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A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer
BBRC Biochemical and Biophysical Research Communications 324 (2004) 999–1003 www.elsevier.com/locate/ybbrc
A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer Enping Xua, Maode Laia,*, Bingjian Lva, Xiaoming Xinga, Qiong Huanga, Xiaoping Xiab a
Department of Pathology and Pathophysiology, Center for Environmental Genomics, School of Medicine, Zhejiang University, Hangzhou 310031, China b Clinical laboratory of Sir Run Run Shaw Hospital, Hangzhou, China Received 25 August 2004
Abstract Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C fi T transition at 1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 1306 C fi T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055–3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT + TT genotypes. Our data suggest that MMP-2 1306 C fi T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population. 2004 Elsevier Inc. All rights reserved. Keywords: Colorectal cancer; Matrix metalloproteinase-2; Single nucleotide polymorphism
Colorectal carcinoma (CRC) is one of the major causes of mortality worldwide. The adenoma–carcinoma sequence has been accepted as the main pathway involving numerous molecular events, such as activation of oncogenes and inactivation of suppressor genes [1]. Recently, with the completion of human genome project, single nucleotide polymorphisms (SNPs) have been the focus in biomedical research. Evidence is accumulating to suggest that the SNPs in several genes are implicated in the pathogenesis of CRC [2,3].
*
Corresponding author. Fax: +86 571 87217497. E-mail address: [email protected] (M. Lai).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.150
The matrix metalloproteinases (MMPs) comprise a family of at least 20 proteolytic enzymes which are capable of selectively degrading a wide spectrum of extracellular matrix proteins [4]. Accumulating evidence uncovered that overexpression of metalloproteinases was associated with tumor invasion, metastasis, and poorer prognosis [5–7]. Recently, several studies have documented the important role of the MMPs in the development of the cancer [8–10]. MMP-2 (gelatinase A) has type IV collagenolytic activity and is constitutively expressed by most connective tissue cells. Numerous studies have demonstrated that MMP-2 played an essential role in promoting tumor invasiveness, and metastasis and the levels of MMP-2 expression can be
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E. Xu et al. / Biochemical and Biophysical Research Communications 324 (2004) 999–1003
correlated with tumor grade [11,12]. Recently a naturally occurring sequence variation in the human MMP-2 gene promoter was reported [13]. This single nucleotide polymorphism C fi T transition at 1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. The CC genotype in the MMP-2 promoter has been reported to be associated with the development of lung cancer and gastric cardia adenocarcinoma [9,10]. However, there are no reports of the association between this polymorphism and the development and progression of colorectal cancer. In this study, we investigated whether this MMP-2 genetic polymorphism was associated with susceptibility to colorectal cancer in Chinese population. We also analyzed MMP-2 expression in colorectal cancer cell lines. Moreover, we examined the relations between the polymorphism and clinicopathological factors among colorectal cancer patients.
