A Pilot Study On Association Between Matrix Metalloproteinases 1,3,7,9 And Adult Astrocytoma

Objectives: Matrix metalloproteinases (MMPs), a family of highly conserved zinc-dependent proteolytic enzymes that degrade many different components of extracellular matrix (ECM) and regulate various cell behaviors, play important roles in tumor development, infiltration, and metastasis. It has been shown that MMPs expression is very low in normal tissues while elevates in tumor tissues with different extent. Growing evidence indicates that single nucleotide polymorphisms (SNPs) in promoters of MMP genes may result in variable transcription and expression of MMPs in different individuals, therefore, influence the development, progression, and outcome of tumors. Gliomas are the commonest primary brain tumors. Despite recent advances in diagnostic imaging, neurosurgical technique, radiation therapy, and chemotherapy, significant improvement in the survival of gliomas has not been achieved. Astrocytoma is the commonest type of brain gliomas and develops from a type of star-shaped cell called astrocyte. The etiology of astrocytoma is unknown. Although high-dose of radiotherapy and chemotherapy are supposed to be exogenous environmental causes, the accumulated data suggests that genetic background also play a role in the development of this tumor. Since it has been reported that the expression of MMP proteins may increase in astrocytoma tissues, we suspected that the SNPs in promoter region of MMP genes may alter susceptibility to astrocytoma via modifying the transcription activity of MMP genes. Recently, McCready et al. reported that the MMP-1 2G/1G SNP may play a role in the development and infiltration of glioblastoma, however, the association between the development of gloma and other MMP polymorphisms has not been reported so far. In addition, the relationship between the MMP polymorphisms and protein expression and mRNA transcription has not been clearly demonstrated. Hence, we conducted a hospital-based case control study on association between the promoter polymorphisms of MMP-1, 3, 7, 9 genes and susceptibility to adult astrocytoma. Additionally, the protein expression and mRNA transcription of the MMP-1, 3, 7, 9 were also evaluated in astrocytoma tumor tissues. This pilot study aims to investigate the relationship between the MMP-1, 3, 7, 9 genes and development and progression of astrocytoma, to help exploring the genetic liability as well as the molecular mechanism of the astrocytoma and provide candidate targets for prevention and control of this tumor type.Part I Polymorphisms in the MMP-1,3,7,9 promoters and susceptibility to adult astrocytomaMethodsThe association between the promoter polymorphisms of MMP-1, 3, 7, 9 genes and susceptibility to adult astrocytoma was tested in a hospital-based case control study. Tissues from 236 cases of adult astrocytoma patients were collected after pathologic diagnosis for extracting DNAs by proteinase K digestion followed by phenol-chloroform purification. Blood samples from 366 healthy subjects were collected for DNA extraction by proteinase K digestion followed by a salting out procedure. The MMP-1 -1607 2G/1G, MMP-3 -1171 5A/6A, MMP-7 -181A/G, and MMP-9 -1562 C/T genotypes were determined by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) assay. Hardy-Weinberg analysis was performed to compare the observed and expected genotype frequencies using Chi-square test. Comparison of the MMP genotype distribution among study groups was performed by means of two-sided contingency tables using Chi-square test or Fisher's exact test. The odds ratio (OR) and 95% confidence interval (CI) were calculated using an unconditional logistic regression model and adjusted by age and sex accordingly.Results1 The overall distribution of the MMP-1 1607 2G/1G SNP allelotype and genotype among astrocytoma patients and healthy controls was significantly different (P=0.002 and P<0.001, respectively). Compared with 2G/2G, the 1G/1G genotype significantly decreased the risk of development of astrocytoma, the age and gender adjusted OR was 0.58 (95%CI=0.42~0.79), while the relationship between the 2G/1G genotype and astrocytoma was not observed (age and gender adjusted OR was 0.74, 95%CI=0.51~1.08). The similar results were obtained when stratified by gender and age at tumor diagnosis (≤45 Years or > 45 Years). When stratified according to the World Health Organization (WHO) astrocytoma classification, the 2G/2G genotype significantly reduced the risk of grade II and III astrocytoma, the age