The Methylation Of DKK3 Gene And Its Regulation In Neuroblastoma

Background and Objective Neuroblastoma is the most common tumor in children.Neuroblastoma nearly half occurred in infants under 2 year of age,at present about the etiology of neuroblastoma true is not clear,some genetic susceptibility factors were found to correlate with the onset of neuroblastoma,in recent years, the study found that some tumor suppressor gene DNA sequence was found not to have the complete gene mutation, gene deletion and a series of changes.Then gradually recognized that epigenetic alterations, especially the methylation status of DNA, which is involved in tumor occurrence and development of the incident.DNA methylation is reversible and can reverse tumor suppressor gene methylation at promoter region and restore its function, thereby inhibiting the growth of tumor.There are two kinds of demethylation drugs: 5- azaC and 5-azaCdR.5- azaC is a kind of demethylation drugs.In the process of DNA replication, the methyltransferase binding and the formation of covalent complexes, re expression by methylation and inactivation of tumor suppressor genes, and to restore the function, plays the role of inhibiting tumor cell growth.The study showed that the DKK3 gene is a newly discovered candidate tumor suppressor gene, which can antagonize the Wnt signal transduction pathway through,DKK3 gene is a member of the Dickkopf family, and Wnt receptor complex competitive binding and selective inhibition of Wnt signaling pathway, its inactivation and silent antagonist genes, which lost its inhibitory effect on the pathway to induce cell unlimited proliferation, and thus lead to tumor.Research shows that DKK3 gene promoter region rich in CpG Island, and the DKK3 gene promoter region hypermethylation, these are the important reasons leading to the expression of DKK3 gene inactivation.At present, many tumor cell lines or malignant tumors, such as gastric cancer, esophageal cancer, colorectal cancer, loss or down-regulation of expression, prostate cancer, breast cancer and lung cancer tissues, but DKK3 in neuroblastoma gene promoter and transcriptional expression condition, the influence of 5-azaC on neuroblastoma cell line NB-1 cells proliferation and DKK3 gene has not been reported at home and abroad.Therefore, we use semi quantitative RT-PCR detection of transcription from the mRNA DKK3 expression level of RNA gene in neuroblastoma tumor tissues and corresponding adjacent to NB in TAC, from the level of DNA in detection of DKK3 gene methylation in the promoter region of TAC organization in NB and the corresponding cancer adjacent condition,to investigate the expression mechanism of inactivation of DKK3 gene in neuroblastoma cells, effects of 5-azaC on growth proliferation and DKK3 gene expression on NB-1 cells, thereby determining whether DKK3 gene neuroblastoma was a candidate tumor suppressor gene, to find a new target for tumor therapy. Material and Method 1. RT-PCR and MSP were used to detect the level of transcription of DKK3 gene in 51 cases of NB and TAC in tissue, determination of methylation status of NB and TAC in tissues of DKK3 gene promoter from DNA molecular level, analysis the relationship between in clinic. 2. The 5-azaC NB-1 cells in treatment 2, 5, 10, 20, 50, 100, 200, 400 mol/L, and to observe its effects on cell growth; using MTT RT-PCR technology application, methylation specific PCR, determination of cell by 5-azaC(5, 10, 20, 50 mol/L) after treatment, DKK3 gene methylation status of DKK3 promoter and mRNA expression. 3. Statistical analysis is the application of the SPSS10.0 statistics software, two sample rate compared using X2 test four table; two sample quantitative data using t test, with P<0.05 as a significant difference compared with the standard; the method of single factor analysis of variance of the mean diversity. Results 1. The positive rate of expression of DKK3 gene of mRNA were 100% and 43.4% in NB.no DKK3 gene promoter methylation status.38.8% appeared in the NB organization of DKK3 gene promoter methylation status,there was significant difference between the 2 groups(P<0.05); mRNA methylation and NB the promoter of the DKK3 gene expression in patients with pathological grading was significantly associated(P<0.05). 2. There was distinct correlation between methylation of DKK3 gene promoter and expression of its mRNA in NB(P<0.05) 3. After treatment of NB-1 cells with different concentrations of 5-azaC, increased with the prolongation of the time of drug action and drug concentration, cell growth was inhibited, in a time and dose dependent(P<0.05); 5-azaC NB-1 cells treated by 48 h 5, 10, 20, 50 mol/L after the drug concentration, the relative expression of DKK3 m RNA followed by 0.199 + 0.015, 0.348 + 0.009, 0.457 + 0.016, 0601 + 0.010, expression intensity and 5-aza C concentration of the drug in a dose-dependent relationship(F=368.458, P<0.01); MSP test results show that, after 5-aza C treatment, DKK3 gene promoter hypermethylation occurs in part region demethylation(Fm=295.342, Fum=189.148; P<0.05). Conclusions 1. Methylation of DKK3 promotor region is an important mechanism of DKK3 gene inactivation, plays an important role in the occurrence of NB, development. 2. 5-aza C can effectively reverse the papillary thyroid cancer cell NB-1 gene aberrant methylation, thereby enhancing the expression of DKK3 gene, inhibit the growth of tumor cells.2. 5-aza C might effectively cause the demethylation of DKK3 gene CpG- rich promoter regions, activate its expression, and inhibit the growth of TPC-1 cells.