Materials and methods Clinical samples. A cohort of 126 unrelated colorectal cancer cases (71 male and 55 female; age range, 26–78 ; mean age, 58.4 years) were accrued from the affiliated hospital, Zhejiang University College of Medicines from January 2000 to September 2003. The histologically normal tissues in the distant margin to the tumor were collected at the time of surgery from patient who were undergoing resection of colorectal tumors. Other clinicopathological data were also reviewed via patients medical records and 116 cases of clinicopathological data were intact. Healthy controls were obtained from people coming for routine check-up without evidence of cancer history or family cancer predispositions and were frequency-matched to the cases on sex and age (±5 years). All patients and controls were well informed. MMP-2 genotyping. The method used to determine the MMP-2 1306 C/T polymorphism was PCR-based denaturing high performance liquid chromatography (DHPLC) and sequencing. Genomic DNA was prepared by digestion with proteinase K and phenol–chloroform extraction from surgically resected normal tissues in the distant margin to the tumor and the peripheral blood of the controls. PCR was carried out in a total volume of 25 ll containing 100 ng DNA, 0.25 lM forward and reverse primers, (5 0 -CTG ACC CCC AGT CCT ATC TGC C-3 0 and 5 0 -TGT TGG GAA CGC CTG ACT TCA G, respectively) [13], 200 lM dNTPs, 2.0 mM MgCl2, and 1.0 U Taq DNA polymerase with 1· reaction buffer (Sangon). The polymerase chain reaction started with 3 min of incubation at 94C, followed by 35 cycles of 30 s at 94 C, 30 s at 59 C, and 30 s at 72 C, and a final elongation of 5 min at 72 C. The amplification was verified on an agarose gel (2%). DHPLC analysis was performed on a Wave DNA Fragment Analysis System (Transgenomic) as previously reported [14]. Briefly, before applied to the DHPLC cartridge, each PCR product was mixed with that of known wild-type (CC), denatured at 95 C for 5 min, and then slowly reannealed by ramping the temperature down to 25 C at a rate of 0.1 C/4 s (45 min) to form homo- and/or heteroduplex. The genotypes of MMP-2 1306 C/T revealed by DHPLC analysis were further confirmed by DNA sequencing. Three different allelic PCR products were directly analyzed with an ABI PRISM 377 automatic sequencer using a dye terminator sequencing kit. To ensure quality control, genotyping was performed with blinding to case control status, and 20% random samples of cases and controls were genotyped twice, the reproducibility being 100%.
Cell culture and MMP-2 genotyping and RT-PCR. Four human colorectal cancer cell lines (SW480, SW620, HCe8693, and HR8348) were used. HCe8693 and HR8348 were kindly provided by Zhejiang Cancer Hospital. SW480 and SW620 were donated by Cancer Institute, Zhejiang University. Cells were grown in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum in an incubator under a humidified atmosphere of 5% CO2 at 37 C. The MMP-2 genotype of each line was also determined by PCR-based DHPLC and sequencing as described above. The mRNA levels of MMP-2 expression were analyzed by semi-quantitative RT-PCR. Total RNA was extracted using Trizole (Invitrogen), and cDNA was synthesized by RevertAid H Minus M-MuLv reverse transcriptase (MBI), in the presence of oligo(dT)18 primers and a ribonuclease inhibitor, in a 20 ll reaction volume, at 42 C for 1 h. The pair of primers used to amplify the cDNA of MMP-2 was: 5 0 -ACC CAT TTA CAC CTA CAC CAA G-3 0 and 5 0 -GTA TAC CGC ATC AAT CTT TTC CG-3 0 . Total volume of 25 ll of each reaction mixture contained 0.5 ll cDNA solution, 0.25 lM forward and reverse primers, respectively, 200 lM dNTP, 2.0 mM MgCl2, and 1.0 U Taq DNA polymerase with 1· reaction buffer (Sangon). Thermal cycling was carried out as follows: 94 C for 3 min, 40 cycles of 30 s at 94 C, 30 s at 60 C, and 30 s at 72 C, and a final elongation of 5 min at 72 C. To examine the quality and quantity of synthesized cDNA, b-actin cDNA was amplified using the primer set: 5 0 -CGC CGC GCT CGT CGT CGA CA-3 0 and 5 0 -GTC ACG CAC GAT TTC CCG CT-3 0 . The thermal profile was 94 C for 3 min, 26 cycles of 30 s at 94 C, 30 s at 60 C, 30 s at 72 C, and a final elongation of 5 min at 72 C. A 10 ll aliquot of each PCR product was electrophoresed in a 2% agarose gel containing ethidium bromide and visualized under UV transillumination. Statistical analysis. v2 test was used to test differences in the genotype distribution between the patients and the controls. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated as the estimate of the relative risk. Relations between the genotype and clinicopathological characteristics of colorectal cancer patients were calculated by FisherÕs exact test. All statistical analyses were performed with the SPSS version 11.0.
