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Association of interleukin-1B (IL-1B) gene polymorphisms with risk of gastric cancer in Chinese population
www.elsevier.com/locate/issn/10434666 Cytokine 30 (2005) 378e381
Association of interleukin-1B (IL-1B) gene polymorphisms with risk of gastric cancer in Chinese population Wei-Hua Zhang a,b, Xun-Ling Wang a, Jun Zhou c, Li-Zhe An a,*, Xiao-Dong Xie a,) b
a School of Life Science, Lanzhou University, Lanzhou, Gansu, China Department of Gastroenterology, The Gansu Provincial Cancer Hospital, Lanzhou, Gansu, China c Department of Pathology, The Gansu Provincial Cancer Hospital, Lanzhou, Gansu, China
Received 20 April 2004; received in revised form 9 February 2005; accepted 25 February 2005
Abstract The incidence of gastric cancer (GC) in China is among the highest in the world. In present work, 154 patients with GC and 166 healthy controls in population of north-western China were investigated to evaluate the genetic associations of IL-1B gene single nucleotide polymorphisms (SNP) and variable number tandem repeat (VNTR) polymorphisms of IL-1RN gene with increased risk of GC. The frequency of IL-1BC3954C/T was significantly higher in GC cases group (25.97%) than that in controls (4.82%) with odds ratio (OR) Z 6.93 (95% confidence interval [CI] 3.13e15.36); the frequencies of IL-1B-31C/T, IL-1B-31C/C and IL-1B-511C/T genotypes were also higher in GC cases group (51.95%, 23.38% and 50.65%) than those in controls (46.99%, 19.88% and 42.77%) with OR Z 1.48 (95% CI 0.88e2.49), OR Z 1.58 (95% CI 0.84e2.95) and OR Z 1.39 (95% CI 0.80e2.41), respectively. The results show that these SNPs of IL-1B gene are associated with significantly increased risk of GC. This is the first report that IL-1BC3954C/ T heterozygote is associated with greatly increased risk of GC. The results of this study did not support the report that IL-1RN*2C genotypes were associated with increased risk of GC in Chinese population. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: IL-1B; IL-1RN; Polymorphism; Risk of gastric cancer; SNP
1. Introduction The mortality of GC in China is among the highest in the world, especially in north-western China [1]. It is considered that GC is a complicated disease of multi-pathogeny including environmental and genetic factors [2].
* Corresponding authors. Tel.: C86 931 8279963/8912560; fax: C86 931 8912561. E-mail addresses: [email protected] (L.-Z. An), xdxie@ lzu.edu.cn (X.-D. Xie). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.02.002
The IL-1 genes cluster consists of 3 linked genes: IL-1A, IL-1B and IL-1RN, mapping to chromosome 2q13e24 that encodes the glycoproteins IL-1a, IL-1b and IL-1 receptor antagonist (IL-1Ra). IL-1a and IL-1b are pro-inflammatory cytokines, whereas IL-1Ra is an anti-inflammatory cytokine and competes with IL-1a and IL-1b for binding to IL-1 receptors. IL-1b is important in starting and enhancing the inflammatory response to Helicobacter pylori infection [3e5] and is also a very powerful inhibitor of gastric acid secretion [6,7], long-time inhibition of gastric acid secretion may lead to gastric mucosa atrophy which is considered as a pre-cancerous lesion. SNPs of IL-1B gene have been
379
W.-H. Zhang et al. / Cytokine 30 (2005) 378e381
reported, including IL-1B-31 representing TeC base transition, IL-1B-511 and IL-1BC3954 both representing CeT base transitions; and there is an 86 bp VNTR polymorphism consisting of 6 alleles (1e6 repeats) in the second intron of IL-1RN gene [8,9]. This work was aimed to evaluate the genetic associations of IL-1B gene SNPs (IL-1B-31, IL-1B-511 and IL-1BC3954) and IL-1RN gene VNTR polymorphisms with increased risk of GC in population of north-western China.
