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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 1  |  Page : 296-300

Association between survivin gene polymorphism and colorectal cancer


Department of Clinical Pathology and General Surgery, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission23-Apr-2018
Date of Acceptance01-Jun-2018
Date of Web Publication17-Apr-2019

Correspondence Address:
Mona Maamoun Ahmed
44-Gamal Abdelnaser Street, Sector-2, Shebin El Kom City, Menoufia Governorate 32512
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_155_18

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  Abstract 


Objective
To study the possible association of genetic polymorphism of survivin gene (rs9904341) with the risk of developing colorectal cancer (CRC) in Egyptian patients and explore the mechanisms of survivin polymorphism in the development of CRC.
Background
Survivin gene is an inhibitor of apoptosis, plays an important role in cell cycle regulation, and may be involved in the development and progression of cancer. Structurally, the human survivin gene comprises four exons and three introns spanning 14.7 kb, which encodes a 16.5 kDa protein. Survivin gene is expressed mostly at the G2/M phase and declines rapidly in the G1 phase of cell cycle. This is largely transcriptionally controlled and involves cell cycle-dependent elements and cell cycle homology regions located in the survivin gene promoter. This mutation can depress cell cycle-dependent transcription of the survivin gene and result in the overexpression of survivin at mRNA and protein levels. Survivin −31G/C polymorphism may be associated with CRC.
Patients and methods
A case–control study was carried out between November 2016 and December 2017 at the Clinical Pathology Department, Faculty of Medicine, in collaboration with the General Surgery Department and Oncology Department, Menoufia University. The case–control study included 100 participants divided into two groups. Group I included 50 diagnosed CRC patients and group II included 50 apparently healthy participants as a control group. PCR-restriction fragment length polymorphism was used for the detection of survivin gene polymorphisms (rs9904341).
Results
Mutant CC genotypes was statistically higher in the CRC group (42%) than in the control group (18%) (P = 0.01). C allele was statistically higher in the CRC group (62%) than in control group (43%) (P = 0.007).
Conclusion
The −31CC genotype of survivin gene is associated with CRC and may be a risk factor for the development of CRC.

Keywords: colorectal carcinoma, genetic, polymerase chain reaction-restriction fragment length polymorphism, polymorphism, survivin


How to cite this article:
Fathy WM, Amar MS, Montaser B, Ahmed MM. Association between survivin gene polymorphism and colorectal cancer. Menoufia Med J 2019;32:296-300

How to cite this URL:
Fathy WM, Amar MS, Montaser B, Ahmed MM. Association between survivin gene polymorphism and colorectal cancer. Menoufia Med J [serial online] 2019 [cited 2019 May 27];32:296-300. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/296/256087




  Introduction Top


Cancer is the abnormal cell growth that causes cell proliferation and morbidity due to multiple gene expressions, and if not treated, it may result in tissue invasion and metastasis leading to death. Gastrointestinal cancers are the most common malignant tumors along with lung and breast cancer in the world [1]. Colorectal cancer (CRC) is considered the third most common cancer worldwide after lung and breast cancers; it is the third most common cancer in men after lung and prostate cancers and the second most common cancer in women after breast cancer [2]. The global annual incidence of CRC is approximately one million cases and its annual mortality is more than 500 000. [3]. Apoptosis, also known as programmed cell death, has an evolutionarily conserved role and it is a necessary process for organ development, tissue remodeling, and suppression of immune response [4]. Inhibitor of apoptosis (IAPs) proteins are a family of proteins with antiapoptotic functions that contribute to the evasion of apoptosis. IAP proteins are expressed at high levels in a variety of human cancers including colon cancer [5]. Survivin is considered as a novel member of the IAP protein family that is involved in both cell division regulation and apoptosis inhibition [6]. The overexpression of survivin is associated with the development of disease including cancers [7]. Survivin gene promotes tumor development and progression by inhibiting apoptosis and increasing cell proliferation [8].

