Variant genes are involving in extra cellular matrix (ECM) turnover, including matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). They have been considered as strong candidates for a genetic susceptibility factor to different diseases and cancer. Degradation of ECM is essential in many physiological processes, for example, during development, growth, and repair of tissue. ¹,²,³ Tumor cell invasion, metastasis and angiogenesis require controlled degradation of ECM. Increased expression of MMP enzymes is associated with invasion and metastasis of different malignances. ⁴ MMPs comprise a family of enzymes that are able to degrade components of ECM. ⁵ At least 26 human MMPs have been identified, which are classified according to their substrate specificity and structural similarities. ² These subgroups follow: Collagenases, gelatinases, stromelysins, matrilysins, and transmembrane MMPs. ⁴,⁵

Colorectal cancer (CRC) is the most common malignancy of the gastrointestinal tract. It still carries a relatively poor prognosis despite recent improvement in early diagnosis, surgical techniques, and chemotherapy. About 40% of patients who undergo operative resection will die within 5 years due to recurrent disease or metastasis. ⁶,⁷,⁸

Expression of the most MMPs is normally low in tissues and they are induced when remodeling of ECM is required. ⁹ MMPs expression in tumor cells is regulated primarily at the transcriptional level, but there is also evidence of modulation of mRNA stability in response to growth factors and cytokines. ⁹,¹⁰,¹¹ There is much evidence that MMPs play essential roles in migration, tumor growth, and development. ⁵,¹²,¹³

The gene coding for MMP-3 is located on the long arm of chromosome-11 ¹⁴ and consists of 10 exons, although there may be other shorter splice forms in addition to its full length transcript. ¹⁵ MMP-3, also known as stromelysin-1, is up-regulated in a variety of tumors and it has been shown to influence tumor initiation and neoplastic risk. ¹¹,¹⁵ MMP-3 is involved in the turnover of the ECM component. This enzyme degrades proteoglycans, collagen III, IV, V, and IX, laminin, fibronectin, gelatin, and elastin. Stromelysin-1 activates other members of the MMP family, such as MMP-1 and MMP-9. ¹⁵,¹⁶,¹⁷ A large number of polymorphisms have been reported in this gene, but the 5A/6A polymorphism in the promoter region has been most extensively investigated as this polymorphism increases the activity of the MMP-3 promoter. ¹⁶,¹⁸,¹⁹ The adenine insertion/deletion polymorphism (5A/6A) at position -1171 of the MMP-3 gene promoter influences transcription factor binding and MMP-3 promoter activity. ¹¹,¹⁶,¹⁸ . In vitro assays of promoter activity showed that the 5A allele had 2- to 4-fold higher promoter activity than the 6A allele. ¹⁰,²⁰ It has been reported that there is strong coloration between the MMP-3 polymorphism with breast, lung, and ovarian cancers. ²¹,²²,²³ There are some reports about correlation between 5A/5A polymorphism allele with progression and invasion activity of tumor cells, for example, in the breast cancer. ¹⁰,¹¹,²³

The aim of this study was to determine whether 5A/6A polymorphism may be associated with initiation and development or/and progression and invasion of tumor cells of CRC in Iranian population.

Subjects

This case-control study includes 120 cancer patients and 100 healthy controls. Median age of cases was 53 years (age range, 32-74 years) and controls were age-matched (΁ 3 years). This research is a retrospective case-control study. Cases were collected from a consecutive incident series with pathologically confirmation using colonoscopy and surgery; including 100 gender, age, and smoking status-matched healthy subjects without any diagnosis or history of cancer and any acute disease, were collected from Omid and Imam Khomeini hospitals, based in Isfahan and Tehran, respectively between 2009 and 2011. All the samples were collected from the patients with their permission. Detailed questionnaires, including clinical and family history, were initially collected. Patients were placed in two categories: with detectable metastasis position category (M ⁺ ) and without detectable metastasis position category (M ^- ). CRC patients consisted of 60 nonmetastasis patients (Stages 1, 2, and 3) and 60 metastasis patients (Stage 4). Healthy control subjects were basically collected from central laboratories in the hospitals (tumor-free volunteer). In the cases of smoking habit, complete information about the former and present smoking habits, the numbers of cigarettes/day and the time of starting and quitting were inquired from each subject. The definition of smoker was considered as described by other related publications. Nonsmokers are classified as people smoking less than 5 cigarettes/day; individuals who formerly or currently smoked 5 or more cigarettes/day for at least 2 years were defined as smokers. ¹¹

DNA extraction

Five milliliters of venous blood from each subject was drawn into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) and stored at 4΀C for short study and -80΀C for long study. Genomic deoxyribonucleic acid (DNA) was extracted using salting out method as published by Miller et al., with some modifications. ²⁴

