Study On The Role Of Matrix Metalloproteinases Polymorphism And Expression In The Pathogenesy Of Endometriosis

Endometriosis (EMs) is a kind of benign but invasive and metastatic gynaecological disease. It is conservatively estimated that the incidence rate can be as high as 25% among women aged 30 to 40 years old, and the rate has increased in recent years. 30% to 50% of women with EMs are suffering from infertility. Although there are various methods to cure EMs such as surgical operation, the result is unsatisfactory, and the 5-year relapse rate is above 40% due to the fact that its aetiology and pathogenesis is not completely understood.EMs is a malignant disease because its'ectopic endometrium may be found in many other organs besides the uterine cavity in histology. Therefore, the key step of EMs occurrence is similar to nicious tumor. As it is known that the implantation and development of endometrium in cavitas pelvis have three main stages such as cell adhesion, extracellular matrix (ECM) remolding and blood vessel forming, which is similar to invasive and metastatic process of tumor. Matrix metalloproteinases (MMPs) play an important role in the ECM remolding.The MMPs are a highly conservative zinc-dependen family of endopeptidases and may degrade most proteins of ECM. Under physiological condition, the expression of MMPs, which exist in various kinds of normal cell, is very rare. However, the expression would increase in the stimulated cytokines, hormones, growth factors and process of cellular transformation, which is implicated in various physiologic and pathologic processes, such as inflammation, embryo occurrence, blood vessel formation, invasion and metastasis of tumor and so on. The expression of MMPs in eutopic endometrium is different in menstrual cycle, and different kinds of MMPs could be found in endometrium stroma, which may play a role in endometrium remolding, blastula implantation and trophoblast invasion. Researches showed that different types of MMPs could be found in ectopic endometrium for endometriosis patients. In addition, the expression of some MMPs in ectopic endometrium was significantly higher than that in eutopic endometrium, which would not be affected by menstrual cycle. MMPs play a very important role in the development of endometriosis. But so far, the mechanism of MMPs over-expression in ectopic endometrium for endometriosis patients is still unclear. Recently, researches of eukaryotic gene expression regulating are largely focused on the transcriptional regulation to study the expression regulation, which illuminates the pathogenesis of diseases and cellular function from the molecular level, and supplies knowledge of prevention and cure for diseases. More and more researches showed that the polymorphisms play an important role in transcriptional regulation and are associated with the susceptibility of some diseases. Thus, we choose MMPs as target gene, which is confirmed to have correlation with MMPs abnormal expression and development of some tumors, and study the association of the polymorphism and expression of MMPs with the risk of endometriosis. Our study includes: (1) comparing the difference of MMP-1, 3, 7 and 9 expressions in eutopic and ectopic endometrium of endometriosis patients. (2) Analyzing the association of single nucleotide polymorphism (SNP) in the promoter regions of the MMP-1, 3, 7, 9 with the risk of endometriosis. (3) Analyzing the relation between polymorphisms about EMs and the expression of mRNA and protein in eutopic endometrium.All in all, by studying the role of four genes (MMP-1, 3, 7, 9) in the development of EMs, we illuminate the pathogenesis by genetic difference and search the possible MMPs susceptible gene of EMs genetically.PartI Comparatiev study of MMPs expression in eutopic and ectopic endometrium of endometriosis patientsObjective: To examine the difference of MMPs expression in eutopic and ectopic endometrium of endometriosis patients and investigate the role of MMPs abnormal expression in the development of EMs. Methods: The MMP-1, 3, 7 and 9 expressions were detected by flow cytometry in eutopic endometrium (n=20) and ectopic endometrium (n=40) for endometriosis patients. Using the t-test to examine the differences of four MMPs expression in eutopic and ectopic endometrium.Results: 1) There was a significant difference in the expression of MMP-1 protein between eutopic and ectopic endometrium of endometriosis patients (P<0.05), the differences were 1.00±0.13 and 1.09±0.16, respectively. 2) The expression of MMP-3 protein in eutopic endometrium (1.00±0.22) was significantly lower than that in ectopic endometrium (1.29±0.23) (P<0.05). 3) There was not a significant difference in the expression of MMP-7 protein between eutopic endometrium and ectopic endometrium of endometriosis patients (P>0.05), which were 1.00±0.16 and 0.94±0.20, respectively. 