Regulation Of P53^N236S On Senescence Induced By Telomere Dysfunction And Environmental Toxicity Factors

Aging is a biological process in which the physiological functions of the human body gradually subsides.The body is accompanied by various diseases caused by degenerative changes in the process of aging,such as cardiovascular diseases and tumors.With the improvement of living standard,the pursuit of health and longevity has become a concern of people.In recent years,the global environment has become increasingly harsh,and the free radicals（environmental toxic factors）that continue to form in the environment continue to stimulate the health of the human body,especially the elderly,and thus have a negative impact on their longevity.Therefore,the environmental effects of toxic factors（such as oxidative damage factors such as peroxides）and genetic factors have further promoted the body’s aging and other aging-related diseases.Werner syndrome（WS）is a human autosomal recessive disorder which is caused by mutation of the RecQ helicase family Wrn gene.Since mouse adult cells retain telomerase activity,the telomere length of mouse cells is long.Therefore,in the establishment of a Wrn syndrome mouse model,it is necessary to simultaneously knock out Wrn and telomerase,and propagate the mouse to the fifth generation（G5DM）.After the telomere is shortened to a certain extent,the premature aging phenotype of human WS can be expressed..Embryonic fibroblasts（MEF）of this G5generation Wrn syndrome mouse rapidly ageing in vitro,and continued culture cells cause some cells to escape senescence.In the early stage,we studied the escape senescence of G5DM cells and found that p53N236S（p53S）mutation occurred in the p53 protein of tumor suppressor gene.Mutant p53S is the key to escape senescence of cells and restart the telomere maintenance mechanism.To reveal the regulation of p53S on Wrn syndrome-induced aging phenotype by comparing the life span,tumor incidence and aging phenotype of G1-G5TM and G1-G5DM mice,and to further study the regulation mechanism at cellular and molecular levels.The results are as follows:1)The effect of p53S on the life span of Wrn syndrome mice.The results showed that the lifespan of G1-G3DM mice was longer than that of G1-G3TM mice in the early algebra of long telomeres.Further analysis showed that the incidence of tumors increased gradually from G1-G3TM mice to the peak of G3TM mice.The lifespan of G1-G3TM mice was shortened by early death from tumors,while that of G4-G5DM mice was shorter by late Algebra with shorter telomeres than that of G4-G5TM mice,and less tumors were observed in G4-G5TM mice.The incidence is very low.These data suggest that the regulation of p53S on aging-related phenotypes depends on the length of telomeres.2)The effect of p53S on aging-related degenerative changes of tissues and organs in Wrn syndrome mice.By comparing the appearance changes of G1-G5D/TM mice and the degenerative changes of senile sensitive tissues,we found that there was no significant difference in body shape and appearance between DM and TM mice in G1-G3 generation,while G4 and G5DM mice were significantly smaller than TM,suggesting that p53S alleviated Wrn syndrome-induced growth retardation in mice.The testicular internal structure of G5DM mice was seriously vacuolar,and the morphology of primary/secondary seminiferous tubules disappeared,while the testicular internal structure of G5TM mice was normal.BrdU incorporation experiment showed that compared with DM mice,G5TM mice had significantly improved the proliferation of small intestinal villus cells.Bone mineral density（BMD）of G5TM mice was significantly higher than that of G5DM mice.These data indicate that p53S delay the aging phenotype of Wrn syndrome and prevent the degeneration of organs and tissues caused by the aging process.3)Tumors and polycystic kidney disease（PKD）are cooccurred in G1-G3TM mice.The analysis of RNA-Seq data of tumor tissues and PKD kidney tissues showed that both tumors and PKD tissues activated mitochondrial energy metabolism pathways,complement pathways and lipid metabolism pathways,suggesting that the regulation of these pathways may be a common factor leading to tumors and PKD.We further found