The Impact Of P21^Waf1/Cip1 Or P16^Ink4a Dysfunction In The Aging Process Of Werner Syndrome Mice And Its Molecualr Mechanism

The aging of population is a global problem,accompanied by the high incidence of age related diseases,which challenges the health care system.At the same time,with the improvement of people's living standards,the healthy and longevous lifestyle has become a new pursuit.The study of aging process and its molecular mechanisms is essential for the prevention and artificial intervention of aging-related diseases,and provides scientific basis for understanding aging-related diseases,such as tumors,organ degeneration and so on.Human Werner syndrome?WS?is an autosomal recessive progeric disease.Mouse model of WS derived from dysfunction provides a good research system for aging and aging-related diseases.Both p21^Waf1/Cip1?p21,encoded by Cdkn1a gene?and p16^Ink4a?p16,encoded by Ink4a gene?are important cyclin-dependent kinase inhibitors,which play important roles in cell cycle control,tumorigenesis and cellular senescence.To test the effect of p21 or p16 deficiency in WS,we crossed WS mice(mTR^-/-Wrn^-/-,double knock out,DKO)with p21^-/-or p16^-/-mice to construct the triple knock out mice?p21-TKO or p16-TKO?.By studying the degenerative changes of bone,testis,small intestine and other tissues.We surprisingly found that the p21-TKO mice displayed accelerated premature aging comparing with DKO mice,while p16-TKO mice attenuated the aging phenotypes.The detailed results are summarized as following:1.Comparing with WS mice,p21-TKO mice were characterized by small size,serious rickets and short lifespan,the p16-TKO mice were normal in size and had a significant longer lifespan.The lifespan of the mice with natural death was collected and the survival curve was drawn.The results show that the p21 deficiency shortened the lifespan and accelerated the aging of Werner Syndrome mice while p16 deficiency rescued it;2.By microCT,we found that comparing with WS mice,the p21-TKO mice manifested severe osteoporosis,while the p16-TKO mice rescued the osteoporosis phenotype in WS mice.We observed the changes in the tissue structure of the small intestine and testis of mice with different genotypes by HE staiing.The results show that,the deletion of p21 caused the disruption of small intestinal villi structure and the depletion of LGR5 positive stem cells in cryspts.The testicular tissue was also depleted.However,with p16 deficiency in WS,the normal small intestine villi structure was restored,with restored LGR5 position stem cells in the cryspts,the testicular tissue became normal.3.By BrdU incorporation,SA-?-Gal staining and TUNEL assay,we detected tissue cell proliferation,senescence,and apoptosis in mice with different genotyges.We found that the small intestine and testis of WS mice with p21 and p16 deficincy showed increased cell proliferation ability.The apoptosis level of WS mice with p21deletion was also increased,and the apoptosis level of WS mice with p16 deletion was less.These data suggested that p21 deletion accelerated the proliferation and apoptosis of WS mice,and p16 deletion attenuated the apoptosis of WS mice.Furthermore,we studied the molecular mechanisms of p21 or p16 deletion in regulating WS aging phenotypes at the cellular level.The results showed that:1.Changes in telomere signal and length of MEFs in mice with different genotypes were detected by PNA-FISH and and TRF-Southern analysis.The date suggests that p16 function is involved in telomere maintenance mechanism involved but not p21 function;2.The p21 deficiency in WS background accelerated the cellular proliferation with high level of DDR.On the other hand,p16 deficiency in WS background rescued the high level of DDR derived from WS,and did not activate either cell cycle inhibitors like p53,p19,p27,Rb,p130 etc,nor cell cycle promoters like E2F1,CDK6,CDK4;3.The protein level of SIRT-1/PGC1-?,FOXO1,FOXO3A was detected.The results showed that,p21 and p16 play an important role in regulating aging process and energy metabolism.These data suggests that p21 protected the stem cell reservoir by regulating cellular proliferation and turnover in a proper rate,p21 loss in WS activated fairly severe DDR responses,might caused abnormal increase of tissue repair.On the other hand,the function of p16 promoted cellular senescence by inhibiting cellular proliferation,the deficiency of p16 released this barrier signal without causing severe DDR.Our study highlights the role of p21,p16 in regulating aging process,provids the basis for selecting target genes in anti-aging drug screening and artificial interference.