Original paper
Increased expression of mRNA specific for c-Met oncogene in human papillary thyroid carcinoma

Introduction
Papillary thyroid carcinoma (PTC) is a differentiated type of thyroid cancer and the most common type of carcinoma of this gland in the Polish population. PTC originates from the thyroid follicular epithelial cell [1]. The molecular pathogenesis of PTC is still poorly understood [2]. Among various genetic factors involved in the pathogenesis of PTC, chromosomal rearrangements or translocations of RET and NTRK1 oncogenes appear to be of crucial importance [3, 4]. It is known that BRAF oncogene somatic mutation (V599E) is the most common genetic alteration in PTC and seems to be an alternative event to RET/PTC rearrangements or RAS mutations in the development of PTC. Moreover, no overlap was found between the changes in question of those genes [5, 6]. The process of constitutive activation of the RAS-RAF-MEK-MAP kinase pathway transmits a mitogenic signal to the nucleus and promotes uncontrolled cell division, very frequently found in human carcinomas. There are also several other mechanisms which may play role in the carcinogenic process in the thyroid. Increased expression of mRNA specific for the enzymes involved in pyrimidine and purine metabolism, e.g. thymidine kinase 1 (TK1), deoxycytidine kinase and thymidine phosphorylase (dCK), has been estimated in PTC [7]. In the molecular background of human PTC, the unregulated activation of intracellular tyrosine kinase (TK) seems to be of crucial value. The c-Met protooncogene, located in chromosome 7q31-34, encodes the Met protein, a TK receptor with high affinity for the hepatocyte growth factor (HGF) [8, 9]. The Met protein has motogenic, mitogenic and morphogenic properties and has been implicated in the invasion process of malignant cells in PTC [10]. Overexpression of the c-Met oncogene has been found in many carcinomas, such as: colorectal, ovarian and non-small-cell lung carcinoma [11]. A lot of studies performed using immunohistochemistry or Western blot analysis have reported increased levels of Met protein in PTC as compared with macroscopically unchanged thyroid tissue [12, 13], but only a few studies have shown high levels of mRNA specific for c-Met in PTC [14]. Activating mutations of the c-Met oncogene are also present in several types of cancer, particularly in human papillary renal carcinoma (in both its hereditary and sporadic forms) [15]. Recently, missense c-Met mutation T1010I in exon 14 was found in differentiated thyroid carcinoma (both papillary and follicular), but its molecular pathological role is not yet fully understood [16]. The aim of the study was to estimate the expression of mRNA specific for the c-Met oncogene in the tissue of human PTC, and to evaluate the possible correlation between the level of c-Met oncogene expression and such parameters as: patient’s age and gender, histopathological variants of the tumour and the assignment of patients to particular stages in the clinical staging system [17].
Material and methods
The procedures used in the study were approved by the Ethical Committee of the Medical University of Lodz, Poland. Thyroid tissue samples (150 mg) were obtained from patients who had been submitted to surgery (total thyroidectomy) for PTC, at the Department of Oncological Surgery, Centre of Oncology, the Maria Sklodowska-Curie Memorial Institute, Gliwice, Poland, and at the Division of Surgery, the Holy Family Municipal Hospital, Lodz, Poland. Immediately after the collection, thyroid tissue was frozen at -70°C. Papillary thyroid carcinoma (18 cases: 11 females, 7 males; the mean age of all studied patients was 52.9±19.4 years) was cytologically diagnosed before the surgery on the basis of fine needle aspiration biopsy (FNAB). Histopathological confirmation, according to WHO classification, was obtained from pathological reports (PTC types were as follows: PTC follicular type – 8 cases, PTC classic type – 10 cases). Oncogene c-Met mRNA expression was measured in 18 pairs of PTC samples and the corresponding macroscopically unchanged thyroid tissue (which served as controls). Clinical and histopathological characteristics of the studied patients are summarized in Table I.
RNA isolation
Total RNA was extracted from thyroid tissue (150 mg) in guanidinium isothiocynate, by means of a commercially available kit [Total RNA Prep Plus (A&A BIOTECHNOLOGY, Gdynia, Poland)]. Tissue samples were stored at -70°C until analysis. RNA concentrations and purity in the final preparations were spectrophotometrically quantified by measuring absorbance at 260 and 280 nm (Ultraspec 2000 UV/Visible Specrophotometer Pharmacia Biotech, Sweden).
