Transcription Regulation Of MDM2 Gene And Methylation Of P53 Between Myospalax Baileyi And Myospalax Cansus

Background and ObjectiveMdm2(Murine double minute 2)is an oncogene that was first discovered in a locus amplified on double minute chromosome in mouse fibroblast.As a negative regulatory factor of cancer suppressor factor p53,MDM2 can bind to p53 and promote p53 translocation from nucleus to cytoplasm via its NES sequence.MDM2 plays an important role in inhibition of p53 transcriptional activity and ubiquitinate p53 for proteasomal degradation.MDM2 is a proto-oncogene,its expression level and posttranslational modification may play an important role in cells for the adaptation of MDM2 over high CO2 and hypoxic microenvironment.Human colorectal carcinoma(RKO)cells express significantly less Mdm2 and concurrently more p53.Other studies have also shown that Mdm2 is down regulated under hypoxia in both primary and transformed cell lines.M.baileyi lives in Qinghai-Tibet plateau burrows at an altitude of about 3300 m(14%O2)therefore enduring meadow of low oxygen and cold environment for its whole life cycle;whereas,M.cansus mainly living in the plain,at lower altitude of about 600m(19.7%O2)for its whole cycle,suffering different concentration of hypoxia.However,as previously proposed by Wei D.B,oxygen partial pressure in the burrow of M.baileyi is higher than that of SD rat{R.norvegicus).In their burrows,oxygen partial pressure is low and carbon dioxide partial pressure is high but their contents fluctuate with the change of seasons,soil types,rain and depth of burrows.Oxygen partial pressure in arterial blood and venous blood of M.baileyi is about 1.5 times and 0.26 times higher respectively compared to SD rat.Partial pressure for carbon dioxide in arterial blood of M.baileyi is 1.5-fold higher than that in SD rat.M.baileyi shows successful adaptation to that extreme environment.These animals have developed different adaptation mechanisms which have help them to acclimatize in hypoxic environment.We previously reported that small mammals such as Ochotona curzonia,Myospalax baileyi and Microtus oeconomus living in Qinghai-Tibet alpine meadow indicates similar adaptations to the extreme environment.This paper mainly discusses the comparison of expression level of MDM2 and its related genes between M.baileyi and M.cansus.We also investigated the transcription activity of Apafl and p21 in MDM2-p53 interaction.Furthermore,we found that the methylation level in p53 promoter in M.baileyi was different from M.cansus.In previous researcher done in S.galili(subterranean rat in Israel),the methylation of p53 promoter decreased p53 gene expression.It gives an implication that p53 promoter methylation may decrease the interaction of MDM2-p53.Research design and MethodsCloning MDM2 CDS domain of M.cansus and analyzing the differences between M.cansus and M.baileyi with molecular biology methods.In animal level;we focused on MDM2 and its related genes about tissue-specific expression between M.baileyi and M.cansus.Furthermore,we simulated the hypoxic environment(7000m,8%O2)with SD rats,in order to compare the tissue-specific expression of SD rats under normoxia and hypoxia.Cell culture:NCI-H1299 cells co-transfected with MDM2 plasmid(from.human,Myospalax baileyi Myospalax cansus,Microtus oeconomus,Microtus ochrogaster)and human p53 plasmid,were cultured under normoxia,hypoxia(1%O2)and high CO2(pH=6.4)respectively.Dual-luciferase reporter assay was used to.investigate that MDM2 function of modulating p53 transactivated or transcriptional repressed target genes.To enable us to predict the possible transcription regulatory regions and target transcription factors,methylation level of p53 promoter in M.baileyi and M.cansus has been analysed.Results1.Cloning MDM2 CDS domain of M.cansus(GenBank:KR149456.1).M.cansus MDM2 CDS area is 1461 bp,encoding 487 amino acid residues.Comparison of M.baileyi and M.cansus shows that the homology ratio is 99.38%.The evolutionary tree analysis shows that MDM2 between M.baileyi and M.cansus is in the same branch.The exchange of Glycine to Serine at position 131 is found in M.cansus.Furthermore,SD rat compared to M.baileyi and M.cansus,the ratio of homology is 81.6%and 82%respectively.2.The MDM2 expression level of spleen in M.baileyi is higher than that in Myospalax cansus;The p53 expression level of spleen and liver of M.baileyi is higher than that of M.cansus;However,M.baileyi p53 expression level is lower than M.cansus in the brain.The p21 expression level of the liver,spleen and lung in M.baileyi is higher than that in M.cansus,but lower in the brain respectively;The puma expression level of liver and lung in M.baileyi is lower than that in M.cansus.3.Under hypoxia for 8 hours,the MDM2 expression level of spleen in SD rats is up-regulated,whereas,the level shows no significant changes in the brain.The p53 expression level of liver and lung is down-regulation under hypoxia,without a significant change in the brain.4.Results by dual-luciferase reporter assay in H1299 cell:Hypoxia increases the transcription activity of Apafl and p21(vs normoxia)after co-transfected hp53 and M.baileyi MDM2.Under hypoxia,through co-transfected p53 and MDM2 plasmid of M.cansus,the expression activity of Apafl increases higher than that of M.baileyi.Under high CO2,activity of Apafl in M.baileyi MDM2-p53 regulation is higher than that of M.cansus.M.baileyi MDM2 responsed to hypoxic and high CO2 environment mainly by a series of activity of Apafl and p21.5.Using MethPrimer to analysis the CpG islands of p53 promoter from the lung of M.baileyi have much difference in the regulation domain.The results exhibit that four of CpG islands are predicted within 2-kb upstream region of M.baileyi p53,the sequence length is 100bp,135bp,358bp and 105bp respectively.Two of CpG islands are predicted within 1959bp upstream region of M.cansus p53,showing 504bp and 108bp respecetively.The CpG islands in M.baileyi is much disperser than that in M.cansus,closed to the transcription start site.However,the CpG islands between-1487?-1181 bp region are much higher than others in both of two species.Among the 307bp of p53 promoter,the CpG islands methylation level is vary different between the two species.Ten of methylation sites showing significant changes in the total of 17 sites.Through PROMO software analysis,40 transcription factors are predicted,33 of them are the same.In the number 1(-1448 bp in the promoter of M.baileyi)and 2(-1446 bp in the promoter of M.baileyi)methylation sites,transcription factors such as COE1,CP2,AP-2 and Yi can bind to these sites respectively.ConclusionsMDM2 of M.baileyi and M.cansus is highly conservative,we found that the exchange of Glycine to Serine at position 131(M.baileyi and SD rat are Glycine).Significant changes of tissue-specific expression were seen in the expression level of MDM2 and p53.MDM2 expression was up-regulated in the spleen of SD rats exposed to hypobaric hypoxia.Further study was conducted to investigate H1299 cell lines under 1%hypoxia for 16h and high CO2 environment.The transcription activity of Apafl of M.baileyi was lower than that of M.cansus under hypoxia,but activity of Apafl of M.baileyi was higher than that of M.cansus under high CO2.Apafl is an apoptosis up-regulated gene by p53.This indicates that MDM2 of M.baileyi plays an important role for its adaptation to hypoxic environment and high CO2 environment.Furthermore,the methylation level of p53 promoter in M.baileyi is higher than that of M.cansus.Above all,our researches suggested that there is a relationship between hypoxic environment and gene expression which has lead to the adaptation of M.baileyi and M.cansus to live under low oxygen in burrows.