The Role Of P53in Cell Senescence,Autophagy And Apoptosis

Cell autophagy (or autophagocytosis) was a process of degradation its own damaged organelles and macromolecule material by the lysosome under the control of autophagy related gene (Atg), and can be activated in a variety of stress and extend the life time of the cell. Cell senescence was cells entered a relatively stable state after cells was out of the cell cycle and irreversibly lost cell proliferation ability. It is the inevitable outcome of the normal adult cells. Cell apoptosis was a process of itself active and orderly death regulated by genes in a variety of stress. Both classical and modernized cellular reprogramming are stressful processes that activate apoptosis and cellular senescence, which are the two primary barriers to cancer development and somatic reprogramming.The p53protein, one of the key nodes in a variety of regulatory networks, played a vital role, in the maintenance of genome integrity and the response to various cellular stress by regulating the cell cycle. P53involved in cell cycle regulation by controlling the control points of the proliferation of cell from cell cycle G1to S. It enforced the DNA damaged cells DNA stopped at G1phase and repaired the damaged DNA. If DNA cannot be repaired, p53induced these cell entering into apoptosis.p53was a common regulatory protein of the senescence, autophagy and apoptosis, but it was unclear that p53inhibited or promoted autophagy during cell senescence. At the same time p53can be used as a kind of apoptosis regulatory proteins, to play an important role of inducing cell apoptosis, but its regulation mechanism was still not fully clear, especially the regulation mechanism of p53together with other cell apoptosis regulation factor needed to be studied. This study tries to reveal the activity mechanism of p53effecting on the regulation of autophagy and the its possible mechanism during the process of cell senescence; and the regulation mechanism of action of p53leading to apoptosis in the tumor celles. This will provide a new basis for the clinical treatment of related diseases. Part Ⅰ. The role of p53in cell senescence and autophagy Objective:Cell senescence is a cell response to a variety of stress, and cell cycle arrest. Autophagy is considered a cell’s survival mechanism under stress state. p53is a common regulatory protein for senescence and autophagy. This experiment would elucidate the function of p53in senescence and autophagy by interfering with p53and by analysis of cell autophagy during the bone marrow mesenchymal stem cells(BMSC) senescence.Method:Bone marrow MSCs were isolated from femurs and tibias of male SD rats, and identified by checking their surface markers by flow cytometry and mesenchymal potential by induced differentiation assays. The BMSCs were the different stages according to the cell’s growth profile (growth curve in each passage, accumulative number of cell doublings (NCD) with subcultures), and senescent (positive for SA-β-Gal staining) cells in total population. The mitochondria morphology changes during the cell senescence were observed by the transmission electron microscope; The expression p53, Lc3A/B、Atg7anf Atg12in BMSCs were evaluated with Western blot and RT-qPCR.Result:When expanded to passage2, BMSC cultures were characterized by their colony-form growth, uniform fibroblast-like morphology, typical surface CD marker expression (91.6%±2.42CD54+,98.86%±0.02944CD90+,0.05667%±0.03859CD34+and1.555%±0.095CD11+) and osteogenic and adiogenic potential. The above characteristics conformed to the accepted the "gold standard" to identify the rat bone marrow MSC.These cells were actively proliferating with only a small number of staining positive cells for SA-β-gal. As the cell subcultured, BMSC proliferation gradually slow down and the staining positive cells for SA-β-gal gradually increased with the increasing expression of P53protein and its downstream proteins. When expanded to passage6(24to25PDs). the cells staining positive rate by SA-β-gal to90%. In the BMSC senescent, the fluorescence signal of LC3increased by immuno-fluorescence, with the mitochondrial swelling and degeneration, and a large number of vacuolated double membrane-like autophagosome by transmission electron microscope. Meanwhile during BMSCs senescence, expression of the Atg7and Atg12genes was assessed by RT-qPCR, in accordance with immunoblot analysis of Atg12and Atg7.The expression level of mRNA and protein of p53could be effectively reduced by shRNA mediated by lentivirus, accopanied by down-regulation of p53downstream cellular senescence regulatory proteins p21. The proportion of the staining positive cell for SA-β-gal and the expression of autophagy related gene were reduced by interfering with the expression of p53.Conclusion:1. The level of autophagy began to increase when SD rats BMSC entering aging state.2. During senescence, p53is not only involved in the regulation of cell senescence but its function are also associated with cell autophagy. Part Ⅱ. The role of p53in cell apoptosisObjective:Apoptosis, or programmed cell death (PCD) was one of the important mechanism to maintain stable cellular internal environment, and was a kind of orderly cell death controlled by the gene. The apoptosis plays a vital role in tumor production and progression. Although the genes mediated directly apoptosis had not been found, some experiments have proved that the p53and RASSF1A are considered related to cell apoptosis. p53not only participated in the regulation of cell autophagy and p53was also confirmed to be a kind of apoptosis regulatory proteins. The purpose of this study was to explore the mechanism of the apoptosis regulatory protein p53participating in apoptosis in the SiHa cells.Method:RASSF1A full-length expression plasmid RASSF1A-pcDNA, p53-pcDNA, the RASSF1A-RNAi interference plasmid were constructed to transfect the SiHa cells respectively or altogether, and identify their transfection rate with the flow cytometry apoptotic cells and, The expression p53and RASSF1A in SiHa cells were evaluated with Western blot and RT-qPCR. The ability of p53DNA-binding to RASSF1A was analyzed with EMSA/Gel shift by the WT/Mut tag probe end labeling method.Result:The level of their protein expression of RASSF1A and p53can be effectively improved respectively, and RASSF1A-siRNA inhibition of RASSF1A protein expression levels by Western blot immunoblot analysis after RASSF1A, p53and RASSF1A-siRNA were transfected into SiHa cells respectively. Flow cytometry combined with Annexin V/PI staining revealed that treatment of RASSF1A-expressing SiHa cells with RASSF1A siRNA inhibits apoptosis by54%, compared to pcDNA. Upon overexpression of RASSF1A, the proportion of apoptotic cells increased from9.965%to18.95%, compared with pcDNA, while in the presence of p53, the proportion of apoptotic cells increased to32.932%. This suggested that both p53and RASSF1A could induce the apoptosis of SiHa cells, and the level of cellular apoptosis was significantly increased.The expression plasmids of p53and RASSF1A were transfected into SiHa cells together, when RASSF1A or p53was transfected into SiHa cells respectively. Flow cytometry combined with Annexin V/PI staining revealed that, p53had no synergies with RASSF1A although the over-expression of p53+RASSF1A might aggravate apoptosis in SiHa cells,(the apoptosis effect of RASSF1A+p53transfection was similar to that of p53transfection individually).Gel-shift assay showed that the complex added the p53antibody formed a supershift band (antibody/p53/RASSF1A). However, single-stranded probes and a mutant RASSF1A probe decreased the formation of the p53/RASSF1A complex. Meanwhile, the experiment showed the the binding and regulation effects was obvious dose-dependent with0-2.5ug of p53.Conclusion: 1. The apoptosis in SiHa cells associated with P53and RASSF1A, But them had no synergy.2. P53may combine with RASSF1A promoter specifically。p53had controlled the range of cell apoptosis probably by this action of RASSF1A, and prevented excessive apoptosis from increasing the damage to the organization. This make the body dealing with the bad environmental stimuli and make cell to establishing an appropriate balance between survival and death.