4) Compared wth ectopic endometrium (1.04±0.11), the expression of MMP-9 protein showed no significant difference in eutopic endometrium (1.00±0.13) (P>0.05).Conclusion: This study is the first to analyze the expression of MMP-1, 3, 7, 9 in eutopic and ectopic endometrium for endometriosis patients by flow cytometry, and the result showed 1) The expression of MMP-1 and MMP-3 protein in ectopic endometrium was significantly higher than that in eutopic endometrium for endometriosis patients, which showed there was a association between abnormal expression of MMPs (MMP-1 and MMP-3) protein and the development of EMs. The over-expression of MMP-1 and MMP-3, known as collagenase and mesenchymal lysine respectively, could increase the activity of ECM degradation, thus the over-expression of MMP-1 and MMP-3 in ectopic endometrium could show puissant activity of ECM degradation and have relation with tissular remolding, hemorrhage and implantation. 2) The expression of MMP-7 and MMP-9 protein showed no significant difference between eutopic and ectopic endometrium of endometriosis patients. PartII Study on the association of polymorphisms of theMMPs with the risk of EMsObjective: By analyzing the differences of MMPs allele and genotype frequencies in endometriosis patients and unrelated healthy women, we attempted to investigate the association of polymorphisms of the MMPs with the risk of EMs.Methods: The genotype frequencies of four polymorphisms in MMPs promoter (MMP-1 -1607 1G/2G, MMP-3 -1171 5A/6A, MMP-7 -181 A/G and MMP-9 -1562 C/T) were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and using case- control study in 143 endometriosis patients and 160 unrelated healthy women. The peripheral blood DNA was used as experimental materials. The age difference of case and matched control was analyzed by the t-test. The difference of the MMP-1, 3, 7, 9 allele and genotype distributions in two study groups was tested by the Chi-square test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model. The haplotype frequencies and linkage disequilibrium coefficient were estimated with the EH and 2ld linkage software respectively.Results: 1) The frequency of the MMP-1 2G allele among endometriosis patients (79%) was significantly higher than those in the healthy controls (66.9%) (P<0.05); The frequencies of the 2G/2G, 1G/2G and 1G/1G genotypes among endometriosis patients (7%, 28% and 65%, respectively) were significantly different from those in healthy controls (16.6%, 32.7% and 50.7%, respectively) (P<0.05); The frequency of the 2G homozygote in endometriosis was significantly higher than in the controls (P<0.05); Compared with the 1G/1G genotype, the 2G/2G or 2G/2G+1G/2G genotype significantly increased the risk of developing endometriosis, the adjusted odds ratios were 3.65 ( 95%CI=1.41~9.43 ) and 3.25 ( 95%CI=1.29~8.23), respectively. 2) The frequencies of the MMP-3 5A and 6A allele among endometriosis patients and healthy controls were 14%, 86% and 20.3%, 79.7%, respectively; No significant difference in the MMP-3 allele distribution was shown between the case and controls (P>0.05); There was no significant difference in 5A/5A, 5A/6A and 6A/6A genotypes distribution between endometriosis patients and healthy women (P>0.05); Compared with the 6A/6A genotype, either the 5A/5A or the 5A/5A + 5A/6A genotype significantly modified the risk of developing endometriosis, the adjusted odds ratio were 2.51(95%CI=0.25~25)and 1.63(95%CI=0.92~2.98), respectively. 3) The combined effect of the MMP-1 and MMP-3 SNP showed that it increased 4-fold higher risk of developing endometriosis, as the 2G and 6A homozygotes were found in the subject at the same time, the adjusted odds ratio was 4.13(95%CI=1.55~10.99). 4) The frequency of the MMP-7 G allele among endometriosis patients (7.3%) was significantly higher than in the healthy controls (2.8%) (P<0.05); The frequencies of the A/A, A/G and G/G genotypes among endometriosis patients (86.0%, 13.3% and 0.7%, respectively) were significantly different from those in healthy controls (94.4%, 5.6% and 0, respectively) (P<0.05); Compared with the A/A genotype, the subjects with G allele significantly increased the risk of developing endometriosis, the odds ratios was 2.71 (95%CI=1.19~6.16). 