Reverse transcription-polymerase chain reaction (RT-PCR)
Reverse transcription was performed by random priming in a reaction mixture (20 µL) containing: 1000 ng of total RNA, 0.3 µL of Random Primer Oligonucleotides (3 µg/µL (Gibco BRL Co.), 2 µL of 0.1 M DTT (Dithiothreitol) (Gibco), 4 µL of 5 × First-Standard Buffer (250 mM Tris-HCl, pH 8.3; 375 mM of KCl; 15 mM of MgCl2) (Gibco), 1 µL of Reverse Transcriptase M-MLV (Moloney Murine Leukemia Virus Reverse Transcriptase) (200 U/µL) (Gibco), 2 µL of dNTP (2500 µM) (Gibco) and 1.0 µL of Inhibitor RNAse (10 U/µL) (Gibco). Reverse transcription was performed in a TRIO-Thermoblock thermocycler (Biometra, Göttingen, Germany) at 37°C for 60 min. The following negative controls for RT were performed: RT reaction without any addition of RNA as the control of RT reagent contamination, and RT reaction without any addition of Reverse Transcriptase enzyme as the contamination control of genomic DNA. The amplification was carried out in a Mastercycler personal (Eppendorf, Hamburg, Germany) in a total volume of 25 µL, containing: 1000 ng of cDNA (RT product), 2.5 µL of 10 × PCR buffer (1 × is 10 mM of Tris-HCL, pH 8.8: 50 mM of KCl, 1.5 mM of MgCl2, 0.1% Triton X-100) (Finnzymes Oy), 2.5 µL of dNTP (250 µM of each) (Gibco), 1 µL of Taq polymerase (DyNAzyme II DNA polymerase) (2U/µL) (Finnzymes Oy), and 25 pmol of each, i.e. 3’ and 5’ PCR primer. The sequences of the synthetic oligonuclotide primers (TIB MOLBIOL, Gdynia, Poland) used for PCR amplification, the reaction conditions under which the amplifications were carried out, and the size of amplicons are presented in Table II. The following negative controls for PCR were performed: a reaction with RT reaction product without any addition of RNA as the control of PCR reagent contamination, and a PCR reaction with RT reaction product without any addition of Reverse Transcriptase enzyme as the control for genomic DNA contamination. The amplification of the control housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as an internal standard. The identity of amplification products with the presumed sequences was proven by digestion with restriction enzyme Hae III (Eurogentec, Brussels, Belgium) to obtain 362 bp and 108 bp fragments using manufacturer’s buffers and protocol.
Analysis of RT-PCR products
RT-PCR products were identified using 8% polyacrylamide (PAA) Tris-borate-ethylenediamine tetraacetate (TBE) gels and visualized by ethidium bromide staining (0.5 mg/mL). Densitometric analyses were conducted using the Scan Pack 3.0 system (Biometra Göttingen, Germany). Results are expressed in ng units and are presented as the ratios of c-Met (pg) expression to GAPDH gene (ng) expression.
Statistical analysis
The data were statistically analyzed using t-test for average values in independent groups, followed by U Mann-Whitney test. Statistical significance was determined at the level of p<0.05. The results are presented as mean ± SEM and ± SD values. Pearson’s test, c2 test and U Mann-Whitney test were performed in order to correlate the level of expression of the c-Met gene in patients with PTC with clinicopathological parameters (age, gender, PTC histopathological variant and assignment to a particular stage in the clinical staging system [17]). For calculations, Statistica (StatSoft, Poland) for Windows 7.0 programme was applied.
Results
Significant differences in c-Met mRNA expression were observed between PTC and macroscopically unchanged thyroid tissue (t-test, p<0.0003). The mean relative expression for c-Met was significantly, three-fold higher in human PTC (Figure 1). Moreover, in the group of 18 pairs – PTC and the corresponding macroscopically unchanged thyroid tissues obtained from the same patient, in most cases expression of the c-Met gene was higher in PTC than in the control tissue (Figure 2). Interestingly enough, in five specimens of macroscopically unchanged thyroid tissue, there was no expression of mRNA specific for the c-Met gene. In two pairs, PTC and macroscopically unchanged thyroid tissue, the level of expression of c-Met mRNA was higher in controls than in PTC samples. In our study, no significant correlation was found between c-Met expression and such characteristics as patient’s age at diagnosis (Pearson test, p=0.126) and patient’s sex (c2 test, p=0.493). We compared expression levels of the c-Met oncogene in different types of PTC (follicular type – 8 cases, and classic type – 10 cases). Interestingly enough, no statistically significant differences were found among histopathological variants of the tumour (t-test, p=0.901; U Mann-Whitney test p=0.789).
