Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 29  |  Issue : 4  |  Page : 912--920

Study of survivin gene expression in hepatocellular carcinoma patients


Hala El-Said1, Tarek Abd El-Hakim2, Manar Obada1, Maather El-Shafey2, Osama Hegazy3, Sabry M Abd El-Mageed1,  
1 Department of Clinical Biochemistry, National Liver Institute, Menoufia, Egypt
2 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Hepatobiliary Surgery, National Liver Institute, Menoufia, Egypt

Correspondence Address:
Sabry M Abd El-Mageed
Department of Clinical Biochemistry, National Liver Institute, Menoufia University, Yassin Abdel-Ghaffar St., Shebin El-kom, Menoufia, 32511
Egypt

Abstract

Objective The aim of this study was to assess the differential expression of survivin gene in tumorous versus nontumorous liver tissue of hepatocellular carcinoma (HCC) patients and determine the possible influence of survivin gene polymorphism −31G/C and +9809T/C on its expression and its association with the risk for HCC in Egyptian patients. Background Mechanisms of hepatic carcinogenesis are not completely understood, but HCC development and progression could be attributed to accumulated genetic alterations as other malignancies. Survivin has functional involvement in apoptosis and proliferation, and is upregulated in malignancy. It attracts considerable interest as a potential diagnostic, prognostic tumor marker and a new target for cancer treatment. Patients and methods This study was performed on 30 HCC patients and 30 unrelated apparently healthy individuals who served as a control group. Genotyping of survivin gene at −31G/C and +9809T/C was performed for HCC cases and controls using TaqMan allelic discrimination Assay. Real-time PCR was performed for survivin mRNA in paired tumorous and nontumorous liver tissue specimens of the HCC patients. Results No significant difference was found between HCC patients and controls as regards different genotypes and alleles of survivin −31G/C and +9809T/C polymorphism. Moreover, none of these genotypes and alleles was associated with risk for HCC. Survivin mRNA was expressed in all tumorous and nontumorous tissue specimens in HCC cases and its expression was significantly higher in tumorous tissues compared with nontumorous tissues by 13 folds. Survivin expression in tumorous tissue was significantly increased with high α-fetoprotein, larger size of the tumor, vascular invasion, absence of cirrhosis, recurrence within 1 year, and low survival rate. The advanced stage of the tumor was the independent variable for survivin expression (P < 0.05). Conclusion Genetic variations of survivin gene had no effect on the susceptibility to HCC in Egyptian patients. Survivin was significantly overexpressed in HCC cases with advanced tumor stage.



How to cite this article:
El-Said H, El-Hakim TA, Obada M, El-Shafey M, Hegazy O, Abd El-Mageed SM. Study of survivin gene expression in hepatocellular carcinoma patients.Menoufia Med J 2016;29:912-920


How to cite this URL:
El-Said H, El-Hakim TA, Obada M, El-Shafey M, Hegazy O, Abd El-Mageed SM. Study of survivin gene expression in hepatocellular carcinoma patients. Menoufia Med J [serial online] 2016 [cited 2020 Mar 29 ];29:912-920
Available from: http://www.mmj.eg.net/text.asp?2016/29/4/912/202510


Full Text

 Introduction



Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and represents the fifth most malignant tumor worldwide and the third most common cause of cancer mortality [1]. In Egypt, the incidence rate of HCC has increased sharply in the last decade [2]. The understanding of the molecular pathways leading to the development of HCC may provide important data to develop new therapies. Similar to other tumor types, an imbalance between unrestrained cell proliferation and impaired apoptosis appear to be a major unfavorable feature of HCC [3]. Some studies have documented the overexpression of antiapoptotic factors such as inhibitors of apoptosis proteins in a variety of solid tumors and cancer cell lines [4].

Survivin, a member of the inhibitor of apoptosis family of proteins, regulates two essential processes. It inhibits apoptosis and promotes cell proliferation [5]. The human survivin gene (BIRC5) consists of four exons and three introns at the telomeric end of chromosome 17 [6]. Its alternative transcription gives rise to five different transcripts: survivin, survivin-ΔEx3, survivin-3β, survivin-2β, and survivin-2α [7].

Although it is expressed at high levels during fetal development, survivin is rarely expressed in normal healthy adult tissues. Overexpression of survivin has been detected in numerous human tumors [8], including colorectal cancer [9], oral squamous cell carcinoma [10], leukemia [11], and breast cancer [12].

