Effects Of Acetylation Of Histone H3on The Regulation Of Nanog In Human Glioblastoma Cells

Objective To detect the effects of acetylation of histone H3on the regulation ofNanog gene in different pathological glioma tissues and U87glioma cell line. HistoneacH3of Nanog gene promoter expressing U87stable cell line was measured byChromatin Immunoprecipitation（ChIP）Real-time PCR. And to investigate the effect ofdecreasing of the acetylation of histone H3in promoter region of Nanog inducedNanog down-regulaterd, which provides a biological basis of Nanog could pay apotential molecular marker for improved pathological grading and a novel candidatetherapeutic target for glioblastoma management.Methods (1) The expression of acetylation of histone H3was detected by Real-timePCR analysis in53cases of glioma tissues and11cases of normal brain tissuesrespectively.(2) U87glioblastoma cells were cultured with different concentrations ofApicidin, Real-time PCR was employed to measure the mRNA expression of Nanogand Western-blot was applied to detect the acetylation of histone H3, ChromatinImmunoprecipitation（ChIP）Real-time PCR was used to measure the level of theacetylation of histone H3in promoter region of Nanog.(3) U87glioblastoma cellswere cultured with different concentrations of Apicidin, the expression level of Nanogwas detected at both mRNA and protein levels by RT-PCR and Western-blotrespectively. In addition, to evaluate its functional role in gliomas, the cell proliferation,cell apoptosis, cell cycle and migration in vitro were examined. Results (1) The relative protein level of H3ac was higher in gliomas compared tonormal brain tissues (F=72.80，P=0.00), Nanog mRNA expression levels inhigh-grade(III, IV) gliomas was higher than the low-grade(I, II) group（t=11.41,P=0.00）.(2) The Western-blot showed that the acetylation of histone H3wasdecreased by(1.87±0.08) fold in Apicidin1.0μmol/L group after48h(P=0.00<0.05) and theNanog was decreased by (2.73±0.13) fold（P=0.00<0.05). The level of the acetylation ofhistone H3in promoter region of Nanog was decreased to (2.31±0.44) fold（P=0.001<0.05）compared with those in control group.(3) Apicidin-treated down-regulation of Nanog inU87glioblastoma cells resulted in suppression of cell proliferation, arrest of cell cycleand inhibition of cell migration in vitro. Moreover, to confirm the effect of Apicidin invivo, tumor xenograft animal model was performed, which showed that Nanogdepletion significantly impaired tumor xenograft growth in nude mice (P<0.05). Thegrowth of U87xenografts was significantly inhibited by the apicidin when comparedwith control group (P<0.05). To confirm the effect of Apicidin induced apoptosis ofU87glioma cells, flow cytometry and transmission electron microscopy methods wereused. The number of the apoptotic cells were significantly higher in U87-treatmentedgroup compared to the the Blank group by flow cytometry.(P<0.05). After thetreatment of apicidin, U87displays a phenotype of apoptotic cells. The nucleicchromatic were aggregated along the margin of the nuclear membrane, and apoptoticbodies were also observed.Conclusion (1) These results suggested that the acetylation of histone H3could be oneof the important mechanisms that regulate its protein expression and influence theoccurrence and development of gliomas.(2) The expression of Nanog can be promotedby decreasing of the acetylation of histone H3in promoter region of Nanog.(3) Thesefindings indicated that Apicidin inhibits cell proliferation, migration capability, inducescell apoptosis and cell cycle arrest, which could be partly due to its inhibitory epigenetic modification effect on Nanog expression in glioma cells.