| Objective: Ovarian cancer is a common malignancy in gynecology. But women with ovarian cancer often are asymptomatic invasion and quick evolution. Furthermore, without the valid diagnosis method in the early stage, 70%～80% of the patients are diagnosed at an advanced stage, which has 5-year survival rates of just 25% to 30%. Though the etiopathogenisis of ovarian cancer is not very clear now, the study showed that 10% patients with ovarian cancer were genetic disorder. Therefor, it is the effective way to improve the survival rate of ovarian cancer to look for predisposing genes, high-risk group and early diagnosis method. Because the matrix metalloproteinases play important roles in all stages of cancer initiation and development, they have been focused as a group of important candidate genes in recent years.The metrix metalloproteinases (MMPs) are a family of highly conserved zinc-dependent proteolytic enzymes that degrade or break down extracellular matrix. The tissue inhibitors of metalloproteinases (TIMPs) are their specific inhibitors. The imbalance in the expression of MMPs and TIMPs plays important role in cancer development and aggression. The relationship between MMP-2, TIMP-2 and cancer is particularly interesting. With the molecularbiological development, studies have found that some single nucleotide polymorphisms (SNPs) located in the promoter of MMP-2 and TIMP-2 genes. They may influence the transcription of the gene and expression of protein and relate to occurrence and development of some tumor. This study was designed to investigate the association between single nucleotide polymorphisms (SNPs) in the promoter region of MMP-2 and TIMP-2 genes and the risk of epithelial ovarian cancer. By this way, we hope to offer some evidences for the prevention and therapy of ovarian cancer at molecular level.Methods: This hospital-based case-control study included 324 ovarian cancer patients and 246 healthy women. Five ml of venous blood from each subject was drawn in vacutainer tubes. Genomic DNA was extracted by using proteinase K digestion followed by a salting out procedure. MMP-2 C-1306T, C-735T and TIMP-2 G-418C SNPs were genotyped by polymerase chain reaction-restrictive fragment length polymorphism (PCR-RFLP) analysis.Statistical analysis was performed using SPSS11.5 software package. Hardy-Weinberg analysis was performed by comparing the observed and expected genotype frequencies in study groups using Chi-square test. Between cases and controls, the t test was used to examine the difference of age and age of menarche and the Chi-square test was used to examine the difference of gravidity and parity. Univariate comparisons of allele and genotype distribution were performed using Chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model. The MMP-2 C-1306T and C-735T haplotype frequencies and linkage disequilibrium coefficient were estimated by using EH linkage software and 2ld software. Tests for the interaction between MMP-2 and TIMP-2 genes were performed using the likelihood ratio test. P<0.05 was considered significant for all statistical analyses.Results1 The distributions of MMP-2 C-1306T,C-735T and TIMP-2 G-418C genotypes among healthy controls were compatible with those expected genotype frequencies from Hardy-Weinberg equilibrium (P=0.87,P=0.75 and P=0.81).2 The genotype and allele distributions of MMP-2 C-1306T were not significantly different in the ovarian cancer group to the control group (P=0.42 and P=0.55). Compared to the T/T+C/T genotype, C/C did not significantly modify the risk of ovarian cancer with odds ratio of 1.07 (95%CI=0.72～1.58). Stratification analysis showed no significant difference between the patient and control groups in allele or genotype distributions of MMP-2 C-1306T according to the pathological type, clinical stage and patient age of epithelial ovarian cancer (P>0.05).3 The frequencies of three genotypes (C/C, C/T and T/T) of MMP-2 C-735T in ovarian cancer patients and healthy controls were 66.7%, 28.0%, 5.3% and 55.9%, 39.2%, 4.9%, respectively. There was significant difference between the two groups (P=0.02). The frequencies of the C allele of the MMP-2 C-735T were significantly higher in ovarian cancer patients (80.7%) than those in healthy controls (75.5%). There was statistical difference between the two groups (P=0.04). Compared with the T/T+C/T genotypes, the C/C genotype significantly increased the risk of ovarian cancer (OR=1.58, 95%CI=1.12～2.23). Stratification analysis showed that subjects carrying C/C genotype were significantly associated with the risk of endometrioid ovarian cancer (OR=1.69, 95%CI=1.03～2.79) and tend to increase risk of the serous ovarian cancer (OR=1.58,95%CI=0.97～2.56) according to histological subtypes. When stratified by clinical stage, individuals with the C/C genotype were significantly associated with the earlier ovarian cancer (OR=1.88,95%CI=1.11～3.17) and moderately increased risk of the advanced ovarian cancer (OR=1.46, 95%CI=0.99～2.15). Furthermore, the increased risk of ovarian cancer was associated with the MMP-2 -735C/C genotype in subjects that were 50 or older, with odds ratio of 1.71 (95%CI=1.14～2.57).4 TIMP-2 G-418C genotype and allele frequencies did not differ between the ovarian cancer group and the control group (P=0.47 and P=0.33). Compared to the C/C+G/C genotypes, G/G did not significantly modify the risk of ovarian cancer with odds ratio of 1.13 (95%CI=0.80～1.62). But TIMP-2 418G/G genotype was associated with a trend for endometrioid ovarian cancer by stratification analysis according to histological subtypes (OR=1.62, 95%CI=0.94～2.78). No association was observed between the polymorphism and clinical stage or age at diagnosis.5 The combined effect of MMP-2 C-1306T and C-735T SNPs was analyzed by EH and 2ld software. The result showed that the C-1306-C-735 haplotype was the most common in the control group, which was 65.7%. But the frequencies of four haplotypes (T-1306-T-735, T-1306-C-735, C-1306-T-735 and C-1306-C-735) of MMP-2 C-1306T and C-735T were not significantly different between the patient and control groups (P=0.24). In addition, linkage disequilibrium analysis showed that the C-1306T and C-735T polymorphisms were in a linkage diseqilibrium (D'=0.78, P=0.00).6 The gene-gene interactions between the TIMP-2 G-418C and MMP-2 C-1306T or C-735T SNPs were analyzed using the likelihood ratio test. Although no significant interactions between TIMP-2 G-418C and MMP-2 C-1306T or MMP-2 C-735T were observed (P=0.74 and P=0.41), the C/CMMP-2 C-735T-G/GTIMP-2 G-418C frequencies of patients (46.3%) were significantly more than those of controls (36.4%) (P=0.02). Among subjects carrying C/CMMP-2 C-735T-G/GTIMP-2 G-418C had 1.67-fold risk of developing ovarian cancer than C/T+T/TMMP-2 C-735T-G/GTIMP-2 G-418C carriers (95%CI=1.10～2.54).Conclusions 1 The MMP-2 C-1306T SNP may have no susceptibility to the epithelial ovarian cancer.2 MMP-2 -735C/C genotype carriers were significantly increased the risk of epithelial ovarian cancer. Furthermore, there were significant association between MMP-2 C-735T polymorphism and tumor histological subtypes, clinical stage and age. It may be a potential risk factor for epithelial ovarian cancer.3 Although the MMP-2 C-1306T and C-735T SNPs showed linkage disequilibrium, there were no association between the haplotypes of C-1306T with C-735T polymorphisms and the risk of epithelial ovarian cancer development.4 The TIMP-2 G-418C SNP may be associated with the risk of different histological subtypes of epithelial ovarian cancer.5 No significant interactions were observed by analyzing gene-gene interactions between the TIMP-2 G-418C and MMP-2 C-1306T or C-735T SNPs. But subjects carrying C/CMMP-2 C-735T-G/GTIMP-2 G-418C may increase the risk of epithelial ovarian cancer and have an additive effect.
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