2. Results A total of 154 GC cases and 166 healthy controls were evaluated in this study from Oct. 2002 to Jan. 2005. All the frequencies of tested genotypes were in HardyeWeinberg equilibrium. There were no statistical differences on genotype frequencies between cancer tissues and para-cancer normal tissues (PCNT) in the cases group (data not shown). The frequency of IL1BC3954C/T genotype was significantly higher in GC cases group (25.97%) than that in controls (4.82%) with OR Z 6.93 (95% CI 3.13e15.36); the frequencies of IL1B-31C/T, IL-1B-31C/C and IL-1B-511C/T genotypes were also higher in cases group (51.95%, 23.38% and 50.65%) than those in controls (46.99%, 19.88% and 42.77%) with OR Z 1.48 (95% CI 0.88e2.49), OR Z 1.58 (95% CI 0.84e2.95) and OR Z 1.39 (95% CI 0.80e2.41), respectively (Table 1). Four alleles (allele 1, 2, 3 and 4) of IL-1RN VNTR were found in studied population, and there were no statistical differences on the frequencies of both IL-1RN*1/*2 and IL-1RN *2/*2 genotypes between the 2 groups.
3. Discussion Since El-Omar et al. [10,11] reported that IL-1B31CC and IL-1RN*2/*2 genotypes were associated with
a significantly increased risk of GC in Caucasians in 2000, some contradictory results from studies in different countries have been reported [12e16]. In present work, IL-1BC3954C/T heterozygote is associated with significantly increased risk of GC in Chinese population. This is the first report that shows IL-1BC3954C/T heterozygote is associated with greatly increased risk of GC. The results also show that carriers of IL-1B-31CC and IL1B-511C/T genotypes have higher susceptibility to GC in Chinese population; this is the same with that in Caucasians, but differs from that in Korean and Japanese populations [11e13]. The study also reveals that alleles of IL-1RN*2C are not associated with increased risk of GC in Chinese population, this is similar to that in Japanese and Korean populations [12,13] but differs from that in Caucasian populations in which IL-1RN*2C were associated with greatly increased risk of GC [10,15,16]. No IL-1BC3954T/T homozygote carrier was found in both groups in the study; this differs from the genotype frequencies of 6.76% in Caucasians [10], whether this is a genetic difference between Asian people and Caucasian populations still need to be proved; and this does not seem to support the hypothesis [10] that IL-1BC3954T/T homozygote may protect against GC. In conclusion, IL1BC3954C/T heterozygote is associated with significantly increased risk of GC, and carriers of IL-1B-31CC and/or IL-1B-511C/T genotypes also have higher susceptibility to GC; otherwise, IL-1RN*2C is not associated with the risk of GC in Chinese population.
4. Materials and methods In a caseecontrol study including 154 patients with GC and 166 healthy controls, samples of GC cases were surgically resected specimens obtained from 4 hospitals in Wuwei city where the incidence of GC (209.8 per 100,000 populations) ranks the first in China [1], Gansu
Table 1 IL-1 genotype frequencies in GC cases and controls in studied Chinese population Locus
Genotype
GC cases (n Z 154) N (%)
Controls (n Z 166) N (%)
IL-1B-31
T/T C/T C/C
38 (24.67) 80 (51.95) 36 (23.38)
55 (33.13) 78 (46.99) 33 (19.88)
1.00 1.48 (0.88e2.49) 1.58 (0.84e2.95)
IL-1B-511
C/C C/T T/T
34 (22.08) 78 (50.65) 42 (27.27)
43 (25.90) 71 (42.77) 52 (31.33)
1.00 1.39 (0.80e2.41) 1.02 (0.56e1.87)
IL-1BC3954
C/C C/T T/T
114 (74.03) 40 (25.97) 0
158 (95.18) 8 (4.82) 0
1.00 6.93 (3.13e15.36) 0
IL-1RN
1/1 1/2 2/2 1/3 1/4
134 (87.01) 19 (12.34) 0 0 1 (0.65)
128 31 5 2 0
1.00 0.59 (0.32e1.08) 0 0 Infinite
(77.11) (18.67) (3.01) (1.21)
OR (95% CI)
380
W.-H. Zhang et al. / Cytokine 30 (2005) 378e381
