| Enzootic nasal tumor(ENT) is a chronic, progressive, infectious disease. Once the clinical symptoms, there is no exception to death, serious threat to the security of a flock of sheep populations. For different positive infected sheep there were no more successful method. To detect the methylation status of DNA methyltransferases(DNMTs),the tumor suppression genes such as MGMT,P73, P53,GADD45G,CHFR and THBS1, proto-oncogenes such as KRAS,NRAS,C-myc, the transcription factors for example CEBPA, and EGFR by the methylation-specific PCR(MSP) in 24 cases of nasal tumor tissues and 20 cases of normal nasal epithelia tissues, and to detect mRNA expression level of the genes by SYBR Green I RT-PCR.We also analysis of the influence of DNMTs to promoter methylation, and the influence of promoter methylation to mRNA expression. To reveal the association between the promoter methylation and the expression level of tumor-related genes, and provide fodder for screening to tumor-specific markers for diagnosis of ENT and further research the epigenetic mechanisms of enzootic nasal tumor virus (ENTV) induced nasal epithelium cell carcinoma. The results of the study are shown below:1. The results of gene promoter methylationOnly THBS1,GADD45G and CHFR were methylated in tumor suppression genes. The methylation expression rate of THBS1 was 79.2 percent in nasal tumor tissues,while there was no methylation in normal nasal epithelia tissues.CHFR was 58.2 in nasal tumor tissues and was 60 percent in normal nasal epithelia tissues. The exhaustive methylation expression rate of GADD45G was 58.5 percent and partial methylation expression rate was 25 percent in nasal tumor tissue, while there was no methylation in normal nasal epithelia tissues. Only C-myc was methylated in proto-oncogenes. The methylation expression rate of C-myc was 54.2 percent in nasal tumor tissues and 1 and there was no methylation in normal nasal epithelia tissues. The methylation expression rate of CEBPA was 79.2 percent in nasal tumor tissues, while there was no methylation in normal nasal epithelia tissues. In our result,the methylation expression rate of EGFR gene was nearly 80 percent in the two tissues. Only DNMT3b gene and DNMT1 gene were methylated in DNMTs. The methylation expression rate of DNMT1 and DNMT3b were20.8%,41.7%, and there was no methylation in normal nasal epithelia tissues.2. The results of transcriptional level expressionThe expression level of THBS1 was 0.38±0.25 in 20 cases of normal nasal epithelia tissues and wasl.05±0.32 19 cases of methylation nasal tumor tissues and was 1.03±0.21 19 cases of unmethylation nasal tumor tissues. The expression level of GADD45G was 0.47±0.72 in 20 cases of normal nasal epithelia tissues, and was 2.48±0.82 in 14 cases exhaustive methylation nasal tumor tissues and was 1.42±0.33 in 6 cases partial methylation nasal tumor tissues. The expression level of C-myc was -0.42±1.90 in 20 cases of normal nasal epithelia tissues,and was-1.37±0.58 inl3 methylation nasal tumor tissues and was -0.98±0.21 in 11 cases of unmethylation nasal tumor tissues. The expression level of CEBPA was 0.85±0.24 in 20 cases of normal nasal epithelia tissues, and was -0.76±0.26 inl9 methylation nasal tumor tissues and was 0.79±0.11 in 5 cases of unmethylation nasal tumor tissues, but the result was different with other tumor research. EGFR did not find transcriptional expression in the control group and experimental group. Only DNMT3b were appeared abnormal expression in DNMTs genes. The expression level of DNMT3b was 1.39±0.88 in 20 cases of normal nasal epithelia tissues. The expression level of DNMT3b was 0.22±0.63 inlO cases of methylation nasal tumor tissues, and was 1.41±0.71 in14 cases of unmethylation nasal tumor tissues.Comprehensively, DNMT1, DNMT3b, C-myc, CEBPA, GADD45G and THBS1 had abnormal methylation. The expression of C-myc and DNMT3b were up-regulated, and the expression of THBS1 gene was down-regulated in nasal tumor tissues. The expression of CEBPA was up-regulated in nasal tumor tissues, but the result was different with other tumor research. EGFR has abnormal methylation, but which had no expression. Through the analysis of the data, we could see that the close correlation between C-myc, CEBPA、GADD45G、THBSland DNMT3b methylation and low mRNA expression suggests.The expression of DNMT3b were up-regulated which connected with the hypermethylation of the genes. The genes methylation of C-myc,GADD45G and THBS1 have high specificity in distinguishing cancers from normal tissues. C-myc、 GADD45G and THBS1 gene has a significant associated with the tumorigenesis and development of ENT. |
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