Results The identification of 1306 C/T polymorphism in MMP-2 by DHPLC was quite a straightforward procedure. As shown in Fig. 1A, samples with intact sequence eluted as single peak, whereas double peaks consisting of the homoduplex and the heteroduplex formations characterized heterozygous CT or homozygous CC samples. To determine whether they are heterozygotes or homozygotes, each sample with double peaks pattern in the first DHPLC were detected without adding wild-type DNA prior to the denaturation step. If the genotype of the sample is homozygote, a single peak pattern should appear in the chromatogram (Fig. 1B). However when the sample is heterozygote, the appearance of the chromatogram should be double peaks.(Fig. 1B). The 1306 C fi T polymorphism was confirmed by DNA sequencing (Fig. 1C). The MMP-2 genotype distribution for cases and controls are shown in Table 1. The genotype frequencies in controls were fit the Hardy–Weinberg law and the allele frequencies in the above healthy controls were consistent with those reported by Yu et al. [9]. A higher frequency of C/C (84.1%) was found in the colorectal cancer
E. Xu et al. / Biochemical and Biophysical Research Communications 324 (2004) 999–1003
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Table 2 Stratified analysis of MMP-2 genotypes by gender MMP-2 genotypes
Cases
Controls
ORa (95% CI)
Male CT, TT CC
14 57
19 52
1.488 (0.678–3.265)
Female CT, TT CC
6 49
15 40
3.063 (1.088–8.619)
a
Fig. 1. Representative DHPLC profiles for the MMP-2 1306 C/T polymorphisms and DNA sequencing analysis. At the first DHPLC, the CC genotype was discriminated from the CT or TT genotypes (A). To determine whether they were heterozygous or homozygous, each sample with double peak pattern in the first DHPLC was detected without adding wild-type DNA prior to the denaturation step. If the genotype of the sample is homozygote, the chromatogram should appear as a single peak. However when the sample is heterozygote, the appearance of the chromatogram should be double peaks (B). The 1306 C fi T polymorphism was confirmed by DNA sequencing (C).
Table 1 Distribution of MMP-2 genotypes and risk estimate MMP-2 genotypes
CT TT CC a
Cases (n = 126) n (%)
Controls (n = 126) n (%)
19 (15.1) 1 (0.8) 106 (84.1)
32 (25.4) 2 (1.6) 92 (73)
P
ORa (95% CI)
<0.05
1.959 (1.055–3.637)
Odds ratio of CC genotype relative to CT + TT genotypes.
patients than in the controls (73%, P < 0.05). There was a significantly elevated colorectal cancer risk for individuals with C/C genotype (OR, 1.959; 95% CI, 1.055– 3.637), When the C/T and T/T genotypes were combined. Moreover, an overall greater increase risk for colorectal cancer risk was observed in women (OR, 3.063; 95% CI, 1.088–8.619) compared with men (OR, 1.488; 95% CI, 0.678–3.625, Table 2) when stratified by gender. Colorectal cancer cell lines SW480, SW620, and HR8348 showed CC genotype, and HCe8693 showed CT genotype. MMP-2 expression in 3 cell lines with
Odds ratio of CC genotype relative to CT + TT genotypes.
Fig. 2. b-Actin expression was identical among the four cell lines, while the MMP-2 expression was higher in cells (SW480, SW620, and HR8348) containing CC compared with cell (HCe8693) with CT genotype.
Table 3 Correlation between the genotype of MMP-2 1306 C/T and clinicopathological characteristics of 116 colorectal patients Genotype CC
CT + TT
Age (years) <60 (n = 62) 660 (n = 54)
53 44
9 10
Gender Male (n = 65) Female (n = 51)
52 45
13 6
Grosstype Protrude type (n = 33) Ulcerative type (n = 70) Infiltrating type (n = 7)
27 59 5
6 11 2
Tumor location Colon (n = 63) Rectum (n = 53)
50 47
13 6
Differentiation Well-differentiated (n = 15) Moderately differentiated (n = 76) Poorly differentiated (n = 25)
7 69 21
6 9 4
pTNM stage Stage I, II (n = 63) Stage III, IV (n = 53)
52 45
10 9
Adventitia/serosa involvement Y (n = 89) N (n = 27)
78 19
8 11
P
0.62
0.314
0.683
0.214
0.155
1.000
0.042
Correlation was analyzed by FisherÕs test. The P values <0.05 were regarded as statistically significant.
