Epigenetic Modifications of PI6 during Cellular Senescence

Objective To understand the epigenetic regulations of P16 during cellular replicative senescence and premature senescence induced by hydrogen peroxide of human embryonic lung fibroblasts(HEFs).Methods The normal HEFs were divided into young cells(22 PDL),mid-aged cells(35 PDL)and replicative senescent cells(49 PDL)during replicative senescence.The synchronously cultured 22 PDL HEFs were exposed for 2 h to 400μmol/L H_2O_2 at half confluence on daily basis.The procedure lasted for 4 consecutive days.And then the treated cells were cultured for another 7 days,called premature senescent cells.The mRNA level of P16 was detected by fluorescence quantitative PCR.The methylation level in the promoter region -846～-639 bp was observed by methylation-specific PCR(MSP).The histone modifications was detected by chromatin immunoprecipitation-QPCR assay,including acetylation for H3,H4 and methylation for H3(Lys4)and H4(Lys20).Results In the process of cellular senescence,the mRNA level of P16 decreased in mid-aged cells,but increased significantly in both replicative and premature senescent cells compared with that of young cells(P0.05).In the-1000 bp of the first exon upstream promotor region of P16,the length of CpG island was 995 bp.The amplified fragment of MSP was 208 bp,located in-846～-639 bp.The relative quantity was 0.42,0.34 and 0.47 of methylated fragment for mid-aged cells,replicative senescent cells and premature senescent cells respectively,and 0.61,0.96 and 0.79 of unmethylated fragment respectively.In the promoter(-685～-489 bp)of P16,the main histone modifications was H4(Lys20)methylation for both replicative and premature senescence,while in the region(-229～-60 bp) was H3,H4 acetylation and H4K20 methylation jointly in replicative senescence,and H3 and H4 acetylation in premature senescence.Conclusion The histone modifications in the promoter region take part in regulating the mRNA expression for P16 during cellular senescence and different mechanisms exist between replicative and premature senescence.