Aim

To study the role of survivin gene polymorphisms (rs9904341) and the danger of developing CRC among Egyptian patients.


  Patients and Methods Top


The case–control study included 100 participants divided into two groups. Group I: 50 diagnosed CRC patients (30 men, 20 females), their ages were 49.10 ± 7.92 years. Group II: 50 apparently healthy age-matched and sex-matched individuals as a control group (25 men, 25 women), their ages were 49.50 ± 5.59 years. This study was carried out between November 2016 and December 2017 at the Clinical Pathology Department, Faculty of Medicine in collaboration with the General Surgery Department and Oncology Department, Menoufia University.

Written informed consents were provided by all participants and agreement was obtained from the ethics committee.

For all participants the followings were done: full history taking, clinical examination, complete blood picture, liver function tests: aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, fecal occult blood test, carcinoembryonic antigen (CEA), cancer antigen 19-9 (CA19-9), and evaluation of survivin gene polymorphism.

Sampling

Under complete aseptic conditions, 7 ml of venous blood was collected. Each blood sample was divided as follows. Tube A: 4 ml was collected in a plain tube, left to clot at 37°C, then centrifuged, clear sera were separated and divided into aliquots, the first one was used to determine liver function tests, kidney function tests by AU 480 auto analyzer (AU 480 chemical analyzer; Beckman Coulter, Inc. Atlanta Vision Center Atlanta, Georgia, USA). The second was used for the measurement of CEA and CA19-9 by Cobas e 411(Hitachi Cobas e 411 High-Technologies Corporation, Tokyo, Japan). The specimens were kept frozen at −20°C until the time of assay, Tube B: 3 ml was collected in sterile vacutainer tubes containing EDTA for DNA extraction and complete blood picture. Stool was collected in sterile cups for Fecal Occult Blood Test. The FOB One Step Fecal Occult Blood Test Device (Feces) is a qualitative, lateral flow immunoassay for the detection of fecal occult blood in humans. The membrane is precoated with an anti-hemoglobin antibody on the test line region of the test. During testing, the specimen reacts with the particle coated with the anti-hemoglobin antibody. The mixture migrates upward on the membrane chromatography by capillary action to react with the anti-hemoglobin antibody on the membrane and generate a colored line. The presence of this colored line in the test line region indicates a positive result, while its absence indicates a negative result. To serve as a procedural control, a colored line will always appear in the control line region. If the control line does not appear, the test result is not valid.

Evaluation of survivin gene polymorphisms

DNA analysis

PCR-restriction fragment length polymorphism method was used to determine the distribution of genotype and allele frequencies of survivin single-nucleotide gene polymorphisms (rs9904341).

DNA extraction

The DNA was extracted using the commercially available Spin column technique (Scientific Zymo Bead Genomic DNA Kits, Quick-g DNA Mini Prep, Murphy Ave. Irvine, CA, U.S.A) for DNA extraction from whole blood, supplied by Zymo Research Corp. (Freiburg, Germany). The eluted genomic DNA was stored in −80°C until amplification PCR.

Primers

The lyophilized primers were supplied by Fermentas Life Sciences (Waltham, Massachusetts, USA). The lyophilized primers were reconstituted by addition of sterile water to a final concentration of 50 pmol/μl and distributed in aliquots and stored at −80°C.

Survivin: forward primer: 5'-GACTACAACTC CCGGCACAC-3'.

And the reverse primer: 5'-TGTAGAGATGC GGTGGTCCT-3'.

PCR amplification

PCR amplification has been performed using the My Taq HS Red Mix master Mix Kit supplied by Bioline USA Inc. (Taunton, Massachusetts, USA), which is a ready-to-use 2 × mix for fast, highly specific, hot-start PCR. My Taq HS Red Mix is powered by antibody-mediated hot start and does not possess polymerase activity during the reaction setup, thus reducing nonspecific amplification. The following protocol is for 25 ml reaction: template 2 ml, primers 1 ml, My Taq HS Red Mix, 2× 12.5 ml, and water (dH2O) up to 25 ml.

