Study On The Mechanism Of TIGAR Gene In Acute Erythrocyte Leukemia

Part Ⅰ Prognostic significance of associated gene mutations in patients with acute myeloid leukemia with normal karyotypeObjective:Acute myeloid leukemia with normal karyotype (NK-AML) is a clinically heterogeneous disease. A variety of gene mutations has showed prognostic significance in NK-AML. However further study of the prognostic factors might contribute to accurate risk stratification of NK-AML. The purpose of this study is to explore the prognostic significance of NPM1, CEBPA, FLT3-ITD and C-KIT gene mutations in NK-AML.Methods:Bone marrow (BM) samples of 87 patients with NK-AML were collected. Mononuclear cells (MNCs) were extracted and genomic DNA was isolated from BM specimens. Chromosome specimens were prepared using the direct method and short-term culture without phytohemagglutinin and the metaphase chromosomes were banded via improved heat treatment Giemsa R-banding method. Incidence of NPM1, CEBPA, FLT3-ITD and C-KIT gene mutations was analyzed using genomic PCR and sequencing in NK-AML. Clinical characteristics such as age, sex, white blood cell count (WBC), hemoglobin (Hb), platelet count (PLT), peripheral blood (PB) and BM blasts rate, complete remission (CR) rate and relapse rate were compared between gene mutant and wild-type patients. Kaplan-Meier method was used to calculate the expected median overall survival (OS) and disease-free survival (DFS), relative risk (HR) and 95% confidence interval (95% CI) in gene mutant and wild-type patients. Curves of OS, DFS and cumulative incidence of relapse rate were drawn and compared between mutant and wild-type patients. Cox regression analysis was performed to estimate the significantly prognostic factors for OS such as age, WBC, Hb, PLT, PB and BM blast proportion, allogeneic hematopoietic stem cell transplantation (allo-SCT), NPM1, CEBPA, FLT3-ITD and C-KIT mutational status.Results:NPM1 and FLT3-ITD mutations were detected in 83 cases. CEBPA and C-KIT mutations were detected in 81 cases. Incidence of NPM1, CEBPA, FLT3-ITD and C-KIT mutation was 24.1%(20/83),23.5%(19/81),13.3%(11/83) and 0.7% (6/81), respectively. NPM1 and CEBPA mutations were concurrent in 4 patients. No patient had NPM1 and C-KIT gene mutations simultaneously. Concurrence of CEBPA, C-KIT and FLT3-ITD mutations were identified in one patient. WBC count of the patients with mutational NPMl was significantly higher than those with wild-type NPM1. Patients with mutant FLT3-ITD or C-KIT had significantly lower PLT, higher PB and BM blast proportion than those with wild-type genes. After excluding the patients giving up treatment and undergoing allo-SCT, patients with NPM1 mutations (without FLT3-ITD mutations) had significantly better OS compared to wild-type patients (P=0.0254) and there was no significant difference in DFS between the two groups. Patients with mutated CEBPA (without FLT3-ITD and C-KIT mutations) had significantly better OS and DFS compared with wild-type patients (P=0.0367 and 0.0179, respectively).There was no significant difference of OS and DFS between patients with mutated and wide-type FLT3-ITD or C-KIT. Patients with CEBPA mutation (without FLT3-ITD and C-KIT mutations) had significantly lower cumulative relapse rate than wild-type patients (P=0.038). Cox regression analysis showed that age (P=0.002) and CEBPA mutations (P=0.017) were independent prognostic factors in OS.Conclusions:NPM1 and CEBPA mutations without FLT3-ITD and C-KIT were associated with better survival than those with wild-type genes in NK-AML. FLT3-ITD and C-KIT mutations were associated with lower PLT count and higher blast proportion in PB and BM.Part Ⅱ Expression and prognostic significances of TIGAR gene in NK-AML patientsPurpose:Molecular abnormalities of NK-AML deserve further study in order to facilitate accurate and in-depth risk stratification. TIGAR (p53-induced glycolysis and apoptosis regulatory factor) is a target gene of p53, which can regulate mitochondrial respiration and suppress p53-related reactive oxygen species (ROS)-mediated apoptosis by inhibition of glycolysis. In this study, we explored expression level of TIGAR mRNA and its relationship with clinicobiological characteristics and prognostic significance in NK-AML.Methods:A total of 87 newly diagnosed NK-AML patients were enrolled in this study. Mononuclear cells (MNCs) were extracted and RNA was isolated from BM specimens. Quantitative PCR was performed to detect TIGAR mRNA expression level. Cycle threshold (Ct) comparative method was used to estimate relative expression level of TIGAR mRNA, using the formula 2(-ΔΔCt). Relative expression level greater than or equal to the median was defined as high expression level of TIGAR (TIGARhigh). Relative expression level below the median was defined as low expression level of TIGAR (TIGARlow). Clinical (age, sex, WBC, Hb, PLT, PB and BM blasts proportion, CR