|Year : 2014 | Volume
| Issue : 2 | Page : 312-316
Detection of survivin 2α gene expression in thyroid nodules
Keyhaneh Kyani1, Esmaeil Babaei2, Mohammad Ali Hosseinpour Feizi2, Somayeh Vandghanooni3, Vahid Montazeri4, Monireh Halimi4
1 Department of Genetics, School of Medicine, Islamic Azad University, Najafabad Branch, Isfahan, Iran
2 Department of Animal Biology, School of Natural Sciences, University of Tabriz, Tabriz, Iran
3 Department of Nanobiomedicine, Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
4 Department of Pathology, Imam Khomeini Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
|Date of Web Publication||14-Jul-2014|
Mohammad Ali Hosseinpour Feizi
Department of Animal Biology, School of Natural Sciences, University of Tabriz, P.O. Box 51666-16471, Tabriz
Source of Support: None, Conflict of Interest: None
Context: Functional studies of the survivin splice variants have been performed almost exclusively in various types of cancer and produced remarkable advances in our understanding of cancer biology and cancer genetics.
Aim: To observation the expression of survivin 2α in thyroid nodules and estimate its potential as a new molecular marker in thyroid nodules screening and malignant thyroid, as well.
Setting and Design: We detected the expression of a splice variant of survivin, survivin 2α, in thyroid nodules.
Materials and Methods: Expression of survivin 2α mRNA was evaluated with specific primers by Hemi-Nested RT-PCR in 77 thyroid nodules including malignant and benign tumors, non-tumoral (goiter and thyroiditis) as well as surgical margin, non-neoplastic normal tissues adjacent to the malignant lesions.
Result: Our data revealed for the first time the expression of survivin 2α in thyroid nodules. It was detected in 85.7% of non-neoplastic surgical margin tissues, 71.4% of non tumoral, 63.2% of tumoral samples. Also, the expression of survivin 2α in benign tumor samples (64.2%) is more than malignant groups (62.8%).
Conclusion: Survivin 2α expression is the highest in non-neoplastic surgical margin rather than other samples and the lowest expression was that of malignancy. According to the results, it can be concluded that survivin 2α protein may be has a vital protective effect throw survivin quenching due to the high expression in normal tissue compared with lesions.
材料和方法：采用半巢式RT-PCR引物对2αSurvivin mRNA的表达进行了评估，分别取自 77个甲状腺结节，包括良性和恶性肿瘤，非肿瘤（甲状腺肿和甲状腺炎）以及手术切缘，非肿瘤旁正常组织的恶性病变。检测survivin剪接变异体及2αSurvivin基因在甲状腺结节中的表达。
Keywords: Hemi-Nested RT-PCR, splice variant, survivin 2α, thyroid cancer
|How to cite this article:|
Kyani K, Babaei E, Feizi MA, Vandghanooni S, Montazeri V, Halimi M. Detection of survivin 2α gene expression in thyroid nodules. J Can Res Ther 2014;10:312-6
| > Introduction|| |
Thyroid cancer is the most common endocrine disease, with worldwide incidence rates generally lower than 3 per 100,000 for men and 5 per 100,000 for women. It has four main histological types: papillary, follicular, medullary and anaplastic. ,,, Thyroid cancers are more obvious among other tumors because many of the tumor-initiating genetic proceedings; such as apoptosis have been identified. In the cell cycle, apoptosis has a main role in tissue homeostasis and any changes including cell signaling or genetic mutations cause changes in apoptosis protein homeostasis that leading to cell division or death. This relief valve occur through an evolutionary conserved cellular program that eliminates infected or unnecessary cells and cells produced in excess or having genetic damage.  It is clear that some oncogenic mutation disrupt apoptosis, leading to tumor initiation progression or metastasis.  Therefore, inhibitors of apoptosis can be involved in tumorigenesis and cancer progression.  Apparently, effective treatment and diagnosis of cancer is dependent on an understanding of this important process.  Survivin, a unique member of the inhibitor apoptosis proteins (IAPs) family is highly expressed in cancers but is undetectable in non-proliferating normal adult tissues suggesting a potential role in tumorogenesis. , A general characteristic that is present in all IAPs including survivin in the presence of a BIR (Baculovirus IAP Repeat) in one to three copies. BIR domains belong to the zinc-finger domain family and typically have a number of invariant amino acid residues, including one conserved histidine and 3 conserved cysteines, which coordinate a zinc ion. They are typically composed of 4-5 alpha helices and a three-stranded beta sheet. The BIR unique structure is found in nearly all proteins with anti apoptosis properties.  Survivin is described as a bifunctional protein inhibiting apoptosis and regulating mitosis. However, the biological functions and contributions to cancer progression of survivin splice variants are controversially discussed. , Transcription from the survivin gene locus yields at least 5 alternative splice variants that, in addition to survivin, have been designated survivin 2B, survivin-ΔEx3, survivin 3B, and survivin 2α.  Survivin 2α is the newly identified isoforms of survivin, structurally truncated BIR domain, consists of 2 exons: Exon 1 and exon 2, as well as a 3΄ 197bp region of intron 2  [Figure 1]. Although highly expressed survivin has been detected in nearly all type of human cancers and clinical outcome have frequently shown that survivin over-expression is associated with neoplasia and metastasis, but in the some studies there have been potent theories that prove survivin's 2B and 2α, spliced variants of survivin, have antagonist effects on the survivin function. ,, In any case, all of these data offers they might be used as the most important diagnostic markers for many types of cancers. Recently, expression and evaluating the potential of the other isoforms of survivin as a diagnostic tumor marker, has been studied and attracted many attentions in study cancer. In this context, our recent researches showed a high expression level of survivin and survivin-ΔEx3 in malignant thyroid tumors compared with benign and non-cancerous nodules ones. 
