The Molecular Mechanism Of P16^INK4a In Regulating The Aging Progress Of Werner Syndrome Mice By Modulating DNA Damage Repair And Mitochondria Biosynthesis

Aging is a natural process in which the structural integrity of the organism gradually decreases,resulting in a decline in the function of the organism and an increase in the risk of death.Aging itself has become the biggest risk factor among almost all the main causes of morbidity and motality in the modern world.Study the molecular mechanism of aging and understanding the potential interventions to prevent aging is beneficial to the diagnosis and treatment of aging-related diseases.Werner Syndrome（WS）is an autosomal recessive genetic disease caused by Wrn gene mutation.The clinical symptoms include aging-related cataract,osteoporosis,skin ulceration,low reproductive system function and so on.The average life expectancy is only 46-48 years.The WS mouse model constructed by knocking out Wrn and telomerase RNA template Terc provides a valuable research system for the study of aging and aging-related diseases.p16^INK4a（abbr.p16,Ink4a gene coding）is one member of the family of cell cycle-dependent kinase（cyclin-dependent kinase,CDK）inhibitors,which regulates cell cycle,tumorigenesis,cell senescence and apoptosis.In order to study the regulatory mechanism of p16 in WS mice,we crossed p16knockout mouse with WS mouse(m TR^-/-Wrn^-/-,double knock out,DKO)to obtain homozygous m TR^-/-Wrn^-/-p16^-/-mouse（triple knock out,TKO,p16-TKO）,and then obtained different generations of mice（G1-G5）by continuous breeding.In previous studies,it was found that the p16 knockout（p16-TKO）mice lived longer than WS（DKO）mice.By measuring bone mineral density,it was found that the symptoms of osteoporosis were rescued and the structures of high proliferative organs such as the small intestine and testis were protected.At the same time,through the detection of tissue proliferation and apoptosis in mice,it was found that p16-TKO mice had better proliferation ability.Through further study,it was found that p16 was involved in maintaining the integrity of telomere.In order to further study the molecular mechanism of p16 in regulating the aging process of WS,this study explored the DNA damage repair and mitochondrial synthesis pathway.First of all,we detected the expression of DNA damage markerγ-H2AX in DKO and TKO cells by immunofluorescence assay.The results showed that there were a large number ofγ-H2AX signals in G5DKO cells,whileγ-H2AX signals decreased significantly in p16-G5TKO cells.This suggests that abnormal telomere function leads to a large number of DNA damages.The knockouting p16 can markedly alleviate the accumulation of DNA damage signals,suggesting that p16mediates cell senescence caused by DNA damage.Then,we detected the expression of p53,MDM2,p21,damage repair proteins（Rad51,RPA,pol E）,Bax,PARP)and proliferation-related protein（PCNA）by Western blot.The results showed that the expression ofγ-H2AX in p16-G5TKO cells was significantly lower than that in G5DKO cells,the expression of total p53 protein increased,the levels of acetylation and phosphorylation decreased,and the expression of p19 and MDM2 decreased.These data suggest that when p16 is deleted,the transcriptional activity of p53decreases and the stress level of DNA injury decreases.At the same time,the cells activated a strong DNA repair pathway.which is reflected by the increased expression of pol E,Rad51 and RPA proteins,decreased expression of apoptosis-related proteins PARP and Bax,and increased expression of PCNA proliferation proteins.Therefore,these molecular regulation after p16 knockout may be the reason for rescueing the aging symptoms of WS mice.The integrity of mitochondrial function is related to the fate of cells,and we measured the number of mitochondria in cells by flow cytometry.The results showed that the number of mitochondria in DKO cells decreased significantly,while the number of mitochondria in TKO cells after p16 knockout did not decrease.We further detected the copy number of CYTB,COX1 and ND1 in mitochondrial genome by Real-time PCR,and found that the copy number in G5DKO cells decreased markedly,while the copy number in p16-G5TKO cells was not significantly different from that in WT cells.Thee data indicates that the loss of p16 function can effectively rescue the decrease of mitochondria in MEF cells of WS mice.By measuring the mitochondrial membrane potential,we also found a significant increase in the quality of mitochondria in p16-G5TKO cells.By measuring the mitochondrial ATP production,we found that after knockout p16,the mitochondria in p16-G5TKO cells produced more ATP than those in G5DKO cells.We also proved that the expression of SIRT1,PGC-1αand FOXO3 was up-regulated in p16-G5TKO.The above data suggest that the expression of p16 in WS cells inhibits mitochondrial biosynthesis and leads to slow cell metabolism,which might faciliate cellular senescence.Knockout of p16 can restore mitochondrial biosynthesis and rescue the symptoms of WS senescence.Stem cells promote tissue homeostasis and regeneration through their unique ability of self-renewal and differentiation.We detected the abundance of HSC（Hematopoietic stem cell）in mouse bone marrow by flow cytometry.We found that the abundance of HSC in G5DKO 2-month-old mice was much lower than that in p16-G5TKO mice of the same age.This phenomenon suggests that the exhaustion of stem cells in WS mice can be rescued after the deletion of p16,indicating that p16 is involved in the homeostasis regulation of stem cells.To sum up,this study showed that in the background of WS,knocking out p16could reduce the activity of p53,thus reduce DDR,and enhance the ability of DNA damage repair,reduce apoptosis and enhance the ability of cell proliferation.It also activated the SIRT1-PGC-1αpathway,improved the quality and quantity of mitochondria,enhanced cell energy metabolism,thus might rescue cellular senescence in WS MEFs.This study explores the molecular mechanism of p16 in regulating the cellular senescence of WS,and sheds new light and provide new targets for anti-aging and anti-tumor strategies.