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CC genotype were much higher compared with cell with CT genotype (Fig. 2). Finally, we analyzed the correlation between the genotypes and clinicopathological features among colorectal cancer patients (Table 3). We only found that colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement in compared with CT plus TT genotypes. However, no significant differences were found in other clinicopathological features.
Discussion In this study, we investigated the role of 1306 C fi T polymorphism within MMP-2 gene promoter in colorectal cancer in the Chinese population. Our results demonstrated a significant difference in MMP-2 allelic variant distribution with a 1.959 exceeding risk of colorectal cancer for C/C homozygous patients and this polymorphism may influence the ability in colorectal cancer invasion. MMPs play an important role in connective tissue remodeling during tissue repair cell migration, angiogenesis, tissue morphogenesis, and growth. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. Recently, there are a number of function-altering polymorphisms found in the promoters of MMP genes [13,15–17]. Some of these promoter polymorphisms have allele-specific affects on the regulation of MMP gene transcription and are associated with the development and progression of coronary heart disease and cancers, and possibly abdominal aortic aneurysms [8–10,17–26]. The MMP-2 possesses proteolytic activity against type IV collagen, a major component of the basement membrane, and is therefore implicated in an extensive array of pathologies including atherogenesis, arthritis, and tumor growth and metastasis. In colorectal cancer, MMP-2 overexpression had been reported to be associated with the tumor invasion and metastasis, leading to poor progression [27–29]. However, there are no reports on the association between the MMP-2 expression and the development of colorectal cancer. Recently, a single nucleotide polymorphism 1306 C fi T in the promoter of MMP-2 gene was reported which disrupts an Sp1-type promoter site (CCACC box) and displayed a strikingly lower promoter activity with the T allele. Sp1 is a ubiquitously expressed transcription factor that binds to GC/GT-rich elements and regulates a variety of genes. The CCACC box has been shown to be essential for SP1 binding and promoter function in several genes by invariably activating transcription [30–32]. The MMP-2 1306 C fi T polymorphism that abolishes Sp1 binding has
the potential to affect the level and specificity of gene transcription. This has been demonstrated in vitro in transient transfection experiments [13]. In this study, we also examined the relations between this SNP and gene expression in cell lines. Levels of expression in cells containing CC were much higher compared with cell with CT genotype. It has been suggested that carcinogenesis was a multicellular and multistage process in which the destruction of the microenvironment was required for conversion of normal tissue to tumor. Although MMPs are not oncogenic or mutagenic, they alter the microenvironment and may affect the process of carcinogenesis [33]. Our data provide the first molecular epidemiological evidence of the MMP-2 promoter polymorphisms associated with the risk of colorectal cancer. Moreover, we observed a higher colorectal cancer risk in women with the C/C genotype than in men with the same genotype. The data suggested a gender-specific effect to the MMP-2 polymorphism. There was one study showing that the activity of MMP-2 may be regulated by sex hormones. The secretion and activation of several MMPs including MMP-2 were inhibited by progesterone, which suggests that the mean level of MMP-2 may be less in women than in men during their lifetime [34]. Several MMPs, including MMP-2, have been reported to play an important role in cancer invasion and metastasis through their overexpression. In this study, 1306 C fi T in the promoter of MMP-2 gene was associated with the depth of tumor invasion, but not associated with TNM classification in colorectal cancer. So this SNP could be of predictive value for cancer invasiveness. In summary, this is the first study to demonstrated that MMP-2 1306 C fi T polymorphism may be associated with the risk of developing colorectal cancer and influence the ability in tumor invasion in a Chinese population, and this SNP could be of predictive value for colorectal cancer prevention and invasiveness.
Acknowledgment This work was supported by Grant 011107607 from Department of Science and Technology of Zhejiang Province, PRC.