rate and relapse rate) and biological characteristics (NPM1, CEBPA, FLT3-ITD and C-KIT gene mutations) were compared between TIGARhigh and TIGARlow patients. Relative expression level of TIGAR mRNA was compared between NPM1, CEBPA, FLT3-ITD, C-KIT gene mutated and wild-type patients. Kaplan-Meier method was performed to calculate the expected median OS and DFS, HR,95% CI of TIGARhigh and TIGARlow patients. Curves of cumulative incidence of relapse rate were drawn to compare the cumulative incidence of relapse between the two groups. Cox regression analysis was used to estimate the significant prognostic factors for OS such as age, WBC, Hb, PLT, PB and BM blast proportion, allo-SCT, NPM1, CEBPA, FLT3-ITD, C-KIT mutational status and especially the expression level of TIGAR mRNA.Results:Median of TIGAR mRNA relative expression level was 0.9751 (0.0022 to 54.2429). CEBPA mutational rate was significantly higher in TIGARlow patients than TIGARhigh patients (P=0.0432). Relapse rate was significantly higher in TIGARhigh patients than TIGARlow patients (P=0.007). There is no significant difference in other clinicobiological characteristics between the two groups. TIGAR mRNA expression level of C-KIT mutational patients was significantly higher than wild-type patients. Survival analysis showed that TIGARlow patients had significantly better OS and DFS than TIGARhigh patients (P=0.0078 and 0.0122, respectively). Cumulative relapse incidence of TIGARhigh patients was significantly higher than TIGARlow patients (P=0.008). Cox regression analysis showed that only high expression of TIGAR mRNA(P=0.005) was independent prognostic factor in OS.Conclusions:TIGAR mRNA expression level had prognostic significance in NK-AML patients. High expression of TIGAR is associated with poor prognosis and low expression suggests a good prognosis.Part Ⅲ Function and mechanism of TIGAR in leukemia cell linesPurpose:TIGAR has anti-apoptotic effect by inhibiting glycolysis in cancer cells but its role in leukemia remains unclear. The purpose of this study is to investigate the function and mechanism of TIGAR in leukemia cell lines.Methods:Quantitative PCR and Western Blot (WB) were performed to detect TIGAR and p53 gene and protein expression levels in HL60, K562 and Jurkat cell lines. Over-expression and lentivirus vector of TIGAR were constructed and stable K562 clones of TIGAR over-expression and HL60 cell clones of TIGAR knock-down were screened. PFKFB3, ROS and glutathione (GSH) protein expression level was detected by WB in cell lines with over-expression and knock-down TIGAR. TIGAR protein expression was detect by WB and cell apoptosis rate was detect by flow cytometry in various cell lines after using synthetic PI3K inhibitor LY294002 to block the PI3K/Akt signaling pathway. TIGAR expression level was detected by WB in experimental and control cells after culturing for 36 h. Nutlin-3a was used to activate p53 in experimental cells. Cell apoptosis was detected by flow cytometry in experimental and control cells after culturing for 36 h.Results:TIGAR, p53 mRNA and protein expression level was higher in HL60 than K562 cell line. PFKFB3 protein expression level significantly decreased in K562 cell line with over-expression TIGAR compared to K562/PCDNA3-HA. Expression level of ROS decreased and expression of GSH increased in K562 cell line with over-expression TIGAR. On the other hand PFKFB3 protein expression level significantly increased in HL60 cells with knock-down TIGAR compared with HL60/NC. Expression level of ROS increased and GSH decreased. There was no significant difference of apoptosis rate in K562/PCDNA3-HA, K562/PCDNA3-TIGAR and HL60/NC cell lines (7.89%,7.56% and 7.82%, respectively), but apoptosis rate of HL60/TIGAR-shRNA2 significantly increased (38.25%). TIGAR expression level of HL60 was reduced after LY294002 was used to inhibit PI3K/Akt signaling pathway (from 1.4577 to 1.0192) and decreased significantly in HL60/p53 after using LY294002 (from 1.5108 to 0.9209). TIGAR expression was reduced in HL60/p53 TIGAR-shRNA2 by LY294002 (from 0.3077 to 0.1911). Apoptosis rate in HL60 increased after using Nutlin-3a (from 8.45% to 12.36%), Knock-down of TIGAR significantly increased cell apoptosis rate in HL60 (from 8.45% to 35.68%). Apoptosis rate increased after p53 was activated in HL60 with knock-down TIGAR (from 35.68% to 45.22%) and significantly increased after using Nutlin-3a in HL60/p53 (from 7.87% to 26.63%). Apoptosis rate in HL60/p53 with knock-down TIGAR significantly increased (from 7.87% to 39.45%) and increased significantly by Nutlin-3a activation (73.22%).Conclusions:TIGAR suppresses p53-associated apoptosis by inhibiting glycolysis, reducing ROS and increasing GSH level in HL60 and K562 cell line. Knock-down of TIGAR make leukemia cell lines sensitive to p53-mediated apoptosis. The PI3K/Akt pathway of TIGAR may be involved in the pathogenesis of TIGAR in leukemia.