【作者】 王涓冬；

【导师】 陈春燕；

【作者基本信息】 山东大学 ， 内科学， 2011， 博士

【摘要】 研究目的：急性髓系白血病(acute myeloid leukemia, AML)是一组以髓系造血细胞的异常增殖、分化障碍、凋亡受阻为特征的恶性克隆性疾病,是成人白血病中最常见的类型。尽管化疗方案的改进、支持治疗的加强、分子靶向药物的应用和造血干细胞移植技术的进展提高了治疗的疗效,但60岁以下的患者五年生存率仅为30-40%,60岁以上的患者五年生存率不到10%。因此,研究白血病发病机制、寻找新的治疗靶点并研发相应的治疗药物成为血液学领域的重要研究课题之一。CIP2A (Cancerous Inhibitor of PP2A, CIP2A),也称为KIAA1524、p90,是一种新近发现的蛋白磷酸酶PP2A (protein phosphatase 2A, PP2A)的内源性抑制剂。CIP2A通过抑制PP2A对c-Myc蛋白的去磷酸化作用使c-Myc蛋白表达升高,从而导致细胞的恶性转化,促进细胞的增殖及锚着非依赖性生长,参与肿瘤的形成。已有研究表明,CIP2A在一些实体瘤如头颈部鳞状细胞癌、结肠癌、胃癌和乳腺癌中过表达,而它在AML中的表达状况及作用尚不明确。本课题通过研究AML患者骨髓标本中CIP2A的表达,以及CIP2A对AML细胞系HL60增殖、分化与凋亡的影响,探讨CIP2A在AML发病中的作用及其机制。材料与方法：1. RT-PCR及Western blot法检测AML患者中CIP2A的表达收集AML患者及健康志愿者的骨髓标本,分离骨髓单个核细胞,提取RNA及蛋白,采用RT-PCR及Western blot的方法分别检测CIP2A在mRNA和蛋白水平的表达。2.用RNA干扰、血清饥饿实验、细胞生长曲线、克隆形成实验、瑞氏染色和流式细胞术研究AML中CIP2A的作用及其机制以AML细胞系HL60为研究对象,用CIP2A特异性siRNA沉默CIP2A的表达,检测HL60细胞增殖、分化与凋亡的相关指标,并观察c-Myc的表达水平及PP2A活性的变化,探讨CIP2A在AML发病中的作用及机制。(1)通过血清饥饿实验和绘制转染CIP2A siRNA后HL60细胞生长曲线、软琼脂克隆形成实验研究CIP2A对HL60细胞增殖的影响；(2)通过瑞氏染色观察转染CIP2A siRNA后HL60细胞形态的变化,并用流式细胞仪检测细胞分化抗原CD11b的表达探讨CIP2A对HL60细胞分化的影响；(3)用流式细胞仪通过Annexin V与PI双染法检测HL60细胞转染CIP2AsiRNA后凋亡率的变化,观察CIP2A对HL60细胞凋亡的影响。(4)检测沉默CIP2A后c-Myc的表达及PP2A活性的变化,研究CIP2A作用的机制。结果：1. CIP2A在AML患者中的表达检测116例AML患者(其中初治组70例,复发组