province in north-western China between Oct. 2002 and Nov. 2003. All of the GC cases were diagnosed histopathologically and none of the patients received any other treatment such as chemotherapy before operation. There were 130 (84.42%) males and 24 (15.58%) females in the cases group, average age was 54.7 years old (36e70 years). The controls were cancer-free healthy volunteers born in the same district matched with age, sex and ethnics from checking-up examinees in the same hospitals. There were 139 (83.73%) men and 27 (16.27%) women in the controls group, average age was 55.2 years old (35e73 years). This study was approved by the Ethics Committee of the Gansu Provincial Cancer Hospital. Genomic DNA of controls was extracted from EDTA anticoagulated peripheral blood according to a standard proteinase K digestion and phenol/chloroform extraction method. Surgically resected GC specimens were separated into cancer tissues and PCNT and stored at ÿ20 C separately. DNA of GC cases was separately extracted (by homogenizer) from both shattered GC tissues and PCNT by the same method. Details of genotyping techniques of IL-1B-31, IL-1B-511, IL1BC3954 and IL-1RN 86 bp VNTR are as follows. 4.1. IL-1B-31 allele genotyping PCR amplification was conducted in a volume of 50 ml with primers designed on the report of El-Omar et al. [10], F: 5#-AGAAGCTTCCACCAATACTC-3# and R: 5#-AGCACCTAGTTGTAAGGAAG-3#. PCR conditions were as follows: initial denaturing was performed at 94 C for 5 min, followed by 35 cycles of denaturing (94 C for 30 s), annealing (55 C, 30 s) and extension (72 C, 1 min), and then a final extension (72 C, 10 min). The PCR products contained a 239 bp fragment which was identified by sequencing using an ABI PRISM 377 sequencer-B2 system (performed by BioAsia Co. Ltd.). PCR products were digested by restriction endonuclease Alu1 (Toyobo Co., Ltd.) at 37 C for 18 h and then analyzed by 2% agarose gel electrophoresis. There were 3 fragments of 239, 137 and 102 bp coding alleles C/C (239 bp), T/T (137 and 102 bp), and C/T (239, 137 and 102 bp), respectively [10]. 4.2. IL-1B-511 allele genotyping PCR amplification with primers F: 5#-GCCT GAACCCTGCATACCGT-3# and R: 5#-GCCAATAG CCCTCCCTGTCT-3#. PCR conditions: initial denaturing (94 C for 10 min), followed by 5 cycles of denaturing (94 C, 30 s), annealing (65 C, 30 s) and extension (72 C, 30 s); then 30 cycles of 94 C for 30 s, 60 C for 30 s and 72 C for 30 s; finally, 5 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 30 s. PCR products were digested by restriction endonuclease Aval (MBI Fermentas Lithuania) at 37 C for 18 h and then
analyzed by 2.5% agarose gel electrophoresis. Alleles were coded as follows: 155 bp for T/T; 88 and 67 bp for C/C; 155, 88 and 67 bp for C/T [12]. 4.3. IL-1BC3954 alleles genotyping Primers F: 5#-GTTGTCATCAGACTTTGACC-3#, R: 5#-TTCAGTTCATATGGACCAGA-3#. PCR conditions: 3 cycles of denaturing (95 C, 2 min), annealing (55 C, 90 s) and extension (74 C, 1 min); followed by 32 cycles of 94 C for 1 min, 55 C for 1 min, and 74 C for 1 min; and a final extension (72 C, 10 min). The PCR products were digested by restriction enzyme Taq1 (MBI Fermentas Lithuania) at 65 C for 16 h and then analyzed by 2.5% agarose gel electrophoresis. There were 3 fragments of 249, 135 and 114 bp coding alleles T/T (249 bp), C/C (135 and 114 bp) and C/T (249, 135 and 114 bp), respectively [17,18]. 4.4. Genotyping of IL-1RN 86 bp VNTR Primers F: 5#-CCCCTCAGCAACACTCC-3# and R: 5#-GGTCAGAAGGGCAGAGA-3#. PCR conditions: initial denaturing (95 C, 2 min), followed by 35 cycles of denaturing (94 C, 1 min), annealing (59 C, 1 min) and extension (70 C, 1 min); and a final extension (72 C, 5 min). The PCR products were analyzed by 2% agarose gel electrophoresis. Alleles were coded conventionally as follows: allele 1 Z 4 repeats, allele 2 Z 2 repeats, allele 3 Z 5 repeats, and allele 4 Z 3 repeats [8]. 4.5. Statistical analysis HardyeWeinberg equilibrium of alleles at individual loci was assessed by c2 statistics. Genotype frequencies were analyzed by odds ratio and 95% CI with statistics software (F:/OddsCCI.htm).
