Abstract
Objective
The aim of this study was to explore the relationships between expression of the NIN1/RPN12 binding protein 1 homologue gene (NOB1) in papillary thyroid carcinoma tissue and clinicopathological variables.
Methods
Expression of NOB1 in papillary thyroid carcinoma, normal thyroid and benign thyroid tumour tissue was evaluated at the mRNA and protein levels by real-time fluorescence quantitative reverse transcription–polymerase chain reaction and immunohistochemistry, respectively. Relationships between immunohistochemical scores and several clinicopathological variables were also examined.
Results
Expression of NOB1 mRNA and protein in papillary thyroid carcinomatissue was significantly higher than in normal thyroid tissue and benign thyroid tissue, while there was no significant difference between normal thyroid tissue and benign thyroid tumour tissue. A high level of NOB1 protein expression was associated with large tumour size and Union for International Cancer Control stage, but was not significantly correlated with sex or age.
Conclusions
Compared with normal thyroid and benign thyroid tumour tissue, papillary thyroid carcinomas showed higher expression of NOB1, which indicates that the expression level of the NOB1 gene in the thyroid may play a key role in the occurrence and development of papillary thyroid carcinoma.
Introduction
Thyroid carcinoma is the most common malignancy of the endocrine system and accounts for ∼1% of all newly diagnosed cancers.1–4 Thyroid cancer can be divided into papillary, follicular, medullary and anaplastic histological types. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer and accounts for >80% of all thyroid malignancies.5,6 It is therefore important to understand the molecular mechanisms responsible for the development, progression and metastasis of PTC, and to develop novel strategies for its early detection, prevention and treatment.
NIN1/RPN12 binding protein 1 homologue (NOB1) is a subunit of the 26 S proteasome and plays a crucial role in protease function and RNA metabolism. 7 The human NOB1 gene is composed of nine exons and eight introns, and is located on chromosome 16q22.1. NOB1 protein, an evolutionarily conserved protein, comprises a PilT N terminus (PIN) domain and a C terminal zinc ribbon domain, and is expressed mainly in liver, lung and spleen. 8 Abnormal expression of NOB1 in leukaemia has been detected. 9 NOB1 is an important regulator of the tumourigenic properties of human hepatocellular carcinoma and could be used as a candidate therapeutic target in human hepatocellular carcinoma. 10 These results suggest that NOB1 may be involved in different tumours, although little information on the expression of NOB1 and its role in other tumours is available. To our knowledge, the correlation between expression of the NOB1 gene and the risk of PTC remains unclear. The aims of the present study were therefore to investigate the expression of NOB1 in PTC, and analyse its association with the occurrence and development of PTC.
Materials and methods
This study was approved by the Ethics Committee of Jilin Hospital. Written informed consent to participate was obtained from all participants included in the study.
Patients and samples
Fresh samples of thyroid tissue were obtained from 50 patients with primary papillary carcinoma, 40 patients with benign thyroid tumours (20 cases of thyroid adenoma and 20 cases of multinodular goitres), and 10 patients with a normal thyroid gland. All participants underwent surgery in the Department of Thyroid Surgery at The First Affiliated Hospital of Jilin University between September 2011 and July 2012. The patients with PTC ranged in age from 23 to 64 years (mean ± SD, 40.1 ± 0.5 years) and included 21 males (42%) and 29 females (58%); 30 patients were <45 years old and 20 patients were ≥45 years old. Diagnosis and staging of PTC was performed according to the Union for International Cancer Control (UICC) classification. 11 Stages I and II were found in 29 (58%) patients and stages III and IV in 21 (42%) patients. Surgical specimens were fixed with 10% neutral-buffered formalin and paraffin wax sections were stained with haematoxylin and eosin.
Quantitative RT–PCR
Total RNA was isolated from frozen thyroid tissue using Trizol® reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purity and concentration of RNA were determined using a dual-beam ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany). Using mRNA as template, single-stranded cDNAs were generated using SuperScript® II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. The NOB1 primer sequences were as follows: sense 5′-ATCTGCCCTACAAGCCTAAAC-3′; antisense 5′-TCCTCCTCCTCCTCCTCAC-3′. The quantitative polymerase chain reaction (PCR) conditions were as follows: 95℃ for 3 min followed by 40 cycles of 95℃ for 15 s and 55℃ for 1 min. A dissociation curve was established after each PCR to verify amplification specificity. Data were analysed using ABI Prism® 7900 Sequence Detection System software (Applied Biosystems, Foster City, CA, USA).