There are several lines of evidence suggesting that survivin is involved in hepatocarcinogenesis. In HCC cells, overexpression of survivin accelerates the G1 checkpoint by releasing p21WAF1/Cip1 from the p21/cyclin-dependent kinase 4 (CDK4) complex and enforcing the formation of a complex between procaspase 3 and p21, which suppresses cell death signaling [13].

Because of this upregulation in malignancy and its functional involvement in apoptosis and proliferation, survivin is currently attracting considerable interest both as a potential cancer biomarker, a prognostic indicator in cancer, and as a new target for cancer treatment [14]. Survivin expression can be deregulated in cancer through several mechanisms, including amplification of the survivin gene, demethylation of survivin exons, increased promoter activity, increased upstream signaling in the phosphatidylinositol 3 kinase, and epigenetic alterations [15].

Polymorphisms of survivin gene may have a functional consequence affecting the production or activity of survivin, thus regulating the individual's susceptibility to tumors such as HCC. Several single-nucleotide polymorphisms have been identified in survivin gene, such as 31G/C (rs9904341) located in the promoter region and +9809T/C (rs1042489) found in 3'-UTR [16].

The aim of this study was to assess the differential expression of survivin gene in tumorous versus nontumorous liver tissue of HCC patients and determine the possible influence of survivin gene polymorphism 31G/C (rs9904341) and +9809T/C (rs1042489) on its expression and its association with the risk for HCC in Egyptian patients.

 Patients and Methods



Study population

This study was conducted at the Medical Biochemistry Department, Menoufia Faculty of Medicine; Clinical Biochemistry Department and Hepatobiliary Surgery Department, National Liver Institute, Menoufia University, during the period from October 2011 to October 2012. This study was approved by the Ethical Committee of Menoufia Faculty of Medicine, and enrollment of the individuals to the study was conditioned with written informed consent obtained from patients. The present study was performed on 30 HCC patients (group I) presented to the Hepatobiliary Surgery Department and 30 age-matched and sex-matched controls (group II). Diagnosis of HCC was based on noninvasive criteria using multislice triphasic spiral computed tomography. HCC patients underwent surgical resection, and were divided into two subgroups according to the origin of the tissue specimen: tumorous (subgroup Ia) and nontumorous (subgroup Ib). Patients were followed up after resection for 1 year to detect recurrence.

Laboratory investigations

Ten milliliter of venous blood samples was collected from patients and controls and divided into three aliquots. Two aliquots (one for serum and the other for blood in a tube containing EDTA) were used for routine laboratory investigations including liver function tests, complete blood count, and serum HBsAg and anti-HCV using fully automated autoanalyzer Synchron CX9ALX (Beckman Coulter Inc., Fullerton, California, USA), Sysmex K-21 (Sysmex Corporation, Kobe, Japan), and immunoassay (Abbott Laboratories, Abbott Park, Illinois, USA), respectively. Serum α-fetoprotein (AFP) concentration was measured using the Automated Chemiluminescence System ACS: 180 (Siemens Medical Solutions Diagnostics Corporation, Holliston, MN., USA). The third aliquot was collected in EDTA-containing tube for genotyping of survivin gene.

Molecular testing

Genotyping of survivin gene polymorphism −31G/C (rs9904341).Genotyping of survivin gene polymorphism +9809T/C (rs1042489)Survivin gene expression (mRNA) for HCC patients.

Tissue specimen collection

Tissue samples were taken from patients with HCC intraoperatively (during resection of tumor) as tumorous and adjacent nontumorous tissue samples and stored separately in cryotubes directly at −80°C to preserve RNA and DNA.

DNA extraction and genotyping

Genomic DNA was extracted from whole blood and tissue samples using Gene JET Whole Blood Genomic DNA Purification Mini Kit (Thermo Scientific, Qiagen, Valenica CA, USA).

Survivin polymorphism −31G/C (rs9904341) and +9809T/C (rs1042489) were genotyped using the TaqMan allelic discrimination (Qiagen, Valenica CA, USA) assay technique that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleotide polymorphism site in a target template sequence. The actual quantity of target sequence is not determined. The allelic discrimination assay classifies unknown samples as follows:

Homozygotes (samples having only allele 1 or allele 2)Heterozygotes (samples having both allele 1 and allele 2).

