Analysis And Significance Of Nanog5’ Untranslated Regions Epigenetic In Gliomas

Objective To detect the methylation status and the histone modifications of Nanoggene5′untranslated regions, to explore their possible mechanism to effect Nanog genetranscription and the function in glioma. To lay a foundation of the further explore intargeting epigenetic regulation of Nanog gene to affect the biological activity ofglioma cellsMethods (1)Methylation specific pcr were conducted to detect Nanog gene promoter’smethylation status in12cases of normal brain tissues,51cases of glioma tissues,22cases of paracancerous tissues.(2)Glioma stem cells were isolated from glioma celI lineU87and cultured in simplified sernm-free neural stem cell medium by nanospheresuspension culture method spheres; methylation specific pcr were conducted todetect Nanog gene promoter’s methylation status in glioma stem cells and glioma celllines U87.(3)The expression of Nanog mRNA was detected by Real-time PCR inglioma and glioma cells.(4) Western blot was applied to detect the acetylation ofhistone H3and the methylation of H3K9in gliomas.(5) ChromatinImmunoprecipitation（ChIP）Real-time PCR was used to measure the level of theacetylation of histone H3and the methylation of H3K9in promoter region of Nanog ingiloma.Results (1)Nanog gene promoter was hypermethylated in gliomas, and the presence was significantly higher in gliomas than that in the paracancerous tissues(58.82%vs.13.63%,χ2=14.852,P=0.000).(2)GSCs were isolated, cultured and purified fromglioma cell line U87; Nanog gene promoter unmethylation state was higher in gliomastem cells compared to U87cell line.(3) Nanog mRNA expression levels inhigh-grade(III+IV) gliomas was higher than the low-grade(I+II) group,and NanogmRNA expression levels in glioma stem cells was higher than U87cell line.(4) Therelative protein level of H3ac was higher in gliomas compared to normal brain tissues（F=72.80，P=0.00）. The relative protein level of H3K9me3was lower in gliomasthan that in the normal brain tissue（sF=84.79,P=0.00）. The acetylation level of histoneH3was higher in promoter region of Nanog in gliomas compared to normal braintissues（F=59.34,P=0.00）. The methylation level of H3K9in promoter region ofNanog was lower in gliomas compared to normal brain tissues（F=F=74.88,P=0.00）.Conclusion Unmethylation state and histone modifications of Nanog gene promoterregions is the important mechanisms that up-regulate its protein expression andinfluence the occurrence and development of gliomas.