoresed in 1% agarose. RNAs were transferred onto Zeta probe membrane (Bio-Rad, Richmond, CA). After prehybridization at 42'C in 50% formamide, 10 X Denhaldt's solution (1% Ficoll, 1% polyvinylpyrrorydone, 1% BSA), 5 X SSPE (20 X SSPE is 3M NaCl, 0.2M sodium phosphate, 2OmM EDTA, pH 7.4), 0.1% SDS, 0.1 mg/ml heat-denatured salmon sperm DNA and 1 pg/ml hybridization was performed at 42OC for 24 h with poly(A), radiolabeled cDNA probes in the same solution described above. The membrane was washed three times at room temparature in 2 X SSC (1 X SSC is 0.15M NaCl, 0.015M sodium citrate), 0.1% SDS, and then further washed three times at 56°C in 0.1 X SSC, 0.1% SDS, and then further washed three times at 56'C in 0.1 X SSC, 0.1% SDS. Air dried membrane was exposed to Kodak XAR-5 film with intensifying screen. 1149
Vol.
174,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
For slot-blot analysis, total RNAs were denatured with 7.5% formaldehyde in 6 X SSC at 60°C for 30 min (8), and then slot-blotted onto nitrocellulose filters each 10 pg/slot using a Manifold II slot-blot apparatus (Schleicher and Schell, Keene, NH). Hybridization and washing were done as described above. After autoradiography, the hybridization signals were quantified by scanning densitometer (DMU-33C DIGITAL DENSITOROL, ADVANTEC).
RESULTS First
of
all,
examine
the
study.
As shown
we performed
specificity in
and TPO hybridized thyroid Although
AND DISCUSSION of
Fig. only
tissue, several
Nothern
cDNA probes
I-A
poly
not with bands,
products
generated
during
the
visible,
the
of
largest
these
sizes
probes
are
respectively.
These from
the
of
TSH-R
and TPO,
In
the
of
sizes
4.9
were
thyroid cDNA libraly, not only with poly
(A)+
for human
of
TSH-R
RNA preparation,
nearly
the
Tg,
hybridization mRNA from
same as those
respectively
which
by
kb,
sequences
kb,
are
recognized
kb and 3.0
reported for
present
mRNA from
molecules
kb and 3.0
cDNA probe
the
that from FRTLS cells. probably the degradation
4.0
previously
in
to
cDNA probes
(A)+
course
approximately
expected
case
the
analysis
used
and l-C,
with
but did smaller
gel
was
cloned
signals were FRTLS cells at
of
the (9-11). from
obtained 8.4 kb
D
C
3.0D
2.0b
H
R
H
H
R
R
H
R
Fia. L. Agarose gel electrophoresis of poly (A)+ RNA samples from human thyroid tissues and FRTLS cells. Poly (A) + RNA was transferred to Zeta probe nylon filters and hybridized with 32P-labeled cDNAs for TSH-R (A) thyrogrobulin, (B) thyroid peroxidase, (C) and P-actin (D), followed by autoradiography. Fragment sizes (in kilobases) are indicated at the left. RNA lanes: H, human thyroid RNA; R,
FRTLS
RNA.
1150
cDNA rat in
Vol.
174,
No.
3, 1991
length,
but
length,
with
that
the
AND
from
respectively
almost kb,
BIOCHEMICAL
respectively
1-B).
rat
(15,
16).
1 shows
the
& 7.5)
expression
carcinoma In contrast, show
tissues levels
levels
of
of TSH-R, Tg, The expression
f 5.1)
significance the
we studied the
neoplastic expression
the
TSH-R
8.4
in
in
TPO
adenoma
comparison
with TSH-R,
between
expression
each
levels
investigated change as
of
histological these
mRNAs in
(data not shown), compared with normal
expression (Figs. 2-B
level of and 2-C). it
thyroid
carcinoma
reason,
the of
mRNA was
~~0.01).
Clinically,
TSH-R is
is
of been
adenoma carcinoma
(n=7) (rk6)
l
10.9
107.2+
14.3
61.4-t
Mean k SEM Values
of
TSH receptor But
the
For
indicate
l
thyroid
assessd
to
thyroid
identical
lOO.O-t5.3
100.0fll.2
82.7 zk 7.5 a
7.6 a ap~0.05
71.8k5.1
to
i34.0f bpcO.Ol
of normals,
1151
p-actin 100.0 fll.2
101.6kl8.2
b
vs. normal
% of the average
peroxidase
86.5 + 3.5 5.2
vs. normal respectively.
Tg the
of
that
been
TSH binding
of
p-actin growth
not.
has
as both
between
TPO or
whether
by TSH or
reported
that
no correlation
important
thyroglobulin
lOO.Ot-
(n=7)
expression
1. mRNA levels of TSH-receptor, thyroglobulin, thyroid peroxidase and p-actin in human thyroid tissues
TSH-receptor normal
the
with
mRNA and that
assay.
has
Table
very
was
influenced
expression cells
between
well-correlated
There
TSH binding
carcinoma
correlation
mRNA and that of Tg (Fig. 2-A) in As shown in Fig. 2-A, the tissues.
thyroid of TSH-R
mRNA (r-=0.74;
means
kb and
tissues.
Then, level
also
kb in
f 7.8 vs. normal 100.0 f 10.9). levels of TPO and p-actin mRNAs
type. Graves' disease (n=3) were but there was no significant thyroid
were
(100.0 t 5.3). In the case of mRNA was decreased only in
of
Additionally,
8.0
9.0
type. decreased
(71.8
(carcinoma 61.4 the expression
no statistical
at
The results suggest that our as probes for detecting human
and carcinoma
normal thyroid the expression
sizes
Tg cDNA,
and j3-actin mRNAs on histological level of Tg mRNA was significantly (82.7
COMMUNICATIONS
tissue
These
and human
cDNAs were able to be used TSH-R, TPO and Tg mRNAs. Table
RESEARCH
human thyroid
(Fig.
same with
BIOPHYSICAL
101.6 +I26
by (2,3)
Vol.
174,
No.
3, 1991
BIOCHEMICAL
A I
AND
BIOPHYSICAL
RESEARCH
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