Acknowledgments This work was supported by grant of major project (3ZS041-A25-013) and Gansu Key Technologies R&D Program (GS022-A41-045) from the Gansu Provincial Science and Technology Committee, China.
References [1] Sun XD, Mu R, Zhou YS, Dai XD, Qiao YL, Zhang SW, et al. Investigation and analysis on mortality of gastric cancer in China between 1990e1992. Chin J Oncol 2002;24(1):4e8 [in Chinese with English abstracts]. [2] Stadtlander CT, Waterbor J. Molecular epidemiology pathogenesis and prevention of gastric cancer. Carcinogenesis 1999;20: 2195e207.
W.-H. Zhang et al. / Cytokine 30 (2005) 378e381 [3] Noach LA, Bosma NB, Jansen J, Hoek FJ, van Deventer SJ, Tytgat GN. Mucosal tumor necrosis factor-alpha, interleukin1beta, and interleukin-8 production in patients with Helicobacter pylori infection. Scand J Gastroenterol 1994;29:425e9. [4] Basso D, Scrigner M, Toma A, Navaglia F, DiMario F. Helicobacter pylori infection enhances mucosal interleukin-1beta, interleukin-6, and the soluble receptor of interleukin-2. Int J Clin Lab Res 1996;26:207e10. [5] Jung HC, Kim JM, Song IS, Kim CY. Helicobacter pylori induces an array of pro-inflammatory cytokines in human gastric epithelial cells: Quantification of mRNA for interleukin-8, -1 alpha/beta, granulocyteemacrophage colony-stimulating factor, monocyte chemoattractant protein-1 and tumour necrosis factoralpha. J Gastroenterol Hepatol 1997;12:473e80. [6] Wallace JL, Cucala M, Mugridge K, Parente L. Secretagogue specific effects of interleukin-1 on gastric acid secretion. Am J Physiol 1991;261:G559e64. [7] Beales IL, Calam J. Interleukin 1 beta and tumour necrosis factor alpha inhibit acid secretion in cultured rabbit parietal cells by multiple pathways. Gut 1998;42:227e34. [8] Tarlow JK, Blakemore AIF, Lennard A, Solari R, Hughes HN, Steinkasserer A, et al. Polymorphism in human IL-1 receptor antagonist gene intron 2 is caused by variable numbers of an 86-bp tandem repeat. Hum Genet 1993;91:403e4. [9] Vamvakopoulos JE, Taylor CJ, Morris-Stiff GJ, Greens C, Metcalfe S. The interleukin-1 receptor antagonist gene: a singlecopy variant of the intron 2 variable number tandem repeat (VNTR) polymorphism. Eur J Immunogenet 2002;29:337e40. [10] El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000;404:398e402.
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[11] El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, et al. The role of interleukin-1 polymorphisms in the pathogenesis of gastric cancer. Nature 2001;412:99. [12] Kato S, Onda M, Yamada S, Matsuda N, Tokunaga A, Matsukura N. Association of the interleukin-1b genetic polymorphism and gastric cancer risk in Japanese. J Gastroenterol 2001;36:696e9. [13] Lee SG, Kin B, Choi WY, Lee IC, Choi JW, Song KY. Lack of association between pro-inflammatory genotypes of the interleukin-1 (IL-1B -31C/C and IL-1RN*2/*2) and gastric cancer/duodenal ulcer in Korean population. Cytokine 2003;21:167e71. [14] Hamajima N, Matsuo K, Saito T, Tajima K, Okuma K, Yamao K, et al. Interleukin 1 polymorphisms, lifestyle factors, and Helicobacter pylori infection. Jpn J Cancer Res 2001;92: 383e9. [15] Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C, et al. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001;121(4):823e9. [16] Ce`u F, Jose` CM, Paul P, Raquel S, So´nia S, Ralph C, et al. Helicobacter pylori and interleukin 1 genotyping: An opportunity to identify high-risk individuals for gastric carcinoma. J Natl Cancer Inst 2002;94:1680e7. [17] Bioque G, Crusius JBA, Koutroubakis I, Bouma G, Kostense PJ, Meuwissen SGM, et al. Allelic polymorphism in IL-1b and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease. Clin Exp Immunol 1995;102:379e83. [18] Hurme M, Santtila S. IL-1 receptor antagonist (IL-1Ra) plasma levels are coordinately regulated by both IL-1Ra and IL-1b genes. Eur J Immunol 1998;28:2598e602.
