【作者】 马秋玲；

【导师】 金洁；

【作者基本信息】 浙江大学 ， 内科学， 2013， 博士

【摘要】 第一部分IDH基因表达在成人核型正常急性髓细胞白血病中的预后分析目的：探讨IDH基因mRNA表达与CN-AML患者预后的关系。方法：采用实时荧光定量PCR技术检测我院232例初发核型正常的急性髓细胞白血病(CN-AML)患者的骨髓单个核细胞、11例正常对照骨髓CD34‘细胞和20例正常对照外周血单个核细胞中IDH1和IDH2基因mRNA表达水平；同时应用PCR后直接测序方法检测CN-AML患者和正常对照的IDH1、IDH2、NMP1、 FLT3-ITD、DNMT3A和CEBPA突变。比较IDH1和IDH2基因mRNA表达水平在CN-AML患者和正常对照之间的差异。分析IDH1和IDH2基因mRNA表达水平与CN-AML患者总体生存(OS)、无事件生存(EFS)和无复发生存(RFS)的关系；并分析IDH1和IDH2基因mRNA表达水平与其它临床指标和基因突变的关系。结果：1、与正常对照相比,CN-AML患者IDH1基因mRNA表达水平高于正常对照骨髓CD34+细胞和正常对照外周血单个核细胞,p值分别为0.01和<0.001；CN-AML患者IDH2基因mRNA表达水平高于正常对照外周血单个核细胞P<0.001。2、米用Kaplan-Meier分析,IDH1基因mRNA低表达组患者3年的OS和EFS分别为50%和31%,IDH1基因mRNA高表达组患者3年的OS和EFS分别为26%和16%,IDH1基因mRNA低表达组的预后明显优于高表达组,其OS和EFS的P值均<0.001。而IDH2基因mRNA表达与CN-AML患者预后没有关系,OS、EFS和RFS差异均无统计学意义,p值均>0.05。3、用Cox回归模型纠正年龄、白细胞数、基因突变等因素,IDH1基因mRNA低表达组的预后明显优于高表达组,其OS和EFS的P值均<0.001。IDH1基因mRNA高表达是CN-AML患者的预后因素,IDH1基因mRNA高表达者预后差。4、通过分析IDH1基因mRNA表达与临床指标和基因突变关系发现：IDH1基因mRNA高表达主要集中M5患者中,其高表达与DNMT3A突变有关系,在DNMT3A突变患者中高表达者占16%,低表达者占5%,两者差异具有统计学意义,P=0.007；IDH1基因mRNA高表达与CEBPA双突变有关系,在CEBPA双突变患者中高表达者占7%,低表达者占17%,两者差异具有统计学意义,P=0.019。结论：我们首次发现IDH1基因mRNA高表达是独立于临床指标和高频基因突变的预后不良的标志物,IDH1基因mRNA表达水平可作为临床预后危险分层。第二部分IDH1基因表达在成人急性髓细胞白血病中的机制研究目的：(1)探索IDH1基因在AML中的作用机制；(2)寻找调控IDH1基因mRNA表达的microRNA。方法：(1)采用siRNA干扰方法沉默THP-1和HL-60/ADR细胞的IDH1基因,MTT法、流式细胞术检测其对生长、周期和凋亡影响,western blot检测凋亡和信号通路蛋白变化。(2)根据IDH1基因mRNA表达量高低从CN-AML中挑选16个患者标本,IDHl基因mRNA高表达和低表达各8例,采用Agilent Microarray芯片(SBC Human miRNA Microarray Release16.0)进行microRNA表达谱芯片分析,寻找与IDH1基因mRNA表达相关的microRNA。荧光定量PCR检测CN-AML患者的microRNA变化,对芯片结果进行验证。结果：(1)沉默THP-1细胞IDH1基因后,小干扰组细胞生长明显低于阴性对照组(p=0.04)；小干扰组48小时凋亡率为37.22±8.27%,明显高于阴性对照组19.50±1.18%(p=0.021),并伴随Caspase-3和PARP剪切带增加,PI3K/AKT/mTOR信号通路抑制,但是小干扰组细胞与阴性对照组细胞相比,周期变化差异无统计学意义(p=0.89)。(2)沉默THP-1细胞IDH1基因后给予临床常用化疗药物如：HHT. ACR和DNR,能增加这些药物对THP-1细胞的敏感性。(3)沉默HL-60ADR细胞IDH1基因48小时后,Caspase-3和PARP剪切带增加,PI3K/AKT信号通路抑制,能部分逆转HL-60/ADR对阿霉素的耐药。(4)芯片结果显示：IDH1基因mRNA高表达组的患者中miR-181家族(miR-181a、miR-181a-2、miR-181b、miR-181c*、miR-181c、miR-181a*和miR-181d)、iR-146a、miR-128、miR-625、miR-25和miR-335低表达,miR-4286、miR-660、miR-107和miR-324-5p高表达,而在IDH1基因mRNA低表达组的患者中上述microRNAs表达相反。(5)荧光定量PCR也证实了在IDH1基因mRNA高表达组的患者miR-181d和miR-181b低表达,miR-4286高表达；而在IDH1基因mRNA低表达组的患者中上述microRNA表达相反。结论：(1)下调IDH1基因表达能降低AML细胞株的增殖,导致细胞凋亡,增加化疗药物的敏感性和逆转耐药。(2)在CN-AML患者中miR-181家族(miR-181d和miR-181b)起到抑癌基因作用,可能抑制IDH1基因mRNA的表达,miR-4286起到癌基因作用,可能促进IDH1基因mRNA的表达。更多还原

【Abstract】 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.更多还原