Abstract
Aims: The purpose of the study was to analyze the relationship of survivin polymorphisms including -31G/C, -625G/C, 9194A/G and 9809T/C with the susceptibility to lung cancer. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to test the polymorphisms of -31G/C, -625G/C, 9194A/G and 9809T/C in 104 patients with lung cancer and 104 healthy controls. Then, linkage disequilibrium and haplotypes were analyzed by HaploView software. The differences of genotype, allele and haplotype frequencies in case and control group were assessed via chi-square test. Odds ratio (OR) with 95% CI were used to evaluate the correlation of survivin polymorphisms with lung cancer. Results: Genotype distribution of each polymorphism site in control group was in agreement with Hardy-Weinberg equilibrium (HWE) (P>0.05). The frequency of -31G/C CC genotype and C allele in case group were much higher than that of controls, respectively (CC: 33.6% vs. 22.1%; C: 57.2% vs. 46.6%) and CC genotype as well as C allele were appeared to be risk factors for lung cancer. Meanwhile, 9194A/G GG genotype could increase the risk for lung cancer (OR=2.86, 95% CI=1.14-7.20). The risk of G allele carriers for lung caner was higher than that of A allele (OR=1.63, 95% CI=1.08-2.47). The haplotypes analysis indicated that CGGC and GCAT were associated with the susceptibility to lung cancer (OR=2.79, 95% CI=1.58-4.92; OR=2.36, 95% CI=1.29-4.30). Conclusions: Survivin -31G/C and 9194A/G polymorphisms were associated with the risk of lung cancer. The CGGC and GCAT haplotypes carriers were more likely to develop lung cancer.
Keywords: Survivin, polymorphisms, lung cancer
Introduction
Lung cancer is one of the most common malignant tumors which jeopardizes people’s health seriously. There are approximately 1.18 million deaths caused by lung cancer every year all over the world. The 5-year survival rate of lung cancer is less than 15% [1]. Early detection and treatment can effectively improve the survival rate. However, due to limited diagnosis methods, over 70% patients are at late or advanced stage when initial diagnosis. Besides, the pathogen of lung cancer are multiple and its progress is complicated. Against the background, genes were extensively studied to explore the pathogenesis of lung cancer and survivin was one of them.
survivin gene locates on chromosome 17q25 with a length of 15 kb. It has been reported that survivin is related to cell apoptosis and cell proliferation [2-4]. Moreover, it plays crucial role in the pathogenesis of hepatocellular carcinoma, gastric cancer, cervical cancer and gallbladder cancer [5-8]. Besides, downregulation of survivin could suppress proliferation and colony formation of non-small-cell-lung cancer cells [9]. The studies also found that survivin could significantly affect the survival rate of lung cancer patients [10]. As we all know, single nucleotide polymorphism (SNPs) are the third generation of genetic markers, which are helpful for the diagnosis, prognosis and treatment of diseases. Although many studies have investigated the effects of survivin polymorphisms on the occurrence of cancers [11-14], there were few studies exploring the association between survivin polymorphisms and lung cancer.
In current study, we selected four polymorphisms sites (-31G/C, -625G/C, 9194A/G and 9809T/C) in survivin gene and analyzed the relationship of them with the risk of lung cancer.
Material and methods
Objects of study
104 patients diagnosed with lung cancer via histopathology were collected from Linyi People’s Hospital, aging from 29 to 67 with an average age of 45. Meanwhile, 104 healthy people without history of genetic disease or cancers whose age were from 31 to 70 with an average age of 52 were taken as controls. All patients did not receive any radiotherapy or chemotherapy treatment before sampling. The controls and cases were matched on the age and gender and they had no blood relationship. The process of sample collection was proceeded in accordance with the national ethics criterion for human genome research. Written informed consents were signed in advance. The study was approved by the Research Ethics Committee of Linyi People’s Hospital.
DNA extraction and genotyping
5 mL venous blood was extracted from each subject and was put into EDTA blood collection tube immediately. Then the samples were stored at -80°C for DNA extraction. DNA was isolated using the improved method of sodium iodide. PCR primers were designed by Primer Premier 5.0 software and synthesized in Shanghai SANGON biological company. The sequences of primers were displayed in Table 1. PCR reaction solution included 1 µl (1×108 ng/L) DNA, 1 µL forward primer, 1 µl reverse-primer, 25 µL Master Mix and 22 µL of ddH2O. The reaction procedure was as follows: initial denaturation at 94°C for 10 min, 35 cycles of denaturation for 30 s at 94°C, annealing at 62°C for 30 s, extension at 72°C for 1 min, and finally extension at 72°C for 5 min. The PCR products were handled with MspI enzyme digestion, and then examined by agarose gel electrophoresis to ascertain the genotypes of each genetic variation.