Association of interleukin-1B (IL-1B) gene polymorphisms with risk of gastric cancer in Chinese population Wei-Hua Zhang a,b, Xun-Ling Wang a, Jun Zhou c, Li-Zhe An a,*, Xiao-Dong Xie a,) b
a School of Life Science, Lanzhou University, Lanzhou, Gansu, China Department of Gastroenterology, The Gansu Provincial Cancer Hospital, Lanzhou, Gansu, China c Department of Pathology, The Gansu Provincial Cancer Hospital, Lanzhou, Gansu, China
Received 20 April 2004; received in revised form 9 February 2005; accepted 25 February 2005
Abstract The incidence of gastric cancer (GC) in China is among the highest in the world. In present work, 154 patients with GC and 166 healthy controls in population of north-western China were investigated to evaluate the genetic associations of IL-1B gene single nucleotide polymorphisms (SNP) and variable number tandem repeat (VNTR) polymorphisms of IL-1RN gene with increased risk of GC. The frequency of IL-1BC3954C/T was significantly higher in GC cases group (25.97%) than that in controls (4.82%) with odds ratio (OR) Z 6.93 (95% confidence interval [CI] 3.13e15.36); the frequencies of IL-1B-31C/T, IL-1B-31C/C and IL-1B-511C/T genotypes were also higher in GC cases group (51.95%, 23.38% and 50.65%) than those in controls (46.99%, 19.88% and 42.77%) with OR Z 1.48 (95% CI 0.88e2.49), OR Z 1.58 (95% CI 0.84e2.95) and OR Z 1.39 (95% CI 0.80e2.41), respectively. The results show that these SNPs of IL-1B gene are associated with significantly increased risk of GC. This is the first report that IL-1BC3954C/ T heterozygote is associated with greatly increased risk of GC. The results of this study did not support the report that IL-1RN*2C genotypes were associated with increased risk of GC in Chinese population. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: IL-1B; IL-1RN; Polymorphism; Risk of gastric cancer; SNP
1. Introduction The mortality of GC in China is among the highest in the world, especially in north-western China [1]. It is considered that GC is a complicated disease of multi-pathogeny including environmental and genetic factors [2].
* Corresponding authors. Tel.: C86 931 8279963/8912560; fax: C86 931 8912561. E-mail addresses: [email protected] (L.-Z. An), xdxie@ lzu.edu.cn (X.-D. Xie). 1043-4666/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2005.02.002
The IL-1 genes cluster consists of 3 linked genes: IL-1A, IL-1B and IL-1RN, mapping to chromosome 2q13e24 that encodes the glycoproteins IL-1a, IL-1b and IL-1 receptor antagonist (IL-1Ra). IL-1a and IL-1b are pro-inflammatory cytokines, whereas IL-1Ra is an anti-inflammatory cytokine and competes with IL-1a and IL-1b for binding to IL-1 receptors. IL-1b is important in starting and enhancing the inflammatory response to Helicobacter pylori infection [3e5] and is also a very powerful inhibitor of gastric acid secretion [6,7], long-time inhibition of gastric acid secretion may lead to gastric mucosa atrophy which is considered as a pre-cancerous lesion. SNPs of IL-1B gene have been
379
W.-H. Zhang et al. / Cytokine 30 (2005) 378e381
reported, including IL-1B-31 representing TeC base transition, IL-1B-511 and IL-1BC3954 both representing CeT base transitions; and there is an 86 bp VNTR polymorphism consisting of 6 alleles (1e6 repeats) in the second intron of IL-1RN gene [8,9]. This work was aimed to evaluate the genetic associations of IL-1B gene SNPs (IL-1B-31, IL-1B-511 and IL-1BC3954) and IL-1RN gene VNTR polymorphisms with increased risk of GC in population of north-western China.