Maxima Probe qPCR Master Mix (2×) (Applied Biosystem, Waltham, MA USA) was used. The probes used were as follows: survivin gene +9809T/C (rs1042489) sequence [VIC/FAM] (GAT GAG AGA ATG GAG ACA GAG TCC C[T/C] G GCT CCT CTA CTG TTT AAC AAC ATG), and survivin gene −31G/C (rs9904341) sequence [VIC/FAM] (CCA TTA ACC GCC AGA TTT GAA TGG C[G/C] G GAC CCG GTT GGC AGA GGT GGC GGC). The genotyping reaction mix was prepared by adding 12.5 μl of master mix, 0.3 μl of each primer, 1.2 μl of each probe, and 4.5 μl of DNAse-free water. For each unknown reaction, 5 μl (0.1 μg/μl) of genomic DNA template was added, and, for negative control reaction (NTC), 5 μl of DNAse-free water was added. The cycling parameters were as follows: initial denaturation step at 94°C for 4 min, 50 cycles of denaturation at 94°C for 30 s, annealing/collection at 50°C for 25 s, and extension at 72°C for 40 s, followed by a final extension step at 72°C for 3 min. PCR was performed using LineGene 9660 (Bioer Technology Co. Ltd, Nagano, Japan).

RNA extraction and cDNA synthesis

Tissue specimens were taken from patients with HCC intraoperatively (during resection of tumor) as tumorous and nontumorous samples and stored separately in cryotubes directly at −80°C to preserve RNA. RNA was extractedusing Gene JET Mammalian Tissue Total RNA Purification Mini Kit protocol (Thermo Scientific). Thereafter, Revert Aid First Strand cDNA was synthesized using kit from Thermo Scientific by mixing random hexamer primer (1 μl), reaction buffer (4 μl), RiboLock RNase inhibitor (1 μl), dNTP mix (2 μl), revert aid RT (1 μl), and nuclease-free water (4 μl). RNA samples (5 μl) were added, mixed, and briefly centrifuged with the previously prepared components and then stored on ice. PCR was performed in a thermal cycler. Incubation was carried out for 60 min at 42°C, followed by termination of the reaction by heating at 70°C for 5 min.

Amplification of cDNA

Amplification of cDNA was performed using SYBR Green I (Thermo Scientific) and Real-Time PCR 7500 (Life Technologies, Applied Biosystems, lincoln Centre Drive Foster City, CA, USA). SYBR Green I from Thermo Scientific is a fluorescent intercalating dye that binds to the double stranded DNA and emits a fluorescent signal upon binding. In qPCR, DNA accumulates and fluorescent signal increases proportionally to the DNA concentration. The excitation and emission maxima of SYBR Green I was at 494 and 521 nm, respectively, which are compatible with the use on any real-time cycler. Survivin forward primer was Sur FmR 5'-CCA CCG CAT CTC TAC ATT C-3' and survivin reverse primer was Sur RmR 5'-GTC TGG CTC GTT CTC AGT GG-3'. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer was GAPDH FmR 5'-CAA CTA CAT GGT TTA CAT GTT C-3' and GAPDH reverse primer was GAPDH RmR 5'-GCC AGT GGA CTC CAC GAC-3'. One tube was used for survivin and another for GAPDH. The tubes contained Maxima SYBR Green qPCR Master Mix (2×) 12.5 μl, forward primer (survivin) 2 μl, reverse primer (survivin) 2 μl, ROX solution 0.1 μl, nuclease-free water 4 μl, and template DNA 5 μl to each tubes of survivin and GAPDH. Cycling conditions were 48°C for 30 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min.

Statistical analysis

Data analysis was performed using SPSS software for Windows (version 16.00; SPSS Inc., Chicago, Illinois, USA). Differences between cases and controls were evaluated using Student's unpaired t-test (for continuous variables) and the χ2-test (for categorical variables). The observed genotype frequencies were compared with expected values calculated from Hardy–Weinberg equilibrium theory (p2+2pq+q2 = 1, wherepis the frequency of the wild-type allele and q is the frequency of the variant allele). Pearson's χ2-test was used to determine whether there was any significant difference in allele and genotype frequencies between patients and controls. The associations between genotypes and the risk for HCC were estimated by computing the odds ratios and their 95% confidence intervals from binary logistic regression analysis. A P value of less than 0.05 was considered statistically significant. All tests were two sided.

 Results



A total of 60 participants were recruited in the present study, including 30 HCC patients (mean age: 55.97 ± 11.11 years; 73.3% male) and 30 unrelated apparently healthy controls (mean age: 54.97 ± 11.59 years; 66.7% male). The studied groups were homogenous in terms of age and sex (P > 0.05) (data not shown).