Discussion
In 1992, Di Renzo et al. reported that Met protein was strongly overexpressed in PTC. The overexpression of the protein was not associated with either amplification or rearangements of the c-Met oncogene and the Met protein did not show structural alterations [12]. The development of immunohistochemical techniques and reagents active on paraffin-embedded material allowed a wide spectrum of immunohistochemical investigations of thyroid carcinomas. In normal thyrocytes, Met protein is either not expressed or expressed at a very low level. In contrast, high Met expression is a frequent abnormality in PTC. It was found that about 90% of PTC cases, including all histological types, over- expressed Met protein [18]. However, recently, Nardone et al. observed higher expression of Met protein in tall cell variant of PTC, which may explain the invasive behaviour of this histological type of PTC [19]. In the present study, we evaluated c-Met oncogene expression in PTC and macroscopically unchanged thyroid tissue by means of the RT-PCR method. We found no mRNA expression specific for the c-Met gene in five specimens of macroscopically unchanged thyroid tissue, while in most studied cases the expression of c-Met was higher in PTC than controls (macroscopically unchanged thyroid tissue). Our results showing the presence of c-Met RNA in normal thyrocytes are in concordance with the results obtained by other authors [20]. Fluge at al. also observed c-Met oncogene overexpression in some cases of PTC by the hybridization method, using complex cDNA probes specific to c-Met RT-PCR products [20]. Recently, the gene expression profile of PTC has been studied using oligonucleotide microarrays method [14, 21]. Among the overexpressed genes, c-Met oncogene has already been recognized to be involved in PTC. Surprisingly, Wasenius et al. documented a significant difference between the methods (RT-PCR vs. cDNA expression array), assessing expression of the c-Met oncogene. In cDNA array analysis, half of the studied PTC cases showed increased c-Met oncogene expression, whereas in RT-PCR analysis as many as 90% of PTC samples showed about six-fold higher c-Met expression than unchanged thyroid tissue [14]. The overexpression of c-Met oncogene may be secondary to the changes observed in other genes involved in the development of PTC. Ivan et al. have shown that introduction of activated RAS and RET oncogenes in thyrocytes results in overexpression of Met protein [22]. This hypothesis seems to be interesting because it suggests the possibility of a relationship between Met protein expression and activation of the RAS or RET signalling pathway (through MAP kinase) in PTC. In order to test the hypothesis that c-Met expression is induced by the activation of other genes known to play a major role in the MAP kinase signalling pathway, we decided to search for RAS, RET and BRAF mutations in the examined series of PTC at our laboratory. We proved that two cases of PTC had contained an activating RAS oncogene mutation. The first mutation was found at codon 61 of N-RAS, the second at codon 31 of K-RAS [23]. Recently, we have found 6 out of 18 cases of rearrangements in RET and NTRK1 oncogenes (rearrangements in NTRK1 gene – 2 cases, and rearrangements in RET gene – 4 cases) [3]. Accidentally, in the case of one patient with a mutation at codon 61 of the N-RAS gene, we detected V600E mutation in gene BRAF [24]. As mentioned before, it is suggested in many studies that BRAF and RAS mutations do not overlap in PTC. Recently it has been shown that RAS mutation does not have any additional effect on the proliferation of thyroid follicular cells and the mutation in the BRAF oncogene is a dominant factor in activating the MAPK signalling pathway [25]. Considering the role of the c-Met gene in PTC, our results may suggest that the overexpression of c-Met is secondary to mutations of RAS or RET oncogenes. In turn, Wasenius et al. [16] have recently reported a change of the sequence in the c-Met oncogene. It is missense mutation T1010I, which replaces an isoleucine residue for a threonine residue in the Met receptor tyrosine kinase. This change has been found in papillary, follicular and medullary thyroid carcinomas, and it is the first report concerning c-Met oncogene sequence changes in the thyroid [16]. Missense c-Met mutations have recently been discovered in papillary renal carcinoma, gastric carcinoma, glioma, childhood hepatocellular carcinoma and small cell lung carcinoma [11]. Although the mechanism of increased expression of the c-Met oncogene in human PTC is probably complex, the one associated with either somatic or inherited mutations seems to play an essential pathological role.
Conclusions
To sum up, increased expression of mRNA specific for c-Met is involved in neoplastic processes in human thyroid, namely in the pathogenesis of PTC.
Acknowledgements
This study was supported by Project No. 3PO5B 009 22 from the State Committee for Scientific Research of Poland.
References
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