Table 1.
Sequences of PCR primers for survivin (-31G/C, -625G/C, 9194A/G and 9809T/C) polymorphisms
| SNP | SNP locus | Primer (5’→3’) |
|---|---|---|
| Rs 9904341 | -31G/C | F: 5’-AAGAGGGCGTGCGCTCCCGACA-3’ |
| R: 5’-AAGAGGGCGTGCGCTCCCGACA-3’ | ||
| rs 8073069 | -625G/C | F: 5’-GTTCATTTGTCCTTCATGCGC-3’ |
| R: 5’-GGCAGAGGGTGCAGTGAGC-3’ | ||
| rs 2071214 | 9194A/G | F: 5’-GAAGAAAGAATTTGAGGAAACCGC-3’ |
| R: 5’-AAACCCTGGAAGTGGTGCAG-3’ | ||
| rs 1042489 | 9809T/C | F: 5’-GCTTA CCAGGTGAGAAGTGAGG-3’ |
| R: 5’-GTATCTGCCAGACGCTTCCTATC-3’ |
F: forward; R: reverse.
Statistical analysis
SPSS18.0 software was used to conductstatistical analysis. Hardy-Weinberg equilibrium (HWE) was taken to test whether the subjects in control group was representative. The differences on genotypes, alleles and haplotypes distribution between cases and controls were detected by chi-square method. The difference was considered to be significant if P<0.05. Odds ratio (OR) and 95% confidence interval (CI) were used to estimate the relationship of survivin polymorphisms with lung cancer susceptibility.
Results
HWE test
The genotypes distribution of -31G/C, -625G/C, 9194A/G and 9809T/C polymorphisms were consistent with HWE (P>0.05 for all), which indicated that the people in control group was representative.
Correlation between survivin polymorphisms and risk of lung cancer
In the analyses, the genotype frequencies of -31G/C CC and 9194A/G GG were significantly higher in cases than controls, which manifested that they were closely related to the risk of lung cancer (OR=2.28, 95% CI=1.05-4.94; OR=2.86, 95% CI=1.14-7.20). Meanwhile, similar results were observed on -31G/C C and 9194A/G G alleles (OR=1.53, 95% CI=1.04-2.25; OR=1.63, 95% CI=1.08-2.47). However, -625G/C and 9809T/C polymorphisms showed no effects on the occurrence of lung cancer (Table 2).
Table 2.
Genotypes and alleles distribution of -31G/C, -625G/C, 9194A/G and 9809T/C
| Genotype/allelotype | Cases n=104 (%) | Controls n=104 (%) | χ2 | P value | OR (95% CI) |
|---|---|---|---|---|---|
| -31G/C | |||||
| GG | 20 (19.2) | 30 (28.8) | - | - | 1.00 |
| GC | 49 (47.1) | 51 (49.0) | 1.087 | 0.297 | 1.44 (0.72-2.87) |
| CC | 35 (33.6) | 23 (22.1) | 4.447 | 0.035 | 2.28 (1.05-4.94) |
| G | 89 (42.8) | 111 (53.4) | - | - | 1.00 |
| C | 119 (57.2) | 97 (46.6) | 4.661 | 0.031 | 1.53 (1.04-2.25) |
| -625G/C | |||||
| GG | 54 (51.9) | 57 (54.8) | - | - | 1.00 |
| GC | 39 (37.5) | 39 (37.5) | 0.033 | 0.855 | 1.06 (0.59-1.88) |
| CC | 11 (10.6) | 8 (7.7) | 0.555 | 0.456 | 1.45 (0.54-3.88) |
| G | 147 (70.7) | 153 (73.6) | - | - | 1.00 |
| C | 61 (29.3) | 55 (26.4) | 0.430 | 0.512 | 1.15 (0.75-1.77) |
| 9194A/G | |||||
| AA | 44 (42.3) | 56 (53.8) | - | - | 1.00 |
| AG | 42 (40.4) | 40 (38.5) | 0.942 | 0.332 | 1.34 (0.74-2.40) |
| GG | 18 (17.3) | 8 (7.7) | 5.256 | 0.022 | 2.86 (1.14-7.20) |
| A | 130 (62.5) | 152 (73.1) | - | - | 1.00 |
| G | 78 (37.5) | 56 (26.9) | 5.328 | 0.021 | 1.63 (1.08-2.47) |
| 9809T/C | |||||
| CC | 32 (30.8) | 33 (31.7) | - | - | 1.00 |
| CT | 52 (50.0) | 54 (51.9) | 0.000 | 0.982 | 0.99 (0.54-1.84) |
| TT | 20 (19.2) | 17 (16.3) | 0.219 | 0.639 | 1.21 (0.54-2.72) |
| C | 116 (55.8) | 120 (57.7) | - | - | 1.00 |
| T | 92 (44.2) | 82 (39.4) | 0.555 | 0.456 | 1.16 (0.78-1.72) |
Association of haplotypes and lung cancer risk
14 haplotypes combinations were established according to the linkage disequilibrium analysis of -31G/C, -625G/C, 9194A/G and 9809T/C with HaploView software. For further analysis, 9 haplotypes with frequency of less than 1% were excluded. The remaining 5 haplotypes accounted for 97.6 of the cases and 96.4% of the controls. The association analyses indicated that CGGC and GCAT haplotypes could increase the risk of lung cancer (OR=2.79, 95% CI=1.58-4.92; OR=2.36, 95% CI=1.29-4.30) (Table 3).
