The Role Of Phosphorylation Sites S15 And S392 Of P53 In Restoreing Its Anti-cancer Activity In Non-small Cell Lung Cancer

p53 inactivation is a common cause of ineffectiveness and drug resistance in lung cancer treatment,which makes it possible to restore p53 anti-cancer activity as a lung cancer treatment strategy.However,it has been found that therapeutic strategies based on restoreing p53 anticancer function are very effective in vitro,but invalid in vivo.The reason is that p53 anticancer function can not be effectively activated.The anti-cancer activity of p53 is related to its phosphorylation site.Previous studies have shown that the phosphorylation of S15 site plays an important role in the anti-cancer function of p53.We found that the phosphorylation of S392 site is related to the anti-cancer function of p53.The specific role of these two phosphorylation sites in restoreing the anti-cancer function of p53 and the interaction between them are still unclear.Therefore,this study intends to study the role of p53 phosphorylation sites S15 and S392 in restoreing the anti-cancer function of p53 in non-small cell lung cancer.The specific research contents are as follows:1.At first,p53 gene,p53(S15A)gene,p53(S392A)gene and p53(S15A-S392A)gene were synthesized directly.Then,the target genes were recombined with p HBAD-EF1-MCS-CMV-GFP vector.The product was transformed into bacterial competent cells,and the bacteria were screened by plate,and the cloned colonies were identified by PCR.The transformed positive clone solution was sent to the sequencing company for sequencing.The sequencing results showed that the vector was successfully constructed,and then the constructed vector plasmid was packaged into adenovirus for subsequent research.The green fluorescence was observed after p53-null NCI-H1299 cells was infected with the constructed adenovirus 24 hours,which indicated that the virus vector was transfected into NCI-H1299 cells.Subsequently,Western blotting showed that the constructed expression vector could successfully express the target gene.These results indicate that adenoviral expression vectors with mutations in p53 and p53 phosphorylation sites have been successfully constructed,which provides a preliminary basis for the study of the role of p53 phosphorylation sites in the reconstruction of p53 anti-cancer function in non-small cell lung cancer.2.Secondly,the inhibition of p53 phosphorylation sites S15 and S392 on the proliferation of NSCLC cells was detected after site activation by p-p53(S15)and p-p53(S392)site activators.The results showed that the phosphorylation of S15 and S392 sites could effectively activate p53 tumor inhibition function.Then,the effects of p53 site S15 phosphorylation on NSCLC cells were determined by Hoechst staining,AIF immunofluorescence staining and subcellular distribution.The inhibition of proliferation is not by inducing cell apoptosis.Finally,the cell cycle was detected by flow cytometry.The results showed that the phosphorylation of S15 and S392 of p53 inhibit cell proliferation by cell cycle arrest.3.Finally,the interaction between p53 S15 and S392 phosphorylation sites was detected.The results showed that there was no significant difference in cell proliferation inhibition between two site activators and single site activator.Western blotting results showed that when Nutlin-3 was added to NCI-H1299 and NCI-H460 cells,the phosphorylation of S15 at p53 site was activated,while S392 at p53 site was activated,indicating that the phosphorylation of the two sites was interacted.In conclusion,we preliminarily studied the inhibitory effect and potential mechanism of p53 phosphorylation sites S15 and S392 on the proliferation of NSCLC cells,clarified the interaction between the two sites,and provided theoretical support and data reference for restoreing the anti-cancer activity of p53.