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A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer Enping Xua, Maode Laia,*, Bingjian Lva, Xiaoming Xinga, Qiong Huanga, Xiaoping Xiab a
Department of Pathology and Pathophysiology, Center for Environmental Genomics, School of Medicine, Zhejiang University, Hangzhou 310031, China b Clinical laboratory of Sir Run Run Shaw Hospital, Hangzhou, China Received 25 August 2004
Abstract Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C fi T transition at 1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 1306 C fi T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055–3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT + TT genotypes. Our data suggest that MMP-2 1306 C fi T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population. 2004 Elsevier Inc. All rights reserved. Keywords: Colorectal cancer; Matrix metalloproteinase-2; Single nucleotide polymorphism
Colorectal carcinoma (CRC) is one of the major causes of mortality worldwide. The adenoma–carcinoma sequence has been accepted as the main pathway involving numerous molecular events, such as activation of oncogenes and inactivation of suppressor genes [1]. Recently, with the completion of human genome project, single nucleotide polymorphisms (SNPs) have been the focus in biomedical research. Evidence is accumulating to suggest that the SNPs in several genes are implicated in the pathogenesis of CRC [2,3].
*
Corresponding author. Fax: +86 571 87217497. E-mail address: [email protected] (M. Lai).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.150
The matrix metalloproteinases (MMPs) comprise a family of at least 20 proteolytic enzymes which are capable of selectively degrading a wide spectrum of extracellular matrix proteins [4]. Accumulating evidence uncovered that overexpression of metalloproteinases was associated with tumor invasion, metastasis, and poorer prognosis [5–7]. Recently, several studies have documented the important role of the MMPs in the development of the cancer [8–10]. MMP-2 (gelatinase A) has type IV collagenolytic activity and is constitutively expressed by most connective tissue cells. Numerous studies have demonstrated that MMP-2 played an essential role in promoting tumor invasiveness, and metastasis and the levels of MMP-2 expression can be
1000
E. Xu et al. / Biochemical and Biophysical Research Communications 324 (2004) 999–1003
correlated with tumor grade [11,12]. Recently a naturally occurring sequence variation in the human MMP-2 gene promoter was reported [13]. This single nucleotide polymorphism C fi T transition at 1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. The CC genotype in the MMP-2 promoter has been reported to be associated with the development of lung cancer and gastric cardia adenocarcinoma [9,10]. However, there are no reports of the association between this polymorphism and the development and progression of colorectal cancer. In this study, we investigated whether this MMP-2 genetic polymorphism was associated with susceptibility to colorectal cancer in Chinese population. We also analyzed MMP-2 expression in colorectal cancer cell lines. Moreover, we examined the relations between the polymorphism and clinicopathological factors among colorectal cancer patients.
Materials and methods Clinical samples. A cohort of 126 unrelated colorectal cancer cases (71 male and 55 female; age range, 26–78 ; mean age, 58.4 years) were accrued from the affiliated hospital, Zhejiang University College of Medicines from January 2000 to September 2003. The histologically normal tissues in the distant margin to the tumor were collected at the time of surgery from patient who were undergoing resection of colorectal tumors. Other clinicopathological data were also reviewed via patients medical records and 116 cases of clinicopathological data were intact. Healthy controls were obtained from people coming for routine check-up without evidence of cancer history or family cancer predispositions and were frequency-matched to the cases