Till now, there has not any other study in the literature investigating been seen about the expression of survivin 2α splice variant in thyroid nodules. However, the aim of this study is detection and evaluating the diagnostic value of survivin 2α gene expression in 77 thyroid nodules including malignant tumors, benign tumors, non-tumoral (goiter and thyroiditis) as well as non-neoplastic normal tissues by Hemi-Nested RT-PCR.
|Figure 1: Schematic representation of exon organization in the survivin splice variants. BIR' = Truncated BIR; UTR = Untranslated region. Perpendicular arrows indicated the site of the stop codon|
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| > Materials and methods|| |
The research conducted in accordance with the Helsinki Declaration and was approved by the ethics committee of hospital. All the specimens were drawn in surgical operation from hospital, after obtaining an informed consent from each patient. The fresh surgically resected samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction.
The histopathological studies were carried out according to the World Health Organization  and tissue samples were organized as malignant tumors, benign tumors, non-tumoral nodules including goiter and thyroiditis and at last non-neoplastic surgical margin tissues for achieving normal tissues from each patient.
For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was extracted from tissues using RNeasy Micro kit (Qiagen, Hilden, Germany) exactly according to the protocol recommended by the manufacturer, the quality and quantity of RNA were assessed respectively by 1% agarose gel electrophoresis (preservation of 18S and 28S rRNA bands) and UV spectrophotometer at the absorbance 260/280, and all samples showed absorbency ratios between 1.8 and 2.0.
Hemi nested RT-PCR reaction
Complementary DNA (cDNA) was synthesized with 5μg of total RNA, 0.5μg Oligo-dT primers and RevertAid TM M-Mulv Reverse transcriptase (Fermentase Co. Lithuania) in a final 20μL reaction volume.
After that, to amplify the cDNA of survivin 2α splice variant (accession number AY 927772) two rounds of PCR reaction were performed. First round of PCR was performed in a final volume of 50 μl on Techne thermal cycler (Bibby Scientific, UK) using survivin 2α specific primers consist of:
HFPN1: 5΄-TGGCAGCCCTTTCTCAAG-3΄ and
PCR conditions included a pre-denaturation 2 min at 94°C, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing for 30 sec at 57°C, extension for 1 min at 72°C and a final extension at 72°C for 5 min. Second round of PCR was performed similar to the one described for the first round except for using 25 cycles of reactions as well as an internal for forward primer (Hemi-Nested RT-PCR):
HFPN2: 5΄-ACCACCGCATCTCTACATTC-3 (as an internal forward primer)
These primers amplified 266 bp segments for survivin 2α. The primer sequences were used for the amplification of β2 m mRNA as an internal control (accession number: NM_004048)
HBF 5΄-CTACTCTCTCTTTCTGGCCTG-3΄ (as a forward primer) and HBR 5΄-GACAAGTCTGAATGCTCCAC-3΄ (as a reverse primer), the reaction was done with identical conditions of first round of PCR of survivin 2α and 30 cycles. These primers amplified a 191 bp segment from β2 m cDNA.
Afterward, PCR products were checked by 1.5% agarose gels containing ethidium bromide and visualized under UV transillumination (UVP, USA). However, to avoid any possible inaccuracy during investigation, the whole process was repeated three times. Also, to ensure data accuracy derived from UV transillumination during photography of the complementary DNA containing gel, the light output, the color and contrast settings in software were considered identical and all photography were repeated 3 times, finally the specimens with clear appropriate DNA bonds considered as positive expression; and negative expression for those with no appropriate DNA bonds, as well.
