Promoter Hypermethylation Of Wnt Pathway Inhibitor Genes In Maliganat Gliomas

Background:Glioma is one of the most common tumors in central nervous system, while its progress mechanism in carcinogenesis is still not clear. Methylation of CpG islands in the promoters of tumor suppressor genes induces inheritable gene silencing, which is one of the basic role of epigenetic events and may be the most common inactivation mechanism of the tumor suppressor genes in cancer. It is evident that Wnt signaling pathway is usually aberrant activated in cancer. It could be activated by overexpression or mutation of Wnts, receptor complex or elements of this pathway. It could be also activated by gene silencing of the extracellular inhibitors of this pathway.Objective:The thesis will research in promoter methylation of the inhibitor genes of Wnt pathway in glioma tissues and glioma cell lines, role of Wnt pathway inhibitors in malignant glioma and effects of demethylation drugs for curing glioma, laying a basis for future use of demethylation drugs in curing glioma.Methods:Methylation-specific PCR, MSP is used to detect the promoter methylation of Wnt patyway inhibitor genes, i.e. SFRP1, SFRP2, SFRP5, DKK1 and WIF-1, in frozen primary gliomas, normal brain tissues and glioma cell lines A172 and U251. With these two lines treated by 5-Aza-2′-deoxycytidine (ADC) and deacetylase inhibitors Trichostatin A (TSA), mRNA varience of these inhibitors and CCND1 is detected by RT-PCR, while proliferation varience and colony varience of the cells by MTT assay and soft argar assay respectively.Results:1. Five genes encoding Wnt pathway inhibitors, including members of the secreted frizzled-related proteins (SFRP1, SFRP2, SFRP5), dickkopf 1(DKK1) and Wnt inhibitor factor 1(WIF-1), were screened for aberrant promoter methylation in 53 primary gliomas of different malignancy grades, compared with normal control. There is at least one of these genes being hypermethylated in 43 of 53 gliomas (83%). The frequency of hypermethylation of SFRP1, SFRP2, SFRP5, DKK1 and WIF-1 was 43.40%, 28.30%, 26.42%, 66.04% and 13.21%, respectively. In WHO II, III grade, the methylation frequency of SFRP1 was significantly higher than in WHO IV grade primary malignant glioma specimens, respectively (II: P<0.01, III: P<0.05); the methylation frequency of SFRP2 is higher than high grade ones(III + IV:P<0.05). The hypermethylation frequency of SFRP2, DKK1 and WIF-1 is more common in this tumor, but no statistical significance. The five genes hypermethylated are found in both glima cell lines.2. Semi-quantitative RT-PCR analysis shows that in cell lines A172 and U251, DKK1 expression is different between control group and treatment group, but without statistical significance. However, treatment with ADC and TSA results in increasing expression of SFRP1. Furthermore, SFRP2, SFRP5 and WIF-1 expression is restored after treatment with ADC and TSA.3. The expression level of CCND1, a well known Wnt/β-catenin downstream target gene, is significantly lower in treatment group than in control group.4. MTT assay and soft-agar assay show that the proliferation and colony formation ability are suppressed after treatment of both A172 (P<0.05) and U251 (P<0.01) with ADC and TSA.Conclusions:1. Hypermethylation fully manifests itself in both of the glioma tissues and glioma cell lines A172 and U251.2. In A172 and U251, when the hypermethylation of Wnt pathway inhibitor SFRP1, SFRP2, SFRP5 and WIF-1 gene was reversed by demethylating drugs, the expression of these genes was remarkably increased. The expression of CCND1 significantly decreased after treatment with drugs. The data suggest that the inactivation of Wnt pathway inhibitor by methylation could cause aberrant activation of the Wnt signaling pathway.3. There was no statistical difference of the mRNA expression level of DKK1 in the cell lines between treatment group and control group. The data suggest that the role of DKK1 in pathogenesis of various cancers needs further study.4. After treatment with demethylating drugs, the proliferation and colony formation ability of A172 and U251 was suppressed. The result shows that DNA demethylating drugs can be used to treat glioma at least partially through restoring the expression of the SFRPs and WIF-1.