Table 3.
Linkage disequilibrium and haplotype analysis of alleles in -31G/C, -625G/C, 9194A/G and 9809T/C
| Haplotype site1-site2-site3-site4 | Patientsa 2n=200 (%) | Controlsa 2n=198 (%) | χ2 | P | OR (95% CI) |
|---|---|---|---|---|---|
| G-G-A-C | 28 (14.0) | 56 (28.3) | - | - | 1.00 |
| C-G-A-C | 10 (5.0) | 8 (4.0) | 3.132 | 0.077 | 2.50 (0.89-7.03) |
| C-G-A-T | 31 (15.5) | 33 (16.7) | 3.457 | 0.063 | 1.88 (0.96-3.66) |
| C-G-G-C | 78 (39.0) | 56 (28.3) | 12.790 | 0.000 | 2.79 (1.58-4.92) |
| G-C-A-T | 53 (26.5) | 45 (22.7) | 7.884 | 0.005 | 2.36 (1.29-4.30) |
Nine haplotypes that had a frequency of less than 1% were excluded from analysis;
controls 30 and cases 20, respectively.
Discussion
Lung cancer is one most common malignancy with a high fatality rate. It is the most fatal cancer in males, while the morbidity and mortality were also high in females [15]. The development of lung cancer is influenced by multiple factors and involves complicated progress. In recent years, it has been demonstrated that genetic polymorphisms show strong effects on the onset of lung cancer [16-18].
Survivin, as a defender against cell apoptosis, can reduce the sensitivity of tumor cells to apoptosis stimulation, increase the survival capability of tumor cells and then affect individuals susceptibility to tumors [12,19,20]. Survivin polymorphisms were widely studied in previous studies and were confirmed to be associated with the risk of many cancers.
George et al. and Marques et al. found that caspase-9 polymorphism combined with survivin -31G/C could increase the risk of pancreatic cancer and renal cell carcinoma [21,22]. Besides, survivin -31G/C polymorphism was a promoter to esophageal cancer as well as prostate cancer and papillary thyroid carcinoma [23-25]. The similar role was found by Li et al. in colorectal cancer for survivin -31G/C based on Chinese population [14]. Besides, Rosato et al. revealed that the -31G/C SNP was related to the metastasis of non small cell lung cancer (NSCLC) [26].
In this study, we analyzed the correlation between four polymorphic locus (-31G/C, -625G/C, 9194A/G and 9809T/C) of survivin gene and the risk of lung cancer through a case-control design. The genotype distribution of each polymorphism locus in control group was conformed to HWE test. The results suggested survivin -31G/C and 9194A/G was involved in pathogenesis of lung cancer among Chinese population, however, survivin -625G/C and 9809T/C were not associated with lung cancer risk. The haplotype analyses indicated that CGGC and GCAT were both risk factors for lung cancer.
In conclusion, survivin polymorphism, especially -31G/C and 9194A/G, could increase the risk of lung cancer. However, there are still some defects in the study. First, the sample size was relatively small. Moreover, the study only investigated the role of survivin gene in the pathogenesis of lung caner, while not considered the effects of environmental factors. A larger scale study with comprehensive design is needed to provide more reliable evidences on the issue.
Disclosure of conflict of interest
None.
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