Characteristics of the HCC group are presented in [Table 1].{Table 1}

The genotype distributions of survivin −31G/C (rs9904341) and +9809T/C (rs1042489) polymorphisms did not significantly deviate from that expected for Hardy–Weinberg equilibrium, and showed no statistically significant difference between HCC patients and controls. Three HCC patients showed change in allele and genotype frequency distribution between tumorous and nontumorous tissue [one case changed from G to C (GG to GC) and two cases changed from T to C (TT to TC)]. The different alleles and genotypes of survivin −31G/C (rs9904341) and +9809T/C (rs1042489) were not associated with risk for HCC [Table 2] and [Table 3].{Table 2}{Table 3}

There was no statistically significant difference among genotypes of survivin at −31G/C (rs9904341) and +9809T/C (rs1042489) in HCC patients as regards liver function tests, complete blood count, AFP, and tumor criteria (data not shown).

Survivin mRNA was expressed in all tissue samples and was significantly increased in tumorous tissue when compared with nontumorous tissue in HCC cases by 13-fold changes (P < 0.001). Survivin expression in group Ia was significantly increased with advanced stage and larger size of the tumor, vascular invasion, and absence of cirrhosis. Moreover, HCC patients with recurrence (the recurrence rate was 40% within 1 year) showed a highly significant increase in survivin expression compared with those who had no recurrence (P < 0.001). Linear regression analysis for independent predictors of survivin expression revealed that only stage of the tumor is the independent predictor of survivin expression (P < 0.05) [Figure 1] and [Table 4],[Table 5],[Table 6].{Table 4}{Table 5}{Table 6}{Figure 1}

Survivin expression in tumorous tissue did not show any significant difference between genotypes of −31G/C (rs9904341) and +9809T/C (rs1042489) polymorphisms in HCC patients (data not shown).

Survivin expression in tumorous tissues had a significant positive correlation with AFP (P < 0.05). Meanwhile, no significant correlation was found as regards other parameters ([Table 7]).{Table 7}

Kaplan–Meier survival analysis of relation of survivin expression and survival of HCC patients revealed that the overall survival rate was 70% and disease-free survival rate was 53.3% within 1 year, and they showed a significant decrease with higher survivin expression. Moreover, survivin expression in tumorous tissues of HCC patients was negatively correlated with time lapsed until recurrence and time lapsed until death [Figure 2]a,[Figure 2]b and [Figure 3].{Figure 2}{Figure 3}

 Discussion



It is widely accepted that apoptosis plays a key role in cell or tissue homeostasis. Dysregulation of apoptosis may induce the accumulation of virtually immortal cells and can ultimately lead to many human disorders, including cancer [10]. Survivin (BIRC5) is a member of the inhibitor of apoptosis protein family. It inhibits the activation of caspase-3 and caspase-7, which are downstream effectors of apoptosis, in cells exposed to apoptotic stimuli. It is overexpressed in most human tumors but not in normal tissues [17],[18].

The aim of this study was to assess the differential expression of survivin gene in tumorous versus nontumorous liver tissue of HCC patients and determine the possible influence of survivin gene polymorphism −31G/C and +9809T/C on its expression and its involvement in the risk for HCC in Egyptian patients.

In the current study, no significant differences were detected in the distributions of genotypes or alleles of survivin −31G/C (rs9904341) and +9809T/C (rs1042489) polymorphisms between HCC patients and controls. Moreover, the frequency distribution of these genotypes were not affected by tumor criteria (presence of cirrhosis, tumor stage and size, number of focal lesions, type of tumor, and vascular invasion), AFP serum level, liver function tests, and hematological indices. Moreover, our study suggested that none of rs9904341 or rs1042489 polymorphisms in survivin gene was associated with risk for HCC.

These results are consistent with the results Bayram et al. [13]andLi et al. [10]. However, another study by Hsieh et al. [19]found that survivin +9809T/C (rs1042489) polymorphic genotype is associated with the risk for HCC and stated that the C allele was significantly associated with the tumor risk.

These diverse results may be attributed to different studied populations, several environmental, geographic, or ethnic differences. Besides, our negative results were possibly due to the relatively small sample size, and therefore additional studies with larger samples are needed to validate our finding.

The current study detected 3/30 (10%) mutations in tumorous tissue compared with nontumorous tissue in HCC patients as regards survivin −31G/C (one case from G to C) and +9809T/C (two cases from T to C).

Jang et al. [20] and Gazouli et al. [21] have revealed that the C allele of survivin −31G/C and +9809T/C predominated in tumorous samples and has a significantly higher transcriptional activity compared with the G allele.