and gender adjusted OR was 0.61(95%CI=0.40~0.93) and 0.41(95%CI=0.20~0.85), respectively, while correlation between the MMP-1 SNP and susceptibility to grade I and IV astrocytoma was not demonstrated.2 The overall genotype and allelotype distribution among astrocytoma patients was not significantly different from that among healthy controls(P>0.05). Compared with 6A/6A, the most common genotype in the study population, both of the 5A/6A and 5A/5A genotype did not influence the risk of astrocytoma development, the age and gender adjusted OR was 1.09(95% CI=0.75~1.57) and 0.95 (95% CI=0.54~1.69), respectively. When stratified by gender, age at tumor diagnosis, and the WHO astrocytoma classification, association between the MMP-3 SNP and susceptibility to astrocytoma was also not observed.3 The overall distribution of the MMP-7 -181 A/G SNP genotypes among astrocytoma patients and healthy controls was significantly different (P = 0.001). Compared with the A/A genotype, the G/G genotype significantly increased the risk to the development of astrocytoma, the age and gender adjusted odds ratio was 2.77 (95% CI = 1.27~6.02), while the MMP-7 A/G genotype also marginally increased the risk of developing astrocytoma (the adjusted OR = 1.69, 95% CI = 1.01~2.84). Stratification analysis showed that the MMP-7 G/G genotype significantly increased the risk of astrocytoma among individuals younger than 45 years compared with the A/A genotype (adjusted OR = 3.16, 95% CI = 1.09~9.16), however, the G/G genotype did not modify the risk of the occurrence of astrocytoma among people older than 45 years (adjusted OR = 1.93, 95% CI = 0.61~6.20). In addition, stratification analysis indicated that the MMP-7 G/G genotype significantly increased the risk of astrocytoma among male subjects (adjusted OR = 3.24, 95% CI = 1.12~9.41). Stratification analysis according to the WHO pathological grading showed that of astrocytomas. A significant higher risk of developing grade II astrocytoma was observed among individuals harboring the MMP-7 A/G genotype compared with those harboring the A/A genotype (adjusted OR = 2.06, 95% CI = 1.05~4.05). In addition, the G/G genotype significantly increased the risk of developing grades II, III, and IV astrocytoma compared with the A/A genotype, the adjusted odds ratio was 2.95 (95% CI = 1.21~7.17), 3.32 (95% CI = 1.15~9.61), and 3.25 (95% CI = 1.10~9.62), respectively, whereas the distribution different among patients with grade I astrocytoma and healthy controls was not observed.4 The overall MMP-9 genotype and allelotype distribution among astrocytoma patients was not significantly different from that among healthy controls(P value was 0.92 and 0.818, respectively). Compared with the C/C genotype, genotypes with the -1562 T allele (C/T +T/T) did not show significant influence on the risk of astrocytoma development (age and gender adjusted OR= 1.09, 95% CI = 0.73~1.64). Stratification by gender, age, and the WHO astrocytoma classification also did not show an association between the MMP-9 SNP and susceptibility to astrocytoma.Conclusions1 The overall analysis and stratification analysis according to gender, age at diagnosis, and histological grade suggested that, the MMP-3 -1171 5A/6A and the MMP-9 -1562 C/T SNPs could not modify the risk of the adult astrocytoma development.2 The MMP-1-1607 2G/1G polymorphism significantly influenced the risk of adult astrocytoma. Compared with 2G/2G, the predominant genotype among the study population, individuals with the 1G/1G genotype significantly decreased the susceptibility to astrocytoma. 3 The MMP-7-181A/G polymorphism significantly influenced the risk of adult astrocytoma development. Individuals harboring the G allele (A/G and G/G genotypes) significantly increased the susceptibility to astrocytoma. This association was more significant among male and people younger than 45 years old.Part II: Relationship between expression of MMP-1,3,7,9 and clinical pathology in adult astrocytomaMethodsParaffin embedded blocks of tumor tissues from 70 pathologic diagnosed and graded astrocytoma cases and of non-tumor tissues from eight brain trauma patients were selected for detecting protein expression. MMP-1, 3, 7, 9 proteins were detected by immunohistochemistry (IHC) using the S-P staining kit. The status of the MMP expressions among histological grading groups was compared using Chi-square test. The correlation of the MMP expression with the pathologic grading was tested by the Kendall's tau-b method with the significance level of 5%.Results1 The MMP-1 protein expression