on sex and age (±5 years). All patients and controls were well informed. MMP-2 genotyping. The method used to determine the MMP-2 1306 C/T polymorphism was PCR-based denaturing high performance liquid chromatography (DHPLC) and sequencing. Genomic DNA was prepared by digestion with proteinase K and phenol–chloroform extraction from surgically resected normal tissues in the distant margin to the tumor and the peripheral blood of the controls. PCR was carried out in a total volume of 25 ll containing 100 ng DNA, 0.25 lM forward and reverse primers, (5 0 -CTG ACC CCC AGT CCT ATC TGC C-3 0 and 5 0 -TGT TGG GAA CGC CTG ACT TCA G, respectively) [13], 200 lM dNTPs, 2.0 mM MgCl2, and 1.0 U Taq DNA polymerase with 1· reaction buffer (Sangon). The polymerase chain reaction started with 3 min of incubation at 94C, followed by 35 cycles of 30 s at 94 C, 30 s at 59 C, and 30 s at 72 C, and a final elongation of 5 min at 72 C. The amplification was verified on an agarose gel (2%). DHPLC analysis was performed on a Wave DNA Fragment Analysis System (Transgenomic) as previously reported [14]. Briefly, before applied to the DHPLC cartridge, each PCR product was mixed with that of known wild-type (CC), denatured at 95 C for 5 min, and then slowly reannealed by ramping the temperature down to 25 C at a rate of 0.1 C/4 s (45 min) to form homo- and/or heteroduplex. The genotypes of MMP-2 1306 C/T revealed by DHPLC analysis were further confirmed by DNA sequencing. Three different allelic PCR products were directly analyzed with an ABI PRISM 377 automatic sequencer using a dye terminator sequencing kit. To ensure quality control, genotyping was performed with blinding to case control status, and 20% random samples of cases and controls were genotyped twice, the reproducibility being 100%.
Cell culture and MMP-2 genotyping and RT-PCR. Four human colorectal cancer cell lines (SW480, SW620, HCe8693, and HR8348) were used. HCe8693 and HR8348 were kindly provided by Zhejiang Cancer Hospital. SW480 and SW620 were donated by Cancer Institute, Zhejiang University. Cells were grown in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum in an incubator under a humidified atmosphere of 5% CO2 at 37 C. The MMP-2 genotype of each line was also determined by PCR-based DHPLC and sequencing as described above. The mRNA levels of MMP-2 expression were analyzed by semi-quantitative RT-PCR. Total RNA was extracted using Trizole (Invitrogen), and cDNA was synthesized by RevertAid H Minus M-MuLv reverse transcriptase (MBI), in the presence of oligo(dT)18 primers and a ribonuclease inhibitor, in a 20 ll reaction volume, at 42 C for 1 h. The pair of primers used to amplify the cDNA of MMP-2 was: 5 0 -ACC CAT TTA CAC CTA CAC CAA G-3 0 and 5 0 -GTA TAC CGC ATC AAT CTT TTC CG-3 0 . Total volume of 25 ll of each reaction mixture contained 0.5 ll cDNA solution, 0.25 lM forward and reverse primers, respectively, 200 lM dNTP, 2.0 mM MgCl2, and 1.0 U Taq DNA polymerase with 1· reaction buffer (Sangon). Thermal cycling was carried out as follows: 94 C for 3 min, 40 cycles of 30 s at 94 C, 30 s at 60 C, and 30 s at 72 C, and a final elongation of 5 min at 72 C. To examine the quality and quantity of synthesized cDNA, b-actin cDNA was amplified using the primer set: 5 0 -CGC CGC GCT CGT CGT CGA CA-3 0 and 5 0 -GTC ACG CAC GAT TTC CCG CT-3 0 . The thermal profile was 94 C for 3 min, 26 cycles of 30 s at 94 C, 30 s at 60 C, 30 s at 72 C, and a final elongation of 5 min at 72 C. A 10 ll aliquot of each PCR product was electrophoresed in a 2% agarose gel containing ethidium bromide and visualized under UV transillumination. Statistical analysis. v2 test was used to test differences in the genotype distribution between the patients and the controls. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated as the estimate of the relative risk. Relations between the genotype and clinicopathological characteristics of colorectal cancer patients were calculated by FisherÕs exact test. All statistical analyses were performed with the SPSS version 11.0.
