Cloning Of Mdm2Gene And Its Effects On Transcriptional Activity Of P53Targeting Genes In The Tibetan Mammals

Background and ObjectiveMdm2(Murine double minute2) had been discovered as an oncogene. Its overexpression and translational modification are important for the cells to adapt to the microenvironment around them. Myospalax Baileyi and Microtus oeconomus inhabit the meadow at3300m altitude on the Qinghai-Tibetan plateau, which is hypoxic and cold through year. The former one lives in underground burrows in the degenerative alpine meadow and the latter one lives in the shrub meadow. Therefore, using the mammals of Qinghai-Tibetan plateau as a model for investigating mdm2has important significance. We cloned the CDS domain of Myospalax Baileyi and Microtus oeconomus and investigated the changes and variation of their sequence preliminarily. We constructed the mdm2expression plasmid of the two mammals from Qinghai-Tibetan plateau and then co-transfected the NCI-H1299cells with the mdm2plasmid and human wild type p53expression plasmid so that we can investigate the functional differences on inhibiting p53transaction because of variation of their mdm2sequence. We co-transfected cells with mdm2and their own p53, comparing the interaction of mdm2-p53from two Qinghai-Tibetan plateau mammals. In the end, we investigated the effects of three sites variation in Microtus oeconomus mdm2to modulate p53transactional function. We also utilized hypobaric chamber to simulate hypoxia and then researched the changes of mdm2and relative genes in various tissue from SD rat.Research design and MethodsCloning the mdm2CDS domain of Myospalax Baileyi and Microtus oeconomus and analyzing the difference between them with molecular biology methods.Cell culture research:NCI-H1299cells co-transfected with mdm2plasmid (from human, Microtus ochrogaster, Myospalax Baileyi and Microtus oeconomus) and p53plasmid were cultured under normoxia and hypoxia (1%O2) respectively. Dual-luciferase reporter assay was employed to investigate mdm2function of modulating p53transactivated or transcriptional repressed target genes.Animal hypoxia model:SD rats were exposed to hypoxia mimicking altitude of7km (10.8%O2,41.02kPa) for1,8and24h. Quantitative Real Time PCR was used to test the expression of mdm2and relative genes in prefrontal cortex, pituitary, liver, and spleen respectively.Results1. Obtaining the mdm2CDS sequence of Myospalax Baileyi (hereafter M.b.) and Microtus oeconomus (hereafter Mo.). The M.b. mdm2CDS contains1464nucleotides, encoding a protein of487amino acids; The M.o. mdm2CDS contains1461nucleotides, encoding a protein of486amino acids. Analysis of M.b. mdm2amino acid with ClustalW showed that M.b. mdm2has an identity of95.47%with Spalax and Phylogenetic tree analysis showed that they are in the same branch. Analysis of M.o. mdm2amino acid with ClustalW showed that M.o. mdm2has an identity of99.18%with Microtus ochrogaster and Phylogenetic tree analysis showed that they are in the same branch. Compared with human mdm2, M.b. mdm2contains a tyrosine-390-phenylalanine variation. Compared with Microtus ochrogaster, M.o. mdm2has a122-valine insertion and two mutations, serine-125-asparagine and arginine-126-lysine.2. A:Dual-luciferase reporter assay using human p53plasmid indicates that:compared with human mdm2, M.b. mdm2and M.o. mdm2significantly inhibit p53transactivation, and M.o. mdm2has a stronger ability to inhibite p53transactivity for IGFBP3, Bax, Hdm2and p21than M.b. mdm2. The inhibition of p53transactivity of Apafl, IGFBP3, Hdm2and p21from M.b. and M.o. mdm2became weak when cells were exposed in hypoxia; B:Dual-luciferase reporter assay using M.b. and M.o. p53plasmid respectively indicates that:compared with human mdm2-p53, M.b. mdm2and M.o. mdm2significantly inhibited p53transactivity, and M.o. mdm2-p53has a stronger function in inhibiting p53transactivity for Apafl, IGFBP3, Puma, Noxa, Hdm2and p21than M.b. mdm2-p53. The inhibition for their own p53transactivation of Apafl and Hdm2from M.b. and M.o. mdm2decreased when cells are exposed in hypoxia.3. Compared with the Microtus ochrogaster mdm2, M.o. mdm2has weaker inhibition for p53transactivation of Apafl, Noxa, Bax and Hdm2. When the cells were exposed to hypoxia, p53transactivation of Apafl, IGFBP3and Hdm2increased and of Bax decreased. 4. Hypoxia changed the expression of mdm2and relative genes in prefrontal cortex, pituitary, liver, and spleen respectively. Mdm2expression has the same model as p53in various tissues under hypoxia.ConclusionsWe have cloned the M.b. mdm2CDS domain (1464nt and487aa) and the M.o. mdm2CDS domain (1461nt and486aa). We discovered codon390variation of M.b. mdm2and codon122,125and126variation of M.o. mdm2. These mutations may affect mdm2-p53interaction and play an important role that the animals on Qinghai-Tibetan plateau can adapt to the environment better.M.o. mdm2has a stronger function in inhibiting human p53transactivation, suggesting that the difference between M.b. mdm2and M.o. mdm2sequence leads to M.o. mdm2has a stronger interaction with p53; the stronger inhibition of M.o. mdm2to its own p53comparing with M.b. indicates divesed adaptive mechanisms of mdm2-p53interaction in Qinghai-Tibetan plateau animals.The expression of mdm2is correlating with expression of p53, suggesting that the mdm2-p53autoregulatory loop under hypoxia is complicated.