that p53S could down-regulate Pkd1/2,Pkhd1 and Hnf1b,which eventually led to the occurrence of PKD.These data revealed that p53 mutations have acquired new functions in regulating PKD pathways,and for the first time confirmed the association between tumors and PKD occurrence at the mouse level.Further,in order to study the molecular mechanism of p53S regulating Wrn syndrome aging and its regulation on telomere length,we used G1,G3,G5DM and TM MEFs to analyze the molecular pathway of p53S regulating Wrn syndrome mice aging at the cellular level.The results showed that:1)p53S interferes with Wrn syndrome-induced cell aging phenotype by regulating cell cycle,cell proliferation,chromosome ploidy and so on.p53S attenuated Wrn syndrome-induced cell growth inhibition.G2/M block is present in p53S and G1TM cells,and chromosomes exhibit polyploidy,breakage,crossover and other aberrations.The data of apoptosis showed that the apoptosis signal of G1TM and G3TM cells was stronger than that of G1DM and G3DM cells.Based on data on telomere length,we conclude that p53S initiates an alternative mechanism for maintaining telomere elongation in Wrn syndrome,alleviating aging due to telomere shortening.The telomere elongation mechanism is usually closely related to tumorigenesis.We still verified the tumorigenicity of G1-G5TM cells,and the TM cells were less tumorigenic.In conclusion,p53S can alleviate the aging phenotype induced by Wrn syndrome-induced telomere dysfunction by enhancing cell proliferation,regulating cell cycle and chromosome ploidy,and initiating telomere elongation mechanism.2)p53S affects the senescence process of Wrn syndrome cells by regulating DNA damage response,DNA replication stress and mitotic-related pathway.To investigate the molecular pathways of p53S regulation of senescence in Wrn syndrome cells,we collected WT,p53S/S,G1DM,G3DM,G5DM,G1TM,G3TM and G5TM MEFs for RNA-seq analysis.We found that wild-type p53-induced DNA damage response pathway activity is gradually enhanced in G1-G5TM cells.At the same time,DNA repair pathway activity was also enhanced in G1-G5TM cells,indicating that p53S promotes DNA damage repair.In addition,cell cycle-associated genes regulated by another oncogene Rb were significantly enriched in p53S and G1TM.p53S promotes the high expression of DNA replication and repair related proteins,which may activate DNA damage repair,telomere maintenance and other mechanisms to protect the normal function of cell function.The data suggest that p53S enhances the stress response in response to weak telomere damage（G1）.According to the results of ssGSEA data analysis,we validated G2/M cycle regulation related genes in p53S cells.The data showed that compared with WT and p53null,Wee1,Birc5,Brca2,Cdc2,Cdk6,PolE,PolQ,Bard1,Gins2,Ube2C,Chk2and Cdc25C genes were significantly higher in p53S cells;Western blot data showed that compared with WT and p53null cells,Wee1,Birc5,Brca2,Cdk6,Cdk6,PolE,PolQ,Bard1,Gins2,Ube2C,CDC25,p-CDC25,Weel,p-Cdc2,Cdk 2 and Cdk6 were highly expressed in p53S MEFs,while Pole,Rad51,Gins,Bard1,Survivin,gamma-H2AX,CHK1/2 and p-CHK1/2 were highly expressed in p53S.The data confirmed that p53S regulates cell cycle and chromosome aberration through G2/M monitoring sites and mitotic spindle monitoring sites,and its biological significance needs further study.Finally,we induced the senescence of cells using peroxide H₂O₂ to investigate the role of p53S in regulating cellular responses to environmental oxidative stress.The results of senescence staining showed that H₂O₂ treatment induced a large number of senescent cells in wild-type（WT）cells,while cells carrying p53S mutations were less senescent,suggesting that the presence of p53S confers resistance to oxidative damage-induced senescence.In conclusion,we found that mutant p53S may rescue the aging process induced by oxidative stress and gene mutation by regulating DNA damage repair and activating telomere maintenance mechanism,thus delaying the aging process of Wrn syndrome mice and prolonging the life span of Wrn syndrome mice.Our study from organism level has proved the balancing between aging and tumors.It provides a new idea for the prevention and treatment of aging-related diseases and tumors.