| Objective:Through the collection of clinical samples from AML patients with early onset from 2015 to 2020 in our hospital,the clinical characteristics and prognostic significance of AML patients with TP53 truncation mutation were retrospectively analyzed,and the biological function of Δ107p53,a novel p53 truncation,was studied at the cellular level,in order to explore the influence of p53 truncation on AML patients.Furthermore,it provides a basis for individualized treatment of AML patients.Methods:1、The clinical features and prognosis of p53 truncated body in AMLTo retrospectively analyze the clinical data of 243 patients(except M3)with newly diagnosed AML admitted to our hospital from March 2015 to January 2020,record their clinical characteristics,immunophenotypes,cytogenetic and molecular biological characteristics,and analyze the patients Survival status and the main factors affecting its prognosis.2、Analysis of the three-dimensional protein structure of truncated Δ107p53Use I-TASSER online tool to predict the full length of p53 protein and the three-dimensional structure of Δ 107p53 protein,and use Pymol software(v1.3.1)to conduct mutation construction and structural comparison analysis of the predicted protein.3、Construction of lentiviral expression system and selection of stable clonesRetrieve the full-length cDNA sequence of the TP53 gene(Gen Bank:NM＿000546)from Gene Bank,follow the deletion site of Δ107p53 and the mutation site of R175H-p53 to obtain the full-length sequence of the CDS region,and insert the sequence into pCDH1-MCS1-In the EF1-copGFP expression plasmid,pCDH1-Δ107p53,pCDH1-R175H-p53,pCDH1-WT-p53 and pCDH1-empty expression vectors were constructed,and the first-generation sequencing method was used to identify whether the target plasmid was successfully constructed.The human promyelocytic leukemia cell line HL60 with deletion of the TP53 gene was selected as the model cell for this experiment.The human embryonic kidney cell line 293 T cells were co-transfected by liposome method,and the virus fluid was collected at 48 h and96h,and infected HL60 cells.The cell models are constructed as follows: Δ107p53 is the experimental group,R175H-p53 is the positive control group,wild-type WT-p53 is the negative control group,and pCDH1-empty is the empty vector control group.The GFP-positive cells were sorted by a flow sorter,stable expression clones were screened by the limiting dilution method,and the expression of the target gene in the cell model was identified by first-generation sequencing and Western blot.4、CCK-8 method and soft agarose clone formation test to detect the effect ofΔ107p53 on the proliferation of HL60 cellsCell Counting Kit-8(CCK8)method was used to detect the proliferation ability of each group of cells.Each group of cells is set with 3 replicate wells,and the absorbance values of each group of cells at 0 hour,24 hours,48 hours,72 hours and 96 hours are measured respectively,and this is used to reflect the proliferation of each group of cells.Soft agarose clone formation test was used to verify the results.Each group of cells was set up with 3 multiple wells,and each well was seeded with 2000 cells.After 2 weeks,the images were scanned and photographed.Image J software was used to analyze the colony size and number.5、Flow cytometry to detect the effect of Δ107p53 on the cell cycle of HL60Collect the above four groups of stable clonal cells,wash them twice with pre-cooled PBS,and then resuspend and count them.Use propidium iodide(PI)to stain the DNA of each group of cells in the logarithmic growth phase,use flow cytometry to detect the fluorescence intensity to reflect the DNA content,and analyze the results with Mod Fit LT software.To calculate the proportion of cells in G0/G1 phase,S phase and G2/M phase.6、RT-qPCR and Western blot to detect the expression of p21 and baxThe Trizol method was used to extract the total RNA in the cells of each group,and the expression of the downstream genes P21 and Bax of p53 was detected by real-time quantitative PCR(RT-PCR).Western blot was used to detect the expression of p53,P21 and BAX,the BCA method was used to quantify the protein,and the Alpha View SA software was used to analyze the protein bands.The clinical features and prognosis of abnormal TP53 spliceosome in AML Results:1、The p53 truncation is significantly related to the poor prognosis of AML patientsAmong the 243 AML patients,there were 213 cases without TP53 mutations,17 cases with point mutations and 13 cases with truncation mutations.Compared with the wild group,the complete remission rate(CR)of patients in the truncated mutation group and the point mutation group was lower(P <0.001),and disease-free survival(DFS)and overall survival(OS)were shorter(P < 0.001).This result suggests that TP53 truncation mutations and point mutations have similar effects on the prognosis of AML patients,both manifested as shorter DFS and OS,and worse prognosis.2、Biological structure analysis of truncated Δ107p53The I-TASSER tool was used to predict the full length of p53 protein and the three-dimensional structure of Δ 107p53 protein.The structure was analyzed and compared with Pymol software(v1.3.1).The results showed that the protein structure of Δ 107p53 changed significantly,and the truncation mutation caused Part of the DNA binding domain(DBD),all nuclear localization signal domain(NLS)and part of the tetramerization domain of p53 are missing.Conformational mutations may eventually affect the normal biological functions of p53.3、Construction of lentiviral expression system and selection of stable clonesThe results of first-generation sequencing showed that the sequences of TP53 have been correctly inserted into the pCDH1-MCS1-EF1-copGFP vector,and the transformation results showed strong expression of GFP,suggesting that the lentiviral expression vectors: pCDH1-Δ107p53,pCDH1-R175H-p53 and pCDH1-WT-p53 were successfully constructed.After HL60 cells were infected with lentivirus and sorted by flow cytometry,each group of cells expressed GFP,and stable expression cell clones were screened.The positive rate of flow cytometry was over 95%.The results of nested PCR and Western blot showed the expression of the target gene,suggesting that each expression vector has been successfully transferred into the HL60 cell line,and each cell model was successfully constructed.4、The truncated body Δ107p53 has an obvious proliferation-promoting effect on HL60 cellsThe results of CCK8 showed that the Δ107p53 group began to proliferate significantly after 48 hours,and the difference was statistically significant compared with the WT-p53 group(P<0.001).The results of the clone formation test showed that the number of Δ107p53 clones and the clone size were higher than those of the control group.The above results suggest that the truncated Δ107p53 has the same cell proliferation effect as R175H-p53.5、The truncated body Δ 107p53 promotes the cell cycle transition from G0/G1 phase to S phaseFlow cytometry detected the ratio of cells in G0/G1,S and G2/M phases.The results showed that the ratio of cells in G0/G1 phase of Δ 107p53 and R175H-p53 decreased compared with WT-p53 group.The proportion of S-phase cells increased,and the difference was statistically significant(P<0.001).This result suggests that both Δ 107p53 and R175H-p53 promote the cell cycle transition from G0/G1 phase to S phase.6、The truncated body Δ107p53 inhibits the expression of p21 and baxThe results of RT-qPCR showed that the expression of P21 and BAX was significantly reduced in the Δ107p53 group compared with the WT-p53 group(P<0.001).The Western blot results are consistent with this result,which suggests that Δ107p53 can inhibit the expression of p21 and bax.Conclusion:1.According to the analysis of clinical data,AML patients with p53 truncated body show shorter DFS and OS,and worse prognosis.2.Cell level experiments have proved that the new truncated body Δ107p53 can promote cell proliferation and inhibit cell apoptosis,which may be achieved by inhibiting the expression of BAX gene,and by inhibiting the expression of P21 gene to promote the cell cycle by G0/ G1 phase transitions to S phase to exert its carcinogenic effect. |
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