2. Results A total of 154 GC cases and 166 healthy controls were evaluated in this study from Oct. 2002 to Jan. 2005. All the frequencies of tested genotypes were in HardyeWeinberg equilibrium. There were no statistical differences on genotype frequencies between cancer tissues and para-cancer normal tissues (PCNT) in the cases group (data not shown). The frequency of IL1BC3954C/T genotype was significantly higher in GC cases group (25.97%) than that in controls (4.82%) with OR Z 6.93 (95% CI 3.13e15.36); the frequencies of IL1B-31C/T, IL-1B-31C/C and IL-1B-511C/T genotypes were also higher in cases group (51.95%, 23.38% and 50.65%) than those in controls (46.99%, 19.88% and 42.77%) with OR Z 1.48 (95% CI 0.88e2.49), OR Z 1.58 (95% CI 0.84e2.95) and OR Z 1.39 (95% CI 0.80e2.41), respectively (Table 1). Four alleles (allele 1, 2, 3 and 4) of IL-1RN VNTR were found in studied population, and there were no statistical differences on the frequencies of both IL-1RN*1/*2 and IL-1RN *2/*2 genotypes between the 2 groups.
3. Discussion Since El-Omar et al. [10,11] reported that IL-1B31CC and IL-1RN*2/*2 genotypes were associated with
a significantly increased risk of GC in Caucasians in 2000, some contradictory results from studies in different countries have been reported [12e16]. In present work, IL-1BC3954C/T heterozygote is associated with significantly increased risk of GC in Chinese population. This is the first report that shows IL-1BC3954C/T heterozygote is associated with greatly increased risk of GC. The results also show that carriers of IL-1B-31CC and IL1B-511C/T genotypes have higher susceptibility to GC in Chinese population; this is the same with that in Caucasians, but differs from that in Korean and Japanese populations [11e13]. The study also reveals that alleles of IL-1RN*2C are not associated with increased risk of GC in Chinese population, this is similar to that in Japanese and Korean populations [12,13] but differs from that in Caucasian populations in which IL-1RN*2C were associated with greatly increased risk of GC [10,15,16]. No IL-1BC3954T/T homozygote carrier was found in both groups in the study; this differs from the genotype frequencies of 6.76% in Caucasians [10], whether this is a genetic difference between Asian people and Caucasian populations still need to be proved; and this does not seem to support the hypothesis [10] that IL-1BC3954T/T homozygote may protect against GC. In conclusion, IL1BC3954C/T heterozygote is associated with significantly increased risk of GC, and carriers of IL-1B-31CC and/or IL-1B-511C/T genotypes also have higher susceptibility to GC; otherwise, IL-1RN*2C is not associated with the risk of GC in Chinese population.
4. Materials and methods In a caseecontrol study including 154 patients with GC and 166 healthy controls, samples of GC cases were surgically resected specimens obtained from 4 hospitals in Wuwei city where the incidence of GC (209.8 per 100,000 populations) ranks the first in China [1], Gansu
Table 1 IL-1 genotype frequencies in GC cases and controls in studied Chinese population Locus
Genotype
GC cases (n Z 154) N (%)
Controls (n Z 166) N (%)
IL-1B-31
T/T C/T C/C
38 (24.67) 80 (51.95) 36 (23.38)
55 (33.13) 78 (46.99) 33 (19.88)
1.00 1.48 (0.88e2.49) 1.58 (0.84e2.95)
IL-1B-511
C/C C/T T/T
34 (22.08) 78 (50.65) 42 (27.27)
43 (25.90) 71 (42.77) 52 (31.33)
1.00 1.39 (0.80e2.41) 1.02 (0.56e1.87)
IL-1BC3954
C/C C/T T/T
114 (74.03) 40 (25.97) 0
158 (95.18) 8 (4.82) 0
1.00 6.93 (3.13e15.36) 0
IL-1RN
1/1 1/2 2/2 1/3 1/4
134 (87.01) 19 (12.34) 0 0 1 (0.65)
128 31 5 2 0
1.00 0.59 (0.32e1.08) 0 0 Infinite
(77.11) (18.67) (3.01) (1.21)
OR (95% CI)
380
W.-H. Zhang et al. / Cytokine 30 (2005) 378e381