PCR cycling conditions

One cycle of initial denaturation at 95°C for 1 min followed by 30 cycles of denaturation at 95°C for 15 s, annealing at 56°C for 15 s and extension at 72°C for 10 s after the last cycle, a final extension of 4 min at 72°C was done. The amplification products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide and visualized on an ultraviolet transilluminator. The PCR products were subjected to digestion by restriction enzyme EcoO109I (NEB-EcoO109I; Bio Lab., New England, New England Biolabs, 240 County Road, Ipswich, MA) supplied by Bio Lab. (NEB-NEB-EcoO109I). The amplification products were separated by electrophoresis on 2% agarose gel stained with ethidium bromide and visualized on an ultraviolet transilluminator. The products in survivin were: C/C at 226 bp and G/G at 13 492 bp and G/C at 22 613 492 bp.

Statistical analysis

The results were collected, tabulated, and statistically analyzed by an IBM compatible personal computer with SPSS statistical package (version 23, Released 2015; SPSS Inc.). IBM SPSS statistics for windows (version 23.0, SPSS Inc., Chicago, IL, USA; IBM Corp., Armonk, New York, USA).

Two types of statistical analyses were done:

  1. Descriptive statistics, for example, was expressed in: number (N), percentage (%), mean (x̄), and SD
  2. Analytic statistics: Student's t test, Mann–Whitney' test, Kruskal–Wallis, χ2 test, Fischer's exact test, and Z test. P value: significant difference if P valueless than 0.05, of nonsignificant difference if P value more than 0.05, and highly significant difference if P value less than 0.001.



  Results Top


The study included 100 participants divided into the two groups: group I included 50 diagnosed CRC patients (30 men, 20 women), their ages were 49.10 ± 7.92 years, group II: 50 apparently healthy age-matched and sex-matched individuals as a control group (25 men, 25 women); their ages were 49.50 ± 5.59 years. There was a statistically high significant difference among laboratory findings that included AST, ALT, and hemoglobin between groups (P < 0.001) [Table 1]. The studied patients had high serum CEA level and serum CA19-9 level, which showed a significant increase when compared with the control group mean (P < 0.001) [Table 2]. The genotype dispersion of survivin single-nucleotide polymorphism showed: the GG wild genotype was higher in the control group (32%) compared with the CRC group (18%) and the CC mutant genotypes were higher in the CRC group (42.0%) than in the control group (18.0%). There was a statistically significant difference between CC and GC (P < 0.001%), no statistical distinction was found between CC and G/G (P = 0.10). Regarding allele frequencies, G allele was more expressed in the control group (57.0%) than in the CRC group (38%), while C allele was higher in the CRC group (62.0%) than in the control group (43.0) [Table 3]. Our result showed that there were association between survivin gene polymorphism and clinic pathologic features of CRC, such as tumor size, lymph node metastasis, and TNM stage [Table 4] and [Figure 1].
Table 1: Laboratory findings of the studied groups

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Table 2: Tumor markers

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Table 3: Genetic polymorphism and alleles

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Table 4: Gene polymorphism and tumor characteristics in colorectal cancer patients

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Figure 1: Agarose gel electrophoresis showing PCR-RFLP analysis of surviving gene after addition restriction enzyme: EcoO109I, 50 bp DNA ladder, lanes 2, 3, 6, 7, 8, and 9 (C/C) band (226 bp), lanes 4 and 5 (G/G) band (92 and 134 bp), lane 1 (G/C) band (226, 134, and92 bp). RFLP, restriction fragment length polymorphism.

