Japanese Journal of Clinical Oncology 32:229-232 (2002)
© 2002 Foundation for Promotion of Cancer Research
Quantitative Analysis of Thymosin Beta-10 Messenger RNA in Thyroid Carcinomas
1 Department of Laboratory Medicine, Osaka University Medical School, Suita, Osaka and 2 Kuma Hospital, Kobe, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Genes that are differentially expressed in benign and malignant tissues are important for the establishment of molecular-based diagnosis of carcinomas. Our recent study on the gene expression profile of thyroid carcinomas revealed an increased expression of thymosin beta-10 mRNA.
Methods: To confirm this, we measured the expression levels of thymosin beta-10 mRNA in 84 thyroid benign and malignant thyroid tissues, including five anaplastic carcinomas, by means of real-time quantitative reverse transcription-polymerase chain reaction.
Results: We found an increased expression of thymosin beta-10 mRNA in thyroid carcinomas, especially in anaplastic carcinomas. Expression levels of thymosin beta-10 mRNA relative to thyroglobulin mRNA in anaplastic carcinomas were greatly increased compared with those in differentiated carcinomas.
Conclusion: These results suggest the usefulness of the quantitative measurement of thymosin beta-10 mRNA in molecular-based diagnosis of thyroid anaplastic carcinomas, but not of differentiated carcinomas.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Recent advances in molecular technology suggest the potential for more efficient and effective molecular-based diagnoses and therapies. In the thyroid, genes that are found to be differentially expressed in benign thyroid tissues and thyroid carcinomas can be directly used as targets for molecular-based diagnosis, i.e. aspiration biopsy reverse transcription-polymerase chain reaction (ABRP) (1–4). By relying on 14–15 base cDNA sequences called tags for gene identification, serial analysis of gene expression (SAGE) can generate a quantitative transcript profile easily (5). We previously described the use of SAGE in providing gene expression profiles in a normal thyroid tissue and thyroid tumors (6). By analyzing these profiles, we found the differential expression of thymosin beta-10 (TB10), an acidic polypeptide originally isolated from a calf thymus, in thyroid carcinomas. In a previous study, Califano et al. described the increased expression of this molecule in thyroid malignancies, especially in poorly differentiated carcinomas (7). It is, however, not clear to what extent thymosin beta-10 (TB10) mRNA is useful for clinical purposes, such as molecular-based diagnosis, because of the limited number of the benign samples they used in their study. Thus, in this study, using 84 benign and malignant thyroid tissues, including five anaplastic carcinomas, we measured the expression levels of TB10 mRNA by means of real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) in order to estimate the usefulness of TB10 mRNA in distinguishing malignant thyroid tumors from those that are benign (8).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Extraction of RNA from Thyroid Tissues
Tissue samples from thyroid tumors or normal thyroid tissues in the opposite lobe of carcinomas were obtained by surgery. All tissues were frozen in liquid nitrogen immediately after resection. Total RNA was extracted according to the method of Chomczynski and Sacchi (9).
SAGE Protocol
The SAGE method was performed as described previously (5). Three micrograms of poly A RNA from each tissue were used to synthesize double-stranded cDNA. Sequence files were analyzed by the SAGE software kindly provided by Dr Kenneth Kinzler (Johns Hopkins University, USA) and the tag sequences were analyzed by the BLAST program of the DNA Data Bank of Japan (Mishima, Shizuoka, Japan).
Real-time Quantitative RT-PCR
RNAs from 21 normal thyroid tissues, 27 papillary carcinomas, 19 follicular adenomas, 12 follicular carcinomas and five anaplastic carcinomas were subjected to real-time quantitative RT-PCR analysis. Reverse transcription was performed using 1 µg of total RNA in an RT mixture containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM dNTPs, 200 U M-MLV reverse transcriptase (Gibco, Gaithersburg, MD), 2 U/µl RNase inhibitor (Takara, Shiga, Japan) and 2.5 µM oligo dT (Gibco) in a total volume of 20 µl at 37°C for 60 min. Real-time quantitative PCR (TaqMan PCR) using an ABI PRISM 7700 Sequence Detection System and a TaqMan PCR Core Reagent Kit (PE Biosystems, Foster City, CA) was performed according to the manufacturer’s protocol. One microliter of the first strand cDNA was used in the following assay. The two primers and one TaqMan probe used for the quantification of TB10, ß-actin and thyroglobulin mRNAs were as follows (10–12):
[TB10F (0.5µM): 5'-AGAAGAACACCCTGCCGACC-3' (bases 134–153)],
[TB10R (0.5µM): 5'-CAAACCGGAGAATTTGGCAG-3' (bases 360–379)] and
[TB-TM (10pmol): 5'-FAM-AAGAGCCACCTGCAAGATGGACACGAGCCA-TAMRA-3' (bases 253–282)];
[ACF (0.5µM): 5'-TGGACATCCGCAAAGACCTG-3' (bases 901–920)],
[ACR (0.5µM): 5'-CCGATCCACACGGAGTACTT-3' (bases 1047–1066)] and
[AC-TM (10 pmol): 5'-FAM-CACCACCATGTACCCTGGCATTGCC-TAMRA-3' (bases 947–971)]; and
[TGF (0.5µM): 5'-GAGAAGAGCCTGTCGCTGAA-3' (bases 7980–7999)],
[TGR (0.5µM): 5'-CAGCTCACTGAACTCCTTGT-3' (bases 8128–8147)], and
[TG-TM (10 pmol): 5'-FAM-TGAGTTCTCACGGAAAGTACCCA-TAMRA-3' (bases 8054–8076)],
respectively. The conditions for the TaqMan PCR were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min. A recombinant pGEM T-vector (Promega, Tokyo, Japan) containing either TB10, ß-actin or thyroglobulin cDNA was constructed by PCR cloning with the same set of primers used in TaqMan PCR and were used as standard samples. Representative amplification blots of TB10 cDNA are shown in Fig. 1. The amplification plots of the PCR reaction were used to determine the threshold cycle (CT). The CT value represented the PCR cycle at which an increase in reporter fluorescence (
Rn) above the line of the optimal value (optimal Rn) was first detected. The initial copy number of the target mRNA was calculated by a plot of the CT against the input target quantity.