5 ) The frequencies of the MMP-9 C and T allele among endometriosis patients and healthy controls were 88.8%, 11.2% and 91.9%, 8.1%, respectively; No significant difference in the MMP3 allele distribution was shown between the case and controls (P>0.05); The frequencies of the C/C, C/T and T/T genotypes among endometriosis patients (78.3%, 21% and 0.7%, respectively) were not significantly different from those in healthy controls (83.8%, 16.2% and 0, respectively) (P>0.05); Compared with the C/C genotype, the T allele did not significantly increase the risk of developing endometriosis, the adjusted odds ratio was 1.41(95%CI=0.79~2.52).Conclusion: This study is the first to investigate the relation between MMPs promoter SNPs and the risk of EMs all over the world. Our result showed 1) There was the association of the MMP-1 promoter -1607 1G/2G with the risk of endometriosis, in other words, 2G allele could significantly increase susceptibility to endometriosis. 2) Although the MMP-3 promoter -1171 5A/6A could not be a layering marking to detect the risk of endometriosis, the 6A/6A and MMP-1 2G/2G genotype could be the conjunctive layering marking. 3) Our data suggest an association of the MMP-7 promoter -181 A/G with the risk of endometriosis, G allele could increase susceptibility to endometriosis. 4) It was not found to the association of the MMP-9 promoter -1562 C/T with the risk of endometriosis. 5) Some polymorphisms in MMPs could be the independent molecule marking to detect the risk of endometriosis.PartIII Association of the mRNA and the protein expression of MMP-1 and MMP-7 with SNPsObjective: To investigate the relation between polymorphisms at MMPs promoter region and the mRNA and protein expression.Methods: The transcriptional activity and protein expression of the MMPs in eutopic endometrium for endometriosis patients, which took with the different genotypes of the MMP-1 and MMP-7, were analyzed by reverse transcription-polymerase chain raction (RT-PCR) and immunohistochemistry, respectively. Wilcoxon signed-rank test was used to measure the difference of mMRA and protein expression in eutopic endometrium with the different genotypes.Results: 1) The relative expression of mMRA in eutopic endometrium for endometriosis patients with the MMP-1 1G/1G, 1G/2G and 2G/2G genotypes was 0.33±0.10, 0.43±0.16 and 0.54±0.24, respectively. The significant difference in the MMP-1 mMRA expression was shown among those (P=0.04). 2) There was significant difference in the relative expression of MMP-7 mMRA in eutopic endometrium for endometriosis patients with G allele (A/G+G/G) (0.37±0.26) and A/A genotype (0.37±0.26) (P=0.01). 3) The MMP-1 protein was mainly expressed in the stromal cells of eutopic endometrium, the expression of MMP-1 protein in eutopic endometrium for endometriosis patients with 2G/2G genotype was significantly higher than those with 1G/1G (P=0.047) and 1G/1G + 1G/2G genotypes (P=0.019). 4) The MMP-7 protein expressed primarily in endothelial cells of eutopic endometrium, compared with A/A genotype, the expression of MMP-7 protein in eutopic endometrium for endometriosis patients with A/G genotype was significantly high (P=0.016).Conclusion: Our study is the first to confirm different transcriptive activity and protein expression for subjects with different genotypes, which result in the difference of eutopic endometrium for different subject, further cause different risk of EMs. 1) There was significant difference in the mMRA and protein expression of MMP-1 in eutopic endometrium for endometriosis patients with the different MMP-1 genotypes, in other words, the mMRA and protein expression of MMP-1 for ones with the 2G/2G genotypes was significantly higher than the others, which show the 2G allele could result in the increasing expression of MMP-1 protein in eutopic endometrium by enhancing gene transcriptional activity. 2) The mMRA and protein expression of MMP-7 in eutopic endometrium for ones with the different MMP-7 genotypes had significant difference, that was to say the subjects with G allele could significantly increase the mMRA and protein expression of MMP-7 in eutopic endometrium, which show the G allele could enhance gene transcriptional activity to result in the increasing expression of MMP-7 protein in eutopic endometrium.In conclusion, our study confirms further that MMPs could play an important role in the development of EMs. In addition, we presume that the role of different MMPs is distinct in various stages of EMs development. The difference of subjects which is resulted by the genetic difference of MMP-1 has the association with the risk of EMs, and the abnormal expression of protein may have relation with the development of EMs; The abnormal expression of MMP-3 has a very important role in the development of EMs; As a protein of endothelial expression, MMP-7 may participate in the development of EMs, endometrial cells with high-expression MMP-7 easily take place ectopic implantation in the blood of menstruate countercurrented and increase the risk of EMs; It is not confirmed in our study that there are the association of MMP-9 with the risk of EMs, but in view of other study, we will further evaluate the role of MMP-9 in the development of EMs.