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  Discussion Top


CRC is the third most common cancer worldwide after lung and breast cancers. CRC affects men and women of all racial and ethnic groups, and is most often found in patients aged 50 years or older [9]. About 5–10% of colon cancer cases are inherited, the remaining occurring sporadically [10]. Causes and mechanisms of colon cancer may be associated with genetic and environmental factors, especially diet [11]. Many studies have shown that increased expression of survivin plays an important role in the development and progression of malignant neoplasms by inhibiting tumor cell apoptosis [12]. With respect to hematological parameters, the studied patients had hemoglobin level and platelet counts lower than that of the control group. While white blood cell counts were in the normal range without any statistically significant differences between patient and control groups, Karagöz et al. [13] reported that white blood cells were decreased in CRC patients compared with healthy participants. The studied patients had high serum ALT level and AST level which showed a significant increase when compared with the control group mean. This increase is due to metastasis to the liver. House et al. [14] and Tomlinson et al. [15] reported that the liver is recognized as the most common site of CRC metastasis because the majority of the intestinal mesenteric drainage enters the hepatic portal venous system. More than 50% of patients with CRC will develop metastatic disease to their liver over the course of their life, which ultimately results in death for more than two-thirds of these patients. The studied patients had high serum CEA level which showed a significant increase when compared with the control group mean (highly significant). Li et al. [16] reported that CEA is overexpressed in CRC and promotes the metastatic capacity of colon cancer cells. The studied patients had high serum CA19-9 level which showed significant increase when compared with the control group mean (highly significant). Azzal et al. [17] reported that CA19-9 is elevated in patients with CRC, and it has been used as tumor markers for CRC. This study aimed to study the impacts of single-nucleotide polymorphisms of survivin gene on CRC susceptibility in two groups: a group of healthy individuals and the CRC group, looking for a possible association that might exist with the risk of developing CRC. The genotype dispersion of survivin rs9904341 single-nucleotide polymorphism was: the CC genotype was higher in the CRC group compared with the control group, in accordance with Xia et al. [18] who found that the expression of survivin in the cancer tissue of CRC patients with the −31CC genotype was significantly higher than that with the GC and GG genotypes. A meta-analysis by Xile and Caizhao [19] suggested that the survivin −31G/C polymorphism might be correlated with an increased risk of CRC, indicating it may serve as a biomarker of disease progression. Another meta-analysis for survivin −31G/C polymorphism based on six studies by Linhua et al. [20] suggested that individuals with survivin −31G/C polymorphism showed an increased risk of CRC in the overall population. Li et al. [21] have determined statistically significant CC genotype frequency compared with GG and GC genotypes for survivin −31G/C in CRC patients. In a southern Chinese population, Huang et al. [22] found that the survivin gene −31G/C polymorphism is associated with sporadic CRC risk, and the G-variant genotype is the independent protective factor against sporadic CRC. Regarding alleles frequencies, G allele was more expressed in the control group than in the CRC group, while C allele was higher in the CRC group than in the control group. This is in concurrence with Xia et al. [18] who found that C allele was significantly higher in CRC patients compared with the healthy control. Nesibe et al. [23] found in his study that mutant C allele was higher in CRC patients but wild G allele was higher in control. Our result showed that there were association between survivin gene polymorphism and clinicopathologic features of CRC, such as tumor size, lymph node metastasis, and TNM stage. Gazouli et al. [24] showed that −31 CC genotype and the −31C allele were significantly more frequent in stage grouping III and IV than in stage grouping I and II. In contrast to our results, carrying G allele for survivin −31C/G has reported increased cancer risk in renal cell carcinoma [25]. Additionally, the GG genotype for survivin −31C/G polymorphism has been shown to be a risk factor in keratocystic odontogenic tumor development [26].


  Conclusion Top


Survivin gene polymorphism (rs9904341) could be implicated in the pathogenesis of CRC and it might contribute to the risk of developing CRC.

Further independent studies using a larger population, studying genotypic phenotypic associations, including both sexes are necessary in order to clarify the relation between survivin gene polymorphism (rs9904341) and pathogenesis of CRC.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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