Exploring The Role Of P53 Mutant Protein P53^N236S In Regulating Akt-mTOR Pathway And Its Role In Tumor Proliferation At The Cellular Leve

P53 tumor suppressor protein is an important molecule in the basic research of cancer.When the DNA damage,p5 3 requires CDKN1A inhibition of G1 phase growth,through the cyclin cyclin B1 to adjust the G2 checkpoint to provide sufficient time for cell DNA repair.If the DNA repair fails,then by regulating puma,Bax and other apoptosis-related proteins to induce apoptosis.However,p53 mutation can not only monitor the integrity of the genome,but also access to oncogene function.The Gain of functional p53 mutations can promote cell proliferation,promote cell transformation,and cause cancer.The mechanism of mutant p53 is divided into four types:a.Mutant p53 directly acts on the target gene promoter to promote or inhibit the transcription of the target gene;b.Mutant p53 binds with other transcription factors or recruits other transcription factors to regulate the transcription of the target gene;C.Mutant p53 can also bind to other transcription factors or cofactors to disintegrate these factors from DNA;d.Mutant p53 binds to the target protein,enhances or closes their function,and indirectly affects the transcription of the target gene.Mutation of p53 to obtain the most potent evidence of oncogene function,derived from the results of transgenic mouse.Mouse with mutant p53 alleles are generally more likely to develop various types of cancers than mouse with wild-type p53 genes.For example,mouse with genotypes of Trp53+/-and Trp53/-will have lymphoma and sarcoma after knockout of intron 4 and exon 5 and exon 26 genes of TP53,respectively,suggesting that We p53 is an important tumor suppressor gene;gene knocked into R172P mutant transgenic mice will suffer from lymphoma and sarcoma;gene knocking R172H mutant transgenic mice will suffer from lymphoma and sarcoma and other malignant tumors,and prone to metastasis,Suggesting that we mutated the acquired function of p53.The p53^N236S（p53S）knockout mouse are the p53-N236S transgenic mouse model constructed in our laboratory.In this study of p5 3-knock-in mouse with genotype p53S/S died about three months time,and almost died of tumors in about ten months.In the previous experiment,the mouse with the genotype p53S/S were subjected to ionizing radiation to detect the apoptosis and cell cycle-related proteins in the thymus tissue of mouse.It was found that genotype p53s/s had lost transcription Regulation of p21,puma and other downstream molecules.The preliminary laboratory study also found that:p53 does not change the total Akt protein expression,can upregulate the phosphorylation of Akt protein activation.This suggests that p53S may interact with the kinase that leads to Akt phosphorylation.We know that Akt can play a key role in the PI3K-Akt-mTOR signaling pathway.PI3K-Akt-mTOR signaling pathway can promote cell proliferation and inhibit apoptosis,but also can affect the invasion of tumor,and the cell survival is very close relationship.In this study,the expression of Akt-TOR pathway-related proteins in p53S/S genotype,p53,P-p53ser15,P-p53ser392,P-AktSer473,mTOR,P-mTORSer2448,p53S/S MEF cells were detected by protein expression in mouse MEF cells.P-mTORSer2481,Rictor,Raptor and other genotypes were WT MEF and the genotype was higher for p53/MEF.In the subsequent MTT assay,we found that within 48 h,p53S/S MEF The cell proliferation rate is the fastest.This suggests that the expression of p53 mutant protein p53 in mouse p53S/SMEF cells increases,and then up-regulation of Akt-m TOR pathway,leading to abnormal cell proliferation.In the next step,the primary tumor cells of mouse spontaneous tumor were cultured with the spontaneous tumor of p53^N236S transgenic mice.We successfully cultivated four primary tumor cell lines with genotype p53S/S,The detection of protein expression and the detection of cell proliferation rate were carried out.The expression of p53 protein and P-p53ser15,P-p53ser392 protein in primary cultured mouse cells was detected by using p53-/-MEF cells as negative control and p53S/SMEF cells as positive control.Akt,P-AktSer473,mTOR,P-mTORSer2448,P-mTORSer2481,Rictor and Raptor were significantly higher than those in the control group.The expression of AktmTOR pathway-related protein The MTT cells were detected by MTT cell proliferation in the mouse model cells.The proliferation rate of the primary cultured mouse tumor cells increased rapidly after 12 hours,compared with the control group.WT MEF cells in the control group had high proliferation rate.This suggests that we may be in the primary culture of tumor cells due to mutant p53 protein p53S expression increased in cells Akt-mTOR pathway is highly activated,causing abnormal cell proliferation.Based on the above results,we hypothesized that if p53S really positively regulates the PI3K-Akt-mTOR signaling pathway in cells,what happens if the expression of p53 in cells is reduced?Therefore,we observed the interference of mouse tumor cells in mice.We found that the protein expression of P-p53ser15 and P-p53ser392 in mouse tumor cells after interfering with p53 was higher than that of untreated mouse tumor cell protein Low expression The protein expression of P-AktSer473,P-AktThr308,mTOR.P-mTORSer2448.P-mTORSer2481,Rictor,Raptor also decreased.Next,it was found that the proliferation rate of mouse tumor cells after interfering with p53 was found in the experiment of detecting cell proliferation rate by MTT assay with uninterrupted mouse tumor cells as a control.This suggests that p53 is a key protein that regulates the Akt-mTOR pathway in mouse tumor cells,which up-regulates the Akt-mTOR pathway,resulting in abnormal cell proliferation,knocking down,causing the expression of related proteins in this pathway.The rate of proliferation also decreased.In summary,this study found that p53S can make mouse MEF cells and mouse tumor cells Akt-mTOR pathway related proteins such as P-AktSer473,mTOR,PmTORSer2448,P-mTORSer2481,Rictor,Raptor expression The amount of upregulation,which led to abnormal cell proliferation.The expression of P-AktSer473,P-AktThr308,mTOR,P-mTORSer2448.P-mTORSer2481,Rictor and Raptor were down-regulated in mouse tumor cells after the successful interference of p53 cells in mouse tumor cells.And the cell proliferation rate also decreased.These results suggest that at the cellular level,p53 can regulate cell proliferation by regulating the Akt-mTOR pathway.Indicating that p53 not only lost the regulation of p53 downstream molecules,but also access to the downstream proliferation of signaling pathway regulation function.The above-mentioned cell level test provided clues for the regulation of latestage p53S in the regulation of Akt-mTOR pathway in mouse tumor tissue.The results of this study show that p53 cells play a very important regulatory role in the process of tumor cell proliferation.The high expression of p53 mutant protein in tumor cells can distinguish it from normal cells and become a specific target for tumor molecular targeted therapy.This paper shows the regulation of p53 on the Akt-mTOR pathway,and provides a new approach for multi-target therapy for tumor targeted therapy.