Results The identification of 1306 C/T polymorphism in MMP-2 by DHPLC was quite a straightforward procedure. As shown in Fig. 1A, samples with intact sequence eluted as single peak, whereas double peaks consisting of the homoduplex and the heteroduplex formations characterized heterozygous CT or homozygous CC samples. To determine whether they are heterozygotes or homozygotes, each sample with double peaks pattern in the first DHPLC were detected without adding wild-type DNA prior to the denaturation step. If the genotype of the sample is homozygote, a single peak pattern should appear in the chromatogram (Fig. 1B). However when the sample is heterozygote, the appearance of the chromatogram should be double peaks.(Fig. 1B). The 1306 C fi T polymorphism was confirmed by DNA sequencing (Fig. 1C). The MMP-2 genotype distribution for cases and controls are shown in Table 1. The genotype frequencies in controls were fit the Hardy–Weinberg law and the allele frequencies in the above healthy controls were consistent with those reported by Yu et al. [9]. A higher frequency of C/C (84.1%) was found in the colorectal cancer
E. Xu et al. / Biochemical and Biophysical Research Communications 324 (2004) 999–1003
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Table 2 Stratified analysis of MMP-2 genotypes by gender MMP-2 genotypes
Cases
Controls
ORa (95% CI)
Male CT, TT CC
14 57
19 52
1.488 (0.678–3.265)
Female CT, TT CC
6 49
15 40
3.063 (1.088–8.619)
a
Fig. 1. Representative DHPLC profiles for the MMP-2 1306 C/T polymorphisms and DNA sequencing analysis. At the first DHPLC, the CC genotype was discriminated from the CT or TT genotypes (A). To determine whether they were heterozygous or homozygous, each sample with double peak pattern in the first DHPLC was detected without adding wild-type DNA prior to the denaturation step. If the genotype of the sample is homozygote, the chromatogram should appear as a single peak. However when the sample is heterozygote, the appearance of the chromatogram should be double peaks (B). The 1306 C fi T polymorphism was confirmed by DNA sequencing (C).
Table 1 Distribution of MMP-2 genotypes and risk estimate MMP-2 genotypes
CT TT CC a
Cases (n = 126) n (%)
Controls (n = 126) n (%)
19 (15.1) 1 (0.8) 106 (84.1)
32 (25.4) 2 (1.6) 92 (73)
P
ORa (95% CI)
<0.05
1.959 (1.055–3.637)
Odds ratio of CC genotype relative to CT + TT genotypes.
patients than in the controls (73%, P < 0.05). There was a significantly elevated colorectal cancer risk for individuals with C/C genotype (OR, 1.959; 95% CI, 1.055– 3.637), When the C/T and T/T genotypes were combined. Moreover, an overall greater increase risk for colorectal cancer risk was observed in women (OR, 3.063; 95% CI, 1.088–8.619) compared with men (OR, 1.488; 95% CI, 0.678–3.625, Table 2) when stratified by gender. Colorectal cancer cell lines SW480, SW620, and HR8348 showed CC genotype, and HCe8693 showed CT genotype. MMP-2 expression in 3 cell lines with
Odds ratio of CC genotype relative to CT + TT genotypes.
Fig. 2. b-Actin expression was identical among the four cell lines, while the MMP-2 expression was higher in cells (SW480, SW620, and HR8348) containing CC compared with cell (HCe8693) with CT genotype.
Table 3 Correlation between the genotype of MMP-2 1306 C/T and clinicopathological characteristics of 116 colorectal patients Genotype CC
CT + TT
Age (years) <60 (n = 62) 660 (n = 54)
53 44
9 10
Gender Male (n = 65) Female (n = 51)
52 45
13 6
Grosstype Protrude type (n = 33) Ulcerative type (n = 70) Infiltrating type (n = 7)
27 59 5
6 11 2
Tumor location Colon (n = 63) Rectum (n = 53)
50 47
13 6
Differentiation Well-differentiated (n = 15) Moderately differentiated (n = 76) Poorly differentiated (n = 25)
7 69 21
6 9 4
pTNM stage Stage I, II (n = 63) Stage III, IV (n = 53)
52 45
10 9
Adventitia/serosa involvement Y (n = 89) N (n = 27)
78 19
8 11
P
0.62
0.314
0.683
0.214
0.155
1.000
0.042
Correlation was analyzed by FisherÕs test. The P values <0.05 were regarded as statistically significant.