Sequencing of PCR product
The specificity of survivin 2α PCR amplification was verified by sequencing. Briefly, products were excited from gels and isolated by gel extraction kit (Qiagen, Hilden, Germany). The purified PCR products were sequenced with the respective forward and reverse primers used in the PCR and analyzed on a DNA sequencer (Model 3100 sequencer; ABI) according to the manufacturer's instructions and matched with the GenBank, NCBI, which was found to be identical (AY 927772).
| > Results|| |
Following the RT-PCR and conventional gel electrophoresis analysis, expression of survivin 2α in tumoral, non tumoral and non-neoplastic samples adjacent cancerous lesions (77 samples) was detected. [Figure 2] showed that upper bands are survivin 2α (266 bp) and the lower bands are a segment of β2 m (191 bp) which was used as an internal control gene.
|Figure 2: RT-PCR analysis of survivin 2α expression in malignant, benign, non-tumoral and non-cancerous samples obtained from margin of carcinoma. Lane 1 corresponds to the DNA marker. N.C = negative control (distilled water used instead of cDNA in PCR reaction); MT = Malignant tumor; BT = Benign tumor; NT = non tumoral samples; M = samples obtained from margin of carcinoma|
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Our data revealed for the first time the expression of survivin 2α in thyroid nodules, also it was demonstrated that its expression increased from tumoral samples to adjacent non-neoplastic tissues. It was detected in 85.7% of adjacent non-neoplastic tissues, 71.4% of non tumoral, 63.2% of tumoral samples with meaningful decreasing rate [Figure 3]. In addition the expression of survivin 2α in benign tumor samples (64.2%) is more than malignant groups (62.8%), statistically.
|Figure 3: Comparison of survivin 2α expression in tumoral, non-tumoral and non-neoplastic samples|
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Also, in this investigation according to our data (not shown) we found that expression of survivin 2α in thyroid nodules were nearly 3 times more in women compared to men.
| > Discussion|| |
Survivin has attracted much attention in cancer researches. Although, most studies have focused on its expression, until now less information are available about the expression of survivin splice variants in the all types of cancers. Therefore, the main aim of this study was the detection of survivin 2α gene expression, a novel splice variant, in thyroid nodules by hemi-nested reverse transcription polymerase chain reaction.
For the first time, survivin 2α was characterized by Caldas et al., in several malignant cell lines and primary tumor. It was proved that 2α interacts physically with survivin and play important function as a natural antagonist of survivin anti-apoptosis activities, but it has some unknown functions. , Survivin 2α expression was detected in several human carcinomas including colon, gastric, prostate and colorectal. Additionally in the several previous researches it was reported that survivin 2α was expressed in normal and para cancerous tissue. ,,,,
In accordance to previous observation, our data showed that survivin 2α was expressed in both normal and tumoral tissues. In addition, we found that this splice variant was expressed in adjacent non-neoplastic tissues more than non tumoral and tumoral samples. Nevertheless, Zhengjiang et al. and Antonacopoulou et al. reported equal expression of 2α in both normal and neoplastic gastric and colorectal tissues respectively. , Functional assays have shown that survivin 2α can attenuate the anti-apoptotic activity of survivin.  It leads to hypothesis that survivin 2α may be has a vital protective effect against cancer throw survivin quenching due to the absence of the BIR as an important functionally domain related to the anti-apoptosis role of survivin.
Due to the lack of enough studies on the subject of survivin 2α expression, the function and the clinical importance of this splice variant remain unclear; therefore further studies are required to identify the potential role and mechanism of this isoform in the development of thyroid cancer.
To the best of our knowledge this is the first report of survivin 2α in human thyroid nodules. An important message yielded from this study is that although the expression of survivin splice variants is used to evaluate prognosis in different cancers, as expected we were found no significant equation in the expression of survivin 2α between none of the groups as an indicator of malignancy. Clearly, in order to draw a better perspective for future studies, other methodology such as Western blot analysis, microarray expression analysis, quantitative RT-PCR and immunohistochemistry are necessary and offered.
| > Conclusion|| |
We detected the survivin 2α gene expression in thyroid nodules. According to the our results anti apoptosis properties of BIR domain can be re-approve due to the its absence in survivin 2α, as quencher of anti-apoptosis properties of survivin. Although in our study, the expression of survivin 2α in tumoral cells was lower than non-neoplastic samples adjacent cancerous lesions and non-tumoral lesions, but it seems maybe the different expression of survivin 2α in these groups cannot be an appropriate criterion in distinguishing of thyroid tumors from non-tumoral lesions. However, further confirmatory and quantitative examination is required to confirm our data.
| > Acknowledgment|| |
Authors would like to thanks the University of Tabriz for financial and technical support of this research project.
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[Figure 1], [Figure 2], [Figure 3]