Effects Of TET2 Gene Transfection On Inhibition Of Acute Myeloid Leukemia Cell Growth And The Sensitivity Of 17-DMAG Treatment And Its Mechanism

In this study,the effects of TET2 gene transfection on the growth of acute myeloid leukemia cells and the sensitivity of 17-DMAG treatment were studied in three parts.In the first part,we retrospectively analyzed the mutation of TET2 in 184 cases of acute myeloid leukemia patients,and studied the clinical characteristics,chemosensitivity and survival time of the patients,so as to provide some reference for the prognosis of AML.In the second part,the effect of 17-DMAG on the proliferation and apoptosis of human leukemia cells and the possible mechanism were investigated by culturing human leukemia cells HL-60 in vitro.In the third part,we treated HL-60 cells ectopic expressing of TET2 gene with 17-DMAG to clarify the relationship between 17-DMAG and TET2 in the development and progression of acute leukemiaThe first part explores the expression of TET2 gene and TET2 gene mutation in acute myeloid leukemia and its clinical significanceOBJECTIVE: To analyze the incidence of TET2 gene mutation in newly diagnosed acute myeloid leukemia and evaluate the clinical characteristics,short-term efficacy and long-term prognosis of TET2 mutant and TET2 wild type.Methods: The expression and mutation of TET2 gene were detected by polymerase chain reaction(PCR)in 184 newly diagnosed patients with acute myeloid leukemia.The mutations of TET2 and NPM1,DNMT3 a,FLT3-ITD and CEBPA were detected by direct sequencing.While patient clinical data was collected,.Induction therapy was performed using a standard dose of DA,IA or decitabine + CAG regimen.To compare the clinical data of TET2 mutant and TET2 wild type patients,to evaluate the therapeutic effect of TET2 mutations and to analyze the relationship between the expression level of this gene and the overall survival rate and event-free survival rate of wild type patients.result: Thirty-six patients with acute myeloid leukemia(non-M3)were detected in 32 cases of TET2 gene mutation,the overall incidence was 17.39%;32 cases of TET2 gene mutations in 26 cases of male,6 female.The median age was 44 years(18 years old);52 cases of negative patients in the group of 110 cases,42 females,the median age of 38.5(18 ~ 87)years old.There was no significant difference in age and sex between TET2 mutant patients and 152 TET2 wild type patients(P> 0.05).Compared with TET2 wild type patients,TET2 mutations have the following clinical characteristics: the type of patients with M4,M5 subtype,peripheral blood platelets lower(P = 0.04);easy to merge DNMT3 a,FLT3-ITD,CEBPA mutation,(P <0.05).However,there was no significant difference between FAB subtype,peripheral blood leukemia,hemoglobin,percentage of bone marrow primitive cells,NPM1 gene mutation and NCCN prognosis risk group(all P> 0.05).Of the 152 patients with TET2-Wt AML,86 patients(86 / 152,56.58%)were treated with complete remission,9 patients(9 / 152,5.92%)were partial remission,and 48 patients(48/151,31.58%,were no remission.Of the 32 patients with TET2-Mut AML,20 patients(20/32,62.5%)were treated with complete remission,4 cases(4 / 32,12.5%)were partial remission,and 8 patients(8/32,25%)were no remission.There was no significant difference beween the three groups(P = 0.37).184 patients were followed up for 12-60 months and the median follow-up was 30 months.Compared with TET2 wild type patients,TET2 mutations showed shorter disease-free survival(DFS)and overall survival(OS)(P <0.05)Conclusion: The mutation rate of TET2 gene in Chinese AML patients is similar to that in western population.TET2 gene mutation is related to specific biological characteristics of AML.The overall survival and disease-free survival of mutant patients are obviously shortened,suggesting that the mutation is harmful to adult patients Classification and treatment of great significance.The second part of 17-DMAG inhibits leukemia cell proliferation and induces apoptosisOBJECTIVE: To observe the effect of 17-DMAG on the proliferation and apoptosis of leukemia cellsmethod: The effects of different concentrations of 17-DMAG on the proliferation of leukemia cells were detected by MTT assay and cell clone formation assay.The apoptosis of leukemia cell line HL-60 cells induced by different concentrations of 17-DMAG was detected by Annexin V / PI staining and flow cytometry.The effect of 17-DMAG on mitochondrial membrane potential of HL-60 cells was detected by JC-1 method.The expression of caspase-3,caspase-9,Bcl and Bax in 17-DMAG leukemia cells treated with 17-DMAG was detected by Western blot.Compared with the control group,the apoptosis of HL-60 cells induced by 17-DMAG was studied.result: 1.Cell clone formation assay showed that 17-DMAG could inhibit the proliferation of leukemia cell line HL-60.MTT