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E. Xu et al. / Biochemical and Biophysical Research Communications 324 (2004) 999–1003
CC genotype were much higher compared with cell with CT genotype (Fig. 2). Finally, we analyzed the correlation between the genotypes and clinicopathological features among colorectal cancer patients (Table 3). We only found that colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement in compared with CT plus TT genotypes. However, no significant differences were found in other clinicopathological features.
Discussion In this study, we investigated the role of 1306 C fi T polymorphism within MMP-2 gene promoter in colorectal cancer in the Chinese population. Our results demonstrated a significant difference in MMP-2 allelic variant distribution with a 1.959 exceeding risk of colorectal cancer for C/C homozygous patients and this polymorphism may influence the ability in colorectal cancer invasion. MMPs play an important role in connective tissue remodeling during tissue repair cell migration, angiogenesis, tissue morphogenesis, and growth. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. Recently, there are a number of function-altering polymorphisms found in the promoters of MMP genes [13,15–17]. Some of these promoter polymorphisms have allele-specific affects on the regulation of MMP gene transcription and are associated with the development and progression of coronary heart disease and cancers, and possibly abdominal aortic aneurysms [8–10,17–26]. The MMP-2 possesses proteolytic activity against type IV collagen, a major component of the basement membrane, and is therefore implicated in an extensive array of pathologies including atherogenesis, arthritis, and tumor growth and metastasis. In colorectal cancer, MMP-2 overexpression had been reported to be associated with the tumor invasion and metastasis, leading to poor progression [27–29]. However, there are no reports on the association between the MMP-2 expression and the development of colorectal cancer. Recently, a single nucleotide polymorphism 1306 C fi T in the promoter of MMP-2 gene was reported which disrupts an Sp1-type promoter site (CCACC box) and displayed a strikingly lower promoter activity with the T allele. Sp1 is a ubiquitously expressed transcription factor that binds to GC/GT-rich elements and regulates a variety of genes. The CCACC box has been shown to be essential for SP1 binding and promoter function in several genes by invariably activating transcription [30–32]. The MMP-2 1306 C fi T polymorphism that abolishes Sp1 binding has
the potential to affect the level and specificity of gene transcription. This has been demonstrated in vitro in transient transfection experiments [13]. In this study, we also examined the relations between this SNP and gene expression in cell lines. Levels of expression in cells containing CC were much higher compared with cell with CT genotype. It has been suggested that carcinogenesis was a multicellular and multistage process in which the destruction of the microenvironment was required for conversion of normal tissue to tumor. Although MMPs are not oncogenic or mutagenic, they alter the microenvironment and may affect the process of carcinogenesis [33]. Our data provide the first molecular epidemiological evidence of the MMP-2 promoter polymorphisms associated with the risk of colorectal cancer. Moreover, we observed a higher colorectal cancer risk in women with the C/C genotype than in men with the same genotype. The data suggested a gender-specific effect to the MMP-2 polymorphism. There was one study showing that the activity of MMP-2 may be regulated by sex hormones. The secretion and activation of several MMPs including MMP-2 were inhibited by progesterone, which suggests that the mean level of MMP-2 may be less in women than in men during their lifetime [34]. Several MMPs, including MMP-2, have been reported to play an important role in cancer invasion and metastasis through their overexpression. In this study, 1306 C fi T in the promoter of MMP-2 gene was associated with the depth of tumor invasion, but not associated with TNM classification in colorectal cancer. So this SNP could be of predictive value for cancer invasiveness. In summary, this is the first study to demonstrated that MMP-2 1306 C fi T polymorphism may be associated with the risk of developing colorectal cancer and influence the ability in tumor invasion in a Chinese population, and this SNP could be of predictive value for colorectal cancer prevention and invasiveness.
Acknowledgment This work was supported by Grant 011107607 from Department of Science and Technology of Zhejiang Province, PRC.
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