province in north-western China between Oct. 2002 and Nov. 2003. All of the GC cases were diagnosed histopathologically and none of the patients received any other treatment such as chemotherapy before operation. There were 130 (84.42%) males and 24 (15.58%) females in the cases group, average age was 54.7 years old (36e70 years). The controls were cancer-free healthy volunteers born in the same district matched with age, sex and ethnics from checking-up examinees in the same hospitals. There were 139 (83.73%) men and 27 (16.27%) women in the controls group, average age was 55.2 years old (35e73 years). This study was approved by the Ethics Committee of the Gansu Provincial Cancer Hospital. Genomic DNA of controls was extracted from EDTA anticoagulated peripheral blood according to a standard proteinase K digestion and phenol/chloroform extraction method. Surgically resected GC specimens were separated into cancer tissues and PCNT and stored at ÿ20 C separately. DNA of GC cases was separately extracted (by homogenizer) from both shattered GC tissues and PCNT by the same method. Details of genotyping techniques of IL-1B-31, IL-1B-511, IL1BC3954 and IL-1RN 86 bp VNTR are as follows. 4.1. IL-1B-31 allele genotyping PCR amplification was conducted in a volume of 50 ml with primers designed on the report of El-Omar et al. [10], F: 5#-AGAAGCTTCCACCAATACTC-3# and R: 5#-AGCACCTAGTTGTAAGGAAG-3#. PCR conditions were as follows: initial denaturing was performed at 94 C for 5 min, followed by 35 cycles of denaturing (94 C for 30 s), annealing (55 C, 30 s) and extension (72 C, 1 min), and then a final extension (72 C, 10 min). The PCR products contained a 239 bp fragment which was identified by sequencing using an ABI PRISM 377 sequencer-B2 system (performed by BioAsia Co. Ltd.). PCR products were digested by restriction endonuclease Alu1 (Toyobo Co., Ltd.) at 37 C for 18 h and then analyzed by 2% agarose gel electrophoresis. There were 3 fragments of 239, 137 and 102 bp coding alleles C/C (239 bp), T/T (137 and 102 bp), and C/T (239, 137 and 102 bp), respectively [10]. 4.2. IL-1B-511 allele genotyping PCR amplification with primers F: 5#-GCCT GAACCCTGCATACCGT-3# and R: 5#-GCCAATAG CCCTCCCTGTCT-3#. PCR conditions: initial denaturing (94 C for 10 min), followed by 5 cycles of denaturing (94 C, 30 s), annealing (65 C, 30 s) and extension (72 C, 30 s); then 30 cycles of 94 C for 30 s, 60 C for 30 s and 72 C for 30 s; finally, 5 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 30 s. PCR products were digested by restriction endonuclease Aval (MBI Fermentas Lithuania) at 37 C for 18 h and then
analyzed by 2.5% agarose gel electrophoresis. Alleles were coded as follows: 155 bp for T/T; 88 and 67 bp for C/C; 155, 88 and 67 bp for C/T [12]. 4.3. IL-1BC3954 alleles genotyping Primers F: 5#-GTTGTCATCAGACTTTGACC-3#, R: 5#-TTCAGTTCATATGGACCAGA-3#. PCR conditions: 3 cycles of denaturing (95 C, 2 min), annealing (55 C, 90 s) and extension (74 C, 1 min); followed by 32 cycles of 94 C for 1 min, 55 C for 1 min, and 74 C for 1 min; and a final extension (72 C, 10 min). The PCR products were digested by restriction enzyme Taq1 (MBI Fermentas Lithuania) at 65 C for 16 h and then analyzed by 2.5% agarose gel electrophoresis. There were 3 fragments of 249, 135 and 114 bp coding alleles T/T (249 bp), C/C (135 and 114 bp) and C/T (249, 135 and 114 bp), respectively [17,18]. 4.4. Genotyping of IL-1RN 86 bp VNTR Primers F: 5#-CCCCTCAGCAACACTCC-3# and R: 5#-GGTCAGAAGGGCAGAGA-3#. PCR conditions: initial denaturing (95 C, 2 min), followed by 35 cycles of denaturing (94 C, 1 min), annealing (59 C, 1 min) and extension (70 C, 1 min); and a final extension (72 C, 5 min). The PCR products were analyzed by 2% agarose gel electrophoresis. Alleles were coded conventionally as follows: allele 1 Z 4 repeats, allele 2 Z 2 repeats, allele 3 Z 5 repeats, and allele 4 Z 3 repeats [8]. 4.5. Statistical analysis HardyeWeinberg equilibrium of alleles at individual loci was assessed by c2 statistics. Genotype frequencies were analyzed by odds ratio and 95% CI with statistics software (F:/OddsCCI.htm).
Acknowledgments This work was supported by grant of major project (3ZS041-A25-013) and Gansu Key Technologies R&D Program (GS022-A41-045) from the Gansu Provincial Science and Technology Committee, China.
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