rate among non-tumor tissues and grade I～IV astrocytoma tissues was 12.5%, 75%, 86%, 90%, 100%, respectively. The MMP-1 expression among the above five groups was significantly different (X2=28.204,P<0.01). The MMP-1 expression was significantly correlated with the disease progression(tb=0.463,P<0.001). Thus, the MMP-1 expression significantly increased with the histological grading.2 The MMP-3 protein expression rate among non-tumor tissues and grade I～IV astrocytoma tissues was 25%, 33%, 45.5%, 35%, 75%, respectively. The MMP-3 expression among the above five groups was not significantly different (X2=20.476,P=0.059). However, the MMP-3 expression was significantly correlated with the disease progressionand significantly increased with the histological grading(tb=0.276,P=0.005).3 The MMP-7 protein expression rate among non-tumor tissues and grade I～IV astrocytoma tissues was 37.5%, 50%, 77.3%, 75%, 100%, respectively. The MMP-7 expression among the above five groups was not significantly different (X2=29.779,P=0.003). The MMP-7 expression was significantly correlated with the disease progression and significantly increased with the histological grading(tb=0.416,P<0.001).4 The MMP-9 protein expression rate among non-tumor tissues and grade I～IV astrocytoma tissues was 0%, 66.7%, 81.8%, 80%, 100%, respectively. The MMP-9 expression among the above five groups was not significantly different (X2=44.038,P<0.001). The MMP-9 expression was significantly correlated with the disease progression and significantly increased with the histological grading(tb=0.427,P<0.001).ConclusionsThe MMP-1,3,7,9 protein expression was significantly correlated to the tumor differentiation and may be useful molecular markers to predicate the prognosis of astrocytoma. Part III Expression and significance of MMP-1,3,7,9 mRNA in astrocytomaMethodsTumor tissues from 39 astrocytoma cases and non-tumor brain tissues from 12 brain trauma patients were frozen in liquid nitrogen in 30 minutes after resection and stored in a -80℃refrigerator. Total cellular RNA was extracted from tested tissues using the one-step RNA extraction kit. The first cDNA strand was generated by a reverse transcription system. The MMP mRNA from tumor and non-tumor brain tissues was quantitatively detected by the real-time fluorescence quantitative polymerase chain reaction (RQ-PCR) and TaqMan probe technology, with GAPDH gene as an internal reference.The MMPs mRNA expression level was calculated by the ratio of MMPs to GAPDH and expressed as mean±standard deviation. Student's T test was used to compare the mRNA expression between the pathologic groups with a significance level of 5%.Results1 The MMP-9 mRNA expression in tumor tissues of astrocytoma was significantly higher than that in non-tumor tissues (p=0.038). The MMP-9 mRNA expression in low differentiation tissues (pathologic gradeⅢ-Ⅳ) was significantly higher than that in both of the high differentiation tissues (pathologic gradeⅠ-Ⅱ) and non-tumor brain tissues ( P value was =0.001 and <0.001, respectively). However, the significant difference between the high differentiation group and non-tumor group was not observed.2 The MMP-1, 3 mRNA expression among low differentiation tissues (pathologic gradeⅢ-Ⅳ),high differentiation tissues (pathologic gradeⅠ-Ⅱ), and non-tumor brain tissues was not significantly different ( P value was 0.26 and 0.419, respectively).Conclusions1 The MMP-9 mRNA expression in low differentiation tissues (histological gradeⅢ-Ⅳ) was significantly higher than that in both of the high differentiation tissues (pathologic gradeⅠ-Ⅱ) and non-tumor brain tissues, suggesting that detection of the MMP-9 mRNA may be used to estimate the tumor histological differentiation and disease prognosis.2 The MMP-1 and MMP-3 mRNA expression among astrocytoma tissues was not significantly different from that in non-tumor brain tissues, indicating that detection of the MMP-1 and MMP-3 mRNA may be not applicable for estimating the biological behavior of astrocytoma.In summary, this pilot study showes that the polymorphisms in promoter regions of the MMP-1, 7 genes are involved in the carcinogenesis of adult astrocytoma and may be used as molecular markers, alone or together with other polymorphic genes, to screen individuals those at risk to develop astrocytoma. The high expression of the MMP-1,3,7,9 proteins can be used as candidate markers to estimate the progression and prognosis of adult astrocytoma. Additionally, detection of the MMP-9 mRNA may be used as an indicator of malignant characteristics of astrocytoma.