The Mechanisms And Clinical Significance Of Isocitrate Dehydrogenase Genes Expression In Adult Acute Myeloid Leukemia

Part one, The prognostic significance of expression of IDH1/2in cytogenetically normal adult acute myeloid leukemia.Purpose:To evaluate the prognostic significance of the expression levels of IDH1/2mRNA in cytogenetically normal AML (CN-AML).Methods:The IDH1/2mRNA expression levels were measured in pretreatment bone marrow mononuclear cells using qRT-PCR in232AML patients with mornal cytogenetic and healthy controls. Genes mutation status of IDH1, IDH2, NMP1.FLT3-ITD, DNMT3A and CEBPA were sequenced as well. Expression differences of IDH1/2mRNA between CN-AML patients and normal controls were compared. The relationship among IDH1/2mRNA expression and overall survival (OS), relapse-free survival (RFS) and event-free survival (EFS) of CN-AML patients were analyzed. At the same time, we analyzed the relationship among IDH1/2mRNA expression and other clinical indicators and gene mutation.Results:1. Compared with normal control, The IDH1mRNA expression level of CN-AML patients was higher than CD34+cells and peripheral blood mononuclear cells of normal control (p=0.01and p<0.001, respectively), IDH2mRNA expression level of CN-AML patients was higher than peripheral blood mononuclear cells of normal control (p<.001).2. After expression of IDH genes were analyzed by Kaplan-Meier method, the3-year overall (OS) and event-free survival (EFS) in low IDH1mRNA expressing group are better than those in high expressing group (50%vs.26%,31%vs.16%, both P values:<0.001, respectively) in232CN-AML cases. We did not find the prognostic value of IDH2mRNA expression levels in CN-AML patients (p>0.05for OS, EFS and RFS).3. After adjusting the impact of clinical data include age, WBC count and highly frequent mutations of genes, the low IDH1mRNA expressing group are better than those in high expressing group (p<0.001for OS and EFS), the expression of higher IDH1mRNA was an independent biomarker and had poor prognosis.4. With analysis the relationship among the IDH1mRNA expression and clinical data, gene mutation, Higher level expression of patients was mainly in M5patients, and closely associated with the higher frequency of DNMT3A mutation and less common in CEBPA mutation(P=0.007and0.019, respectively).Conclusion:To our knowledge, we provide the first evidence that higher IDH1mRNA expression was an independent biomarker after adjusting the impact of clinical variables and highly frequent mutations of genes in CN-AML and may refine their molecular risk classification. Part two, The mechanisms of isocitrate dehydrogenase1genes expression in adult acute myeloid leukemiaPurpose:(1) To explore the mechanism of IDH1in AML(2) To seek that microRNAs regulate IDH1mRNA expression.Methods:(1) Silencing the IDH1gene of THP-1and HL-60/ADR using small interfering RNA (siRNA), cell growth, cycle and apoptosis were examined by MTT and flow cytometry, apoptosis and signaling pathway of proteins were detected by western blot(2) According to the expression level of IDH1mRNA, we select specimens of16patients from CN-AML(8from high expression group and8from low expression group) and conducted a microRNA expression analysis using SBC Human miRNA Microarray Release16.0to seek microRNA associated with IDH1mRNA expression. qRT-PCR was used to validate the result of the microRNA chip.Results:(1) IDH1gene expression of THP-1cells was silenced by siRNA, cultivated for7days, cell viability of siRNA IDH1group was lower than that of negative control group(p=0.04). Apoptotic rate of siRNA IDH1group was37.22±8.27%, higher than that of negative control group (19.50±1.18%, p=0.021). Accordingly, Caspase-3and PARP were activated and PI3K/AKT/mTOR pathway was inhibited in siRNA IDH1group. However, down-regulated of IDH1expression has no effect on cell cycle in THP-1cell as compared to negative control group (p=0.89).(2) After silencing IDH1, THP-1cells increase the sensitivity to chemotherapeutic drugs such as HHT, ACR and DNR.(3) After silencing IDH1of HL-60/ADR for48hours, Caspase-3and PARP were activated and PI3K/AKT pathway was inhibited. The sensitivity of HL-60/ADR to doxorubicin was increased by silenceing IDH1.(4) Result of chip:Patients with high IDH1mRNA expression had low expression of microRNAs, which were miR-181family (miR-181a, miR-181a-2, miR-181b, miR-181c*, miR-181c miR-181a*and miR-181d), miR-146a, miR-128, miR-625, miR-25and miR-335, and had low expression of4microRNAs, which were miR-4286, miR-660, miR-107and miR-324-5p. The microRNA expression were contrary in patients with low IDH1mRNA expression.(5) qRT-PCR confirmed that, low expression of miR-181family(miR-181b and miR-181d) and high expression of miR-4286in samples with high IDH1mRNA expression; above microRNA had opposite expression level in sample with low IDH1mRNA expression.Conclusion:(1) Silenceing IDH1of THP-1and HL-60/ADR, decreased AML cell proliferation, induced apoptosis, increased sensitivity to chemotherapeutic drugs in THP-1, and reversed drug resistance of HL-60/ADR.(2) miR-181family has played the role of tumor suppressor genes, inhibiting the expression of IDH1mRNA in the CN-AML patients. miR-4286has played the role of oncogenes, promoting the expression of IDH1mRNA in the CN-AML patients.