The Mechanism Of HCMV Involving In Glioma Development Through Affecting The Methylation Level Of ATF5

Objective 1.To study the relationship between HCMV infection and gliomas through detecting the expression of IE2 gene in gliomas and normal tissues.2.To analysis the methylation and expression level of ATF5 in clinical glioma tissues and to explore the effects of HCMV infection on the methylation status and expression level of ATF5 in gliomas.3.To establish the cell models of HCMV infection,analysis the methylation and expression level of ATF5,and to explore the effects of HCMV infection on the methylation status and expression level of ATF5 in gliomas.4.To explore the effects of SAM on the methylation status and the expression level of ATF5 during HCMV infection and the effects of SAM and HCMV on the expression level of TET1,DNMT1,DNMT3 A and DNMT3 B.Methods 1.To obtain glioma tissues and clinicopathological information.2.IE2 m RNA expression level was detected using RT-PCR,and the correlation between HCMV and glioma was analyzed.3.To predict Cp G islands and design bisulfite-conversion-based methylation PCR primers of the promoter region of the ATF5 gene using online tools.Genomic DNA was isolated from clinical tissues,treated with bisulfite,PCR was performed,and separated PCR products were cloned into TA-cloning vectors for DNA sequencing,and analysis the ATF5 methylation level in gliomas and normal tissues.4.Quantitative RT-PCR was also performed to detect ATF5 m RNA expression in 35 cases of glioma and 5 normal tissues.The effects of HCMV infection on the methylation status and expression level of ATF5 were also analyzed.5.To analysis the correlation between ATF5 methylation and the characteristics of the clinical pathology.6.To obtain HELF,primary glioblastoma cells,and HCMV,establish cell models of HCMV infection in vitro,and treat cells with SAM.7.To analysis the ATF5 methylation level using BSP sequencing.The effects of HCMV infection and SAM on the methylation status and expression level of ATF5 were also analyzed.8.Quantitative RT-PCR and Western Blot was also performed to detect the expression of ATF5 and methylation related genes(TET1,DNMT1,DNMT3 A,DNMT3B).9.CCK-8 was used to detect the effects of HCMV infection and SAM on the proliferation of glioma cells.Results 1.35 cases of glioma tissues and 5 normal tissues with acute brain trauma were obtained,and glioma of pathologic grade ?-?was classified as low grade one(well differentiated glioma)and that of pathologic grade ?-? as high grade one(poorly differentiated glioma).The average expression levels of IE2 m RNA were significantly higher in glioma than those in the normal tissues(P<0.05),and it's also higher in high-grade glioma than those in low-grade glioma,while it's low in normal tissues.2.Statistical analysis showed that the percentage of methylation of the promoter region of the ATF5 gene was 87.78%,73.89% and 47.70% in normal tissues,low grade glioma and high grade glioma,respectively.3.Quantitative RT-PCR results showed that the average expression levels of ATF5 m RNA were significantly higher in glioma than those in the normal tissues(P<0.05),and it's also higher in HCMV positive gliomas than in HCMV negative gliomas.These results support the hypothesis that the expression level of ATF5 is associated with its methylation status.4.Among the patients examined,the correlation between ATF5 methylation and clinical stages was found to be statistically significant,while it was not significantly correlated with tumor size,gender,and others.5.Astrocytes,U87,and primary glioblastoma cells are permissible for HCMV infection.6.Statistical analysis showed that the percentage of methylation of the promoter region of the ATF5 gene was 96.30% in astrocytes for 0h,and it was 46.30% for HCMV infection 6h,the methylation ratio significantly decreased(P<0.05).In U87 and primary glioblastoma cells,the percentage of methylation of the promoter region of the ATF5 gene significantly decreased for HCMV infection 72 h compared with control(P<0.05),and it was increased during SAM and HCMV + SAM treatment.7.RT-PCR and Western Blot showed that HCMV infection can increase the m RNA and protein level of ATF5,while it was decreased during SAM treatment.At the same time,TET1 expression levels rise,DNMT1 expression level decreased,but DNMT3 A and DNMT3 B did not change significantly.8.CCK-8 showed that HCMV infection can promote tumor cells proliferation,and SAM can inhibit tumor cells proliferation and have no significant effects on astrocyte proliferation.Conclusions 1.The expression of IE2 m RNA was higher in glioma and was significantly correlated with clinical stages.2.The methylation and expression level of ATF5 gene was significantly correlated with clinical stages and its expression level is associated with its methylation status.3.HCMV maybe participate in the development of glioma through raising TET1 expression level,reducing DNMT1 expression level,and affecting the methylation status of ATF5,and activated the transcription of ATF5,which plays an important role in promoting tumor cells proliferation and inhibiting tumor cells apoptosis.4.SAM can inhibit tumor cells proliferation and promote apoptosis of tumor cells through raising the methylation level of ATF5,which maybe regulated by TET1 expression level.