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Statistical Analysis
Statistical analysis of differences between the groups was carried out using the Mann–Whitney U-test. P values of <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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According to SAGE analysis, the tag count of TB10 mRNA was greatly increased in papillary, follicular and anaplastic carcinomas compared with a normal thyroid tissue and a follicular adenoma (Table 1). In SAGE analysis, the count of each tag sequence is considered to represent the expression level of the corresponding mRNA (5).
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By real-time quantitative PCR, the copy numbers of TB10, ß-actin and thyroglobulin cDNAs were measured (Table 2). No statistically significant difference among the groups was observed in the copy numbers of ß-actin cDNA which was used as an internal control. The relative expression levels of TB10 mRNAs to ß-actin were calculated. The relative expression level of TB10 mRNA was greatly increased in anaplastic carcinomas. It was also increased in papillary carcinomas and some of the follicular carcinomas, but there was no statistically significant difference between follicular adenomas and follicular carcinomas. When the cut-off line was set at 0.05, 1/21 (4.8%), 6/27 (22.2%), 0/18 (0.0%), 5/12 (41.6%) and 5/5 (100%) samples were above the cut-off level in normal thyroids, papillary carcinomas, follicular adenomas, follicular carcinomas and anaplastic carcinomas, respectively (Fig. 2).
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Further, the expression level of TB10 mRNA relative to thyroglobulin mRNA was measured. Owing to the greatly decreased expression levels of thyroglobulin mRNA in anaplastic carcinomas, they were greatly increased in five anaplastic carcinomas. Increased values were also observed in papillary carcinomas (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Thymosins are divided into three classes (
, ß, 
) based on their isoelectric point (13,14). The ß-class thymosins are structurally related, highly conserved acidic polypeptides. The precise biological function of this protein is still unknown; however, recent studies suggest that the expression of thymosins is associated with growth and differentiation of many cell types and it is believed that TB10 has some effect on the cytoskeleton because it binds to G-actin (15). Previous reports have indicated an enhanced TB10 mRNA expression in a variety of human tumors such as colon, breast and renal carcinomas, melanomas and ovarian germ-cell tumors and a correlation was found between TB10 mRNA levels and the progression steps of the carcinogenesis process (16). In particular, it seems that TB10 may play a role in the process of thyroid tumor development, since TB10 mRNA is highly over-expressed in human and experimental thyroid tumors and its expression is particularly abundant in undifferentiated thyroid carcinomas. In this study, a marked increase in the expression of TB10 mRNA was observed in five anaplastic carcinomas, whereas its increased expression was observed in only some of the differentiated carcinomas. The increased levels of TB10 mRNA expression in anaplastic carcinomas were even more evident when the expression levels of TB10 mRNA were compared with those of thyroglobulin mRNA. Thus, the increased expression of TB10 mRNA might be useful in establishing a molecular-based diagnosis of anaplastic carcinomas but it may not be so in diagnosing differentiated thyroid carcinomas.
Because a considerable number of differentiated thyroid carcinomas do not over-express TB10 mRNA, TB10 may not play an important role in the expression of cancerous characteristics such as invasion or metastasis in these cancer cells. The mechanisms that regulate TB10 mRNA expression have been studied in osteosarcoma and hematopoietic cells (17,18); however, the molecular basis of TB10 mRNA expression in thyroid tissues is poorly understood. A previous study demonstrated that in the thyroid cell, TB10 mRNA expression is regulated by thyroid stimulating hormone (TSH) and other mitogenic pathways such as the protein kinase C (PKC) and insulin pathways and that mRNA expression of TB10 is markedly enhanced in human goiters compared with normal thyroids (19). Taking these facts into consideration, we suggest that over-expression of TB10 mRNA may be closely related to the proliferation rate of thyroid tumor cells and that its expression levels may be an indicator of tumor growth.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This research was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Scientific Research on Priority Areas, 2001, No.13216068 and Scientific Research B, 2001–2, No. 13557227.
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FOOTNOTES |
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+ For reprints and all correspondence: Toru Takano, Department of Laboratory Medicine, Osaka University Medical School, D2, 2–2, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ttakano@labo.med.osaka-u.ac.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Received November 26, 2001; accepted April 17, 2002
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