Roles Of EPO,EPOR And DNA Methylation Of EPO In The Hypoxic Tolerance Of HPC Mice

Objective The aim of this study is to explore the expression changes of EPO and EPOR and the relationship between them and DNA methylation of EPO in neuroprotection of hypoxic preconditioning(HPC)mice using animal model.Methods Adult male mice(body weigh 18.0-22.0 g)were randomly divided into DNA methyltransferase(DNMTs)inhibitor group(10 ?mol/L 5 ?L 5-aza-cdR was injected into lateralventricles)and control group(isopyknic 0.1% BSA).Then they were exposed to hypoxia for 4 runs(HPC),1 run(hypoxia),and 0 run(blank)one day later after the injection.Hippocampus were isolated after mice exposed to hypoxia 0-4 days;The mRNA levels of EPO,EPOR and the three kinds of DNA methyltransferase enzymes(DNMT1 ? DNMT3 A and DNMT3B)were examined using Real-time PCR.Western blot was used to examine the protein levels of EPO and EPOR in the mouse hippocampus after them exposured to HPC.Fragments of the EPO gene promoters(0-320 bp)was cloned and inserted into PGL3 basic vector.The expression of luciferase before and after the HPC was detected by dual-luciferase(LUC)reporter assay system.The activity of DNMT1,DNMT3 B and total DNMT in hippocampus of mice were measured by DNA Methyltransferase Activity Screeing Assay.Results(1)The mRNA level of DNMT3 B was down-regulated by acute repeated HPC and after continuous hypoxia(P<0.05).The mRNA level of DNMT3 A and DNMT1 was down-regulated after one and three days HPC respectively(P<0.05).But these changes weren't affected by the treatment of 5-aza-cdR.(2)The mRNA level of EPO was up-regulated two days after HPC and treated by injecting 5-aza-cdR respectively(P<0.05).(3)The EPOR mRNA level was down-regulated in early phase(0 day after HPC treatment)(P<0.05)and up-regulated in later phase(1-4 days after HPC treatment).Remarkably,these two changes weren't affected by the treatment of 5-aza-cdR.(4)The protein level of EPO and EPOR increased(P<0.05)and these changes coincides with the level of transcription.Additionally,the protein expression of EPOR was also increased after 5-aza-cdR treatment(P<0.05).(5)Luciferase activity assay showed that the promoter activity of EPO was enhanced in HPC group(P<0.05).(6)HPC treatment had no significant effect on the activity of DNMT1 in hippocampus of mice,but decreased the activity of DNMT3 B and total DNMT.Conclusions The decreased expression of these three kinds of DNA methyltransferase enzymes(DNMT1,DNMT3 A and DNMT3B)may lead to low methylation of some gene promoter sequences,which can regulate the expression of these genes.Therefore,the increased expression of EPO and EPOR in HPC may be related to the methylation extent of the EPO promoter by DNMT mediated and the change of the enzyme activity.