Immunohistochemistry
To detect the expression and localization of NOB1 protein in thyroid tissue, immunohistochemistry was performed using an SP reagent kit (Tiangen, Beijing, China) according to the manufacturer’s instructions.
The degree of immunoreactivity was assessed as described by Campo et al. 12 Immunoreactivity was measured semiquantitatively using a scale from −to +++, where −represents absolutely no immunostaining, + represents <25% reactive cells, ++ represents 25–50% of reactive cells, +++ represents >50% of reactive cells. Values of − and + were considered to indicate negative staining; values of ++ and +++ were considered to indicate positive staining. Five cases with discordant results were re-evaluated to obtain agreement.
Statistical analyses
Data were expressed as mean ± SD. Student’s t-test was used to evaluate the significance of differences in mRNA and protein level expression of the NOB1 gene in different groups. The relationships of NOB1 protein expression with clinical and pathological variables, including sex, mean age, UICC stage and lymph node metastasis in PTC, were examined using Pearson’s χ2-test. Statistical analyses were performed with SPSS® software, version 13.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was accepted at a P-value of < 0.05.
Results
Quantitative PCR showed that NOB1 mRNA was significantly (P < 0.05) overexpressed in PTC (45 678.23 ± 2884.25 copies/µg RNA) compared with thyroid adenoma tissue (24 651.89 ± 13 758.72 copies/µg RNA), normal thyroid tissue (23 425.67 ± 15 789.64 copies/µg RNA) and multinodular goitre tissue (22 379.39 ± 14 331.67 copies/µg RNA). There were no significant differences among the thyroid adenoma, normal thyroid and multinodular goitre samples.
Immunohistochemistry for NOB1 protein expression revealed little immunostaining in thyroid tumour cells (and stromal fibroblasts of thyroid adenoma and multinodular goitre) in any of the cases examined, and no staining was found in normal control samples (Figure 1). NOB1 was immunolocalized mainly to the carcinoma cells; its expression was significantly (P < 0.05) upregulated in the thyroid carcinoma group (45 positive out of 50 samples, 90%) compared with thyroid adenoma (five of 20, 25%), multinodular goitre (three of 20, 15%) and normal tissue (none of 10, 0%) groups. There were significant associations between NOB1 protein expression and UICC stage, tumour size and lymph node metastasis (P < 0.05, Table 1). However, NOB1 protein expression was not significantly associated with sex or age.
Immunohistochemical staining showing expression of NOB1 protein in (a) normal thyroid tissue, (b) thyroid adenoma, (c) multinodular goitre and (d) thyroid carcinoma tissue. Associations between NOB1 protein expression and clinicopathological characteristics of papillary thyroid cancer (PTC). 2-test was used for statistical analysis. UICC, Union for International Cancer Control;
11
NS, not significant.
Discussion
The present study demonstrated that the expression level of NOB1 protein was higher in PTC than in normal thyroid tissue and benign thyroid tumour tissue. Enhanced production of NOB1 protein has been reported in many human carcinomas,13,14 which is consistent with our finding that the expression of NOB1 protein was increased in thyroid carcinomas. Several recent studies have reported that repression of the NOB1 gene inhibits the growth of hepatocellular carcinoma 10 and ovarian cancer. 15 From these findings, we infer that NOB1 plays a key role in PTC invasion and metastasis.
In conclusion, NOB1 protein and mRNA expression were significantly upregulated in thyroid carcinoma tissue, in comparison with thyroid adenoma and normal thyroid tissues and multinodular goitre tissue. These results imply that NOB1 plays a key role in tumour invasion and metastasis; they also suggest that increased NOB1 expression might be a useful diagnostic marker and might also become a potential target in the treatment of thyroid carcinoma.
Footnotes
Declaration of conflicting interest
The authors had no conflicts of interest to declare in relation to this article.
Funding
The authors gratefully acknowledge the financial support provided by the Health Bureau of Jilin (2008Z017).