These mutations (10%) showed no statistically significant difference between subgroups Ia and Ib; this can be attributed to the relatively small sample size. However, this result indicates the importance of a large scale study to confirm or disregard this observation.

The present study found that survivin mRNA was expressed in all tissue specimens (tumorous and nontumorous) in HCC patients. Moreover, survivin expression showed a significant overexpression in tumorous tissue compared with nontumorous tissue (13-fold changes).

Kannangai et al. [22], Montorsi et al. [23], Augello et al. [24], Ikeguchi et al. [25], and Jin et al. [8] found similar results. Meanwhile, Yang et al. [26] and Cui et al. [27] showed that survivin expression was found in 78 and 64% of tumorous tissue samples, respectively, and was not found in nontumorous tissue samples in HCC patients. Different results were obtained from a study of Chau et al. [28], who stated that survivin protein was more abundant in nontumor liver tissues than in HCC samples. Different sample sizes and different ways of assessment of survivin expression may affect the results.

The molecular basis for the cancer-specific overexpression of survivin has yet to be fully elucidated. Different studies have suggested that it may originate from the amplification of the survivin locus, demethylation of the survivin promoter and exons, and increased promoter activity mediated by a variety of oncogenic pathways [15]. However, survivin expression in nontumorous tissues may be due to the presence of regeneration nodules and the role of survivin in normal cell cycle [5].

In the present study, different alleles and genotypes of survivin gene polymorphism at −31G/C (rs9904341) and +9809T/C (rs1042489) did not affect survivin expression in the tissue specimen of HCC patients. Li et al. [10] found similar results and identified that these polymorphisms probably had nothing to do with the stability of survivin mRNA or its translational efficiency.

Normal shedding of cells initiates the apoptosis process, but the overexpression of survivin exerts an antiapoptotic effect, which leads to a high rate of cell proliferation. Therefore, survivin may play an important role in the progression of HCC and may facilitate metastatic spread throughthe blood stream, and hence the expression of survivin in the primary lesion can be an indicator of metastasis and the prognosis of HCC [29],[30].

In this regard, our finding supported that survivin may be positively correlated with high risk for disease progression, recurrence, and poor prognosis in HCC as we detected that survivin expression in tumorous tissue in HCC patients was significantly increased with advanced stages, larger size of the tumor, vascular invasion, and absence of cirrhosis. The stage of the tumor is an independent predictor of survivin expression. Moreover, survivin expression in tumorous tissue showed a highly significant increase in HCC patients who had recurrence during 1 year after resection compared with patients who had no recurrences and was positively correlated with serum AFP level, which means that its high expression is associated with poor prognosis in our studied population.

These observations were consistent with those of Jin et al. [8], Chen et al. [31], and Augello et al. [24], who reported that there was a significant association between survivin mRNA level in HCC and higher tumor stage, tumor size, vascular invasion, recurrence rate, and poor survival. Moreover, Kannangai et al. [22] found that mRNA appears to be overexpressed to a greater degree in HCC that arise in chronic liver disease (noncirrhotic). In cirrhotic patients the liver cells are changed to fibrous tissue unlike chronic liver cases in which there are regeneration nodules and active replicating cells. These results are contradictory to the results of Yang et al. [26], Cui et al. [27], and Hui et al. [32], who stated that in HCC patients there was no significant association between survivin mRNA level and stage of tumor, vascular invasion, and cirrhosis.

Meta-analysis of 14 studies that correlated the expression of survivin with the overall survival and disease-free survival of HCC patients indicated that high survivin expression in HCC tissue specimens was significantly correlated with poor survival in HCC patients [33]. Similar observation was reported by many other studies [26],[32],[34].

This observation can be attributed to the ability of survivin to inhibit apoptosis, promote proliferation, and increase angiogenesis. Thus, survivin is likely to be causally involved in tumor progression, and hence increased levels would be expected to predict a poor prognosis.

 Conclusion



No specific genotype or allele of survivin gene at −31G/C (rs9904341) and +9809T/C (rs1042489) showed an increased risk for HCC. Survivin was expressed in all tumorous and nontumorous tissue specimens in HCC cases and showed overexpression in tumorous tissues compared with nontumorous tissues. Increased survivin expression in tumorous tissue in HCC patients was correlated with poor prognosis and low survival rate suggesting that survivin plays an important role in HCC progression by promoting cell proliferation; it may be a prognostic marker and may be considered as a new target in the treatment of HCC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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