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

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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

COMMUNICATIONS

P

The mRNA levels of thyrotropin receptor, thyroglobulin and thyroid peroxidase in neoplastic human thyroid tissues.

Expression levels of thyrotropin receptor (TSH-R), thyroglobulin (Tg) and thyroid peroxidase (TPO) mRNA in normal and neoplastic human thyroid tissues...
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