assay showed that 17-DMAG inhibited the proliferation of leukemia cell line HL-60 in a time and dose-dependent manner.Apoptosis of HL-60 cells induced by 17-DMAG detected by Anexexin V / PI staining and flow cytometry showed that 17-DMAG induced apoptosis of HL-60 cells in a time and dose-dependent manner.The results showed that the green fluorescence gradually decreased with the increase of 17-DMAG concentration,indicating that 17-DMAG could decrease mitochondrial membrane potential in HL-60 cells.The expression of Bax,c-caspase-3 and c-caspase-9 was significantly higher than that of the control group(P <0.05).The expression of anti-apoptotic proteins survivin and Bcl-2 was significantly decreased(P <0.01).Conclusion: 1.17-DMAG could inhibit the proliferation of HL-60 cells.With the increase of drug concentration and the prolongation of the time,the cell growth inhibition rate increased gradually.It was confirmed that the proliferation inhibition of HL-60 cells was time-dependent and dose-dependent.2.17-DMAG can induce apoptosis of HL-60 cells.3.17-DMAG-induced apoptosis of HL-60 cells may be through the mitochondrial pathway,with the increase of caspase-9 and caspase-3 protein expression,down-regulation of Bcl-2 and up-regulation of Bax protein expression.The third part of the TET2 gene can enhance the effect of 17-DMAG on the apoptosis of human acute myeloid leukemia HL-60 cell line and the signal pathway pathway PI3 K / AKT signalOBJECTIVE: To study the effect of TET2 gene overexpression on the proliferation and apoptosis of acute acute myeloid leukemia HL-60 cell line;To investigate the effect of TET2 gene combined with 17-DMAG on the proliferation and apoptosis of human leukemia cell line HL-60 and the influence of signal pathway on PI3 K / AKT signal,and to explore the treatment of acute myeloid leukemia.method: 1.Transfection of TET2 lentiviral vector(p WPT-PURO-GFP-TET2)into leukemia HL-60 cell line to increase the expression of TET2 in leukemia HL-60 cell line.To investigate whether the expression plasmid was successfully transfected into the cells or not,human leukemia HL-60 cell line transfected with p WPT-PURO-GFP-TET2 was examined by fluorescence microscopy on day 3 after infection.TET2 was detected by Western blot.The cell growth curve of leukemia cell line HL-60 transfected with TET2 was detected by MTT assay.The apoptosis of leukemia cells was measured by flow cytometry before and after transfection with Annexin V / PI double staining method.The effect of 17-DMAG on the indrement and apoptosis of leukemia cell line HL-60 cells transfected with TET2 was detected by MTT assay.5.The use of Annexin V / PI staining combined with flow cytometry to detect TET2 overexpression increased the sensitivity of leukemia HL-60 cells to 17-DMAG.The effect of 17-DMAG on PI3 K / Akt / CREB / Bcl-2 signaling pathway in HL-60 leukemia cell line transfected with TET2 was detected by western blot.result: 1.Fluorescence microscopy revealed that lentiviral p WPT-PURO-GFP-TET2 transfected human leukemia HL-60 cell lines,with more than 80% of cells expressing GFP.Western blot was used to detect the expression of TET2 protein.The results showed that the expression of TET2 protein in HL-60 cell line transfected with TET2 vetor was much larger than that of empty vector transfection group.The results of MTT assay showed that the cell growth of the transfected group was significantly slower than that of the control group.(P <0.05).The apoptosis of leukemia cells was detected by flow cytometry using Annexin V / PI double staining method.Apoptosis of HL-60 cell line transfected with TET2 vector was significantly increased(P <0.05).4.17-DMAG effect on the transfection of TET2-expressing leukemia HL-60 cells after MTT method showed that transfection of TET2 gene,or by adding 1.6μmol·L17-DMAG,can significantly inhibit HL-60 cell proliferation rate,especially The inhibitory effect of TET2 combined with 17-DMAG on cell proliferation was the strongest(P <0.05).The results of Western blot showed that TET2 protein was significantly increased in leukemia after 17-DMAG transfection of leukemia HL-60 cells transfected with TET2 vector(P <0.05).Flow cytometry showed that TET2 overexpression could enhance the apoptosis of HL-60 cells induced by 17-DMAG.7.17-DMAG promotes the apoptosis of HL-60 leukemia cells by inhibiting PI-3K / Akt signaling pathway,mainly through the increased expression of Bax,caspase-3 and caspase-3,and the decreased expression of Bcl-2 and survivin.Conclusion: 1.TET2 overexpression can cause leukemia cell HL-60 slow growth,increased apoptosis,may be a tumor suppressor gene of leukemia.2.TET2 can promote the apoptosis of HL-60 cells in combination with 17-DMAG and inhibit cell proliferation.The effect of TET2 and 17-DMAG on the growth of HL-60 cells was achieved by PI3 K / AKT signaling pathway.