MMP-3 SNP genotyping

The MMP-3 genotype was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The PCR primers were designed in previous study and checked by OLIGO 7 primer analysis software. A mutation from A to G at the second nucleotide close to the 3′end of the forward primer was made to create a PsyI recognition site in the case of the 5A allele (shown "g" in the forward primer sequences). Primers sequences are shown in Table 1. PCR reaction was performed in a 25 ΅l volume containing 100 ng of DNA template (genomic DNA), 2.5 ΅l of 10Χ PCR buffer, 200 mM MgCl ₂ , 1 unit of taq DNA polymerase (Cinnagen, Iran), 0.2 mM dNTPs, and 200 pM forward and reverse primers. The PCR cycling conditions were 3 min at 94΀C followed by 30 cycles including: 30 s at 94΀C, 30 s at 65΀C, 30 s at 72΀C, and final step at 72΀C for 10 min to complete extension of all PCR fragments. A 5 ΅l PCR product was digested overnight at 37΀C in a 15 ΅l reaction containing 5 units of PsyI enzyme (fermentase- Germany). After digestion, the digested fragments were subjected to electrophoresis on a 3% agarose gel stained with ethidium bromide. As expected, the 5A alleles would be represented by DNA bands of 97 and 32 bp (the small one could not be detected on the agarose gel electrophoresis) and the 6A alleles by a DNA band of 129 bp. For a negative control distilled water was used instead of DNA in the reaction system for each PCR.{Table 1}

Statistical analysis

Statistical analyses were performed using the SPSS 12 Software package. Hardy-Weinberg analyses were performed to compare the observed and expected genotype frequency using the χ΂ test. The odd ratio (OR) and 95% confidence interval (95% CI) were calculated using an unconditional logistic regression model and adjusted for age and sex. They were also calculated as an index of the association of genotype and risk of CRC initiation and metastatic activity. All statistical tests were two-sided, and a P value of less than 0.05 was considered significant.

At the time of CRC diagnosis, patients were aged between 32 and 74 years, with a mean of 53 years, and controls were age-matched (΁ 3 years). The MMP-3 genotype was successfully determined in 120 patients and 100 healthy controls Figure 1. There were no significant difference in the frequency of the MMP-3 genotype according to gender and age.Figure 1

Stromelysin-1 genotyping using PCR-RFLP method. 1 and 5, 5A/5A genotype. 2, 3, 6, and 7, 6A/5A genotype. 4, 6A/6A genotype. L, 50 bp DNA Ladder

Many studies show association of MMP polymorphism with initiation of cancer and especially with invasion risk and metastatic activity of different types of cancers such as breast, lung, and colorectal. ¹⁰,¹¹,²¹ Our previous studies showed the relationship of MMP-1 polymorphism with invasion risk of CRC ⁸ and breast cancer in Iranian population. ¹³ We also demonstrated a strong association of MMP-3 polymorphism with tumor cell progression and metastasis of breast cancer in Iranian population. ¹² The most of cancer deaths are resulting from tumor spread and metastasis, the process that is accompanied by degradation of components of the basal membrane and the ECM. ⁵ It has been demonstrated that those individuals carrying the 5A allele (5A/5A and 5A/6A genotypes) of MMP-3 associate with tumor cell progression and metastatic activity. ²⁵ It has been suggested that the higher transcriptional activity of 5A allele may enhances tumor invasiveness. ¹⁰,²⁶ Our present statistical analyses did not show strong association between the 5A allele and M ^- CRC group by comparison M ^- versus healthy control group at the time of diagnosis (OR = 0.96, 95% CI, 0.47-1.98). Our present results suggest that MMP-3 plays significant roles in spread and cell progression of CRC metastatic activity. Therefore, over expression of MMP-3 due to 5A promoter polymorphism may be a facilitating factor for CRC invasion and metastasis rather than initiation of CRC. The 5A allelic and 5A/5A genotypic frequencies in patient were 67.91% and 45%, respectively. The results showed that 5A/5A genotype and those individuals carrying the 5A allele were frequent in M ⁺ group and therefore, could be correlated to CRC invasion and metastasis (χ² = 7.49, P = 0.006).

Present study demonstrated a weak association between MMP-3 promoter polymorphism and the initiation of CRC. But it demonstrated an important statistical association between those individual carrying the 5A allele and tumor cell progression and metastasis of CRC. Our results show that 5A allele may be a moderate risk factor for invasion/ progression and metastatic activity of CRC. Therefore, MMP-3 genotype determination could be a moderate genetic marker to identify prone for development of tumor cell progression and invasion of CRC. However, it is conceivable that detection of cases with high potential for development of invasion and metastatic activity and then prevention of their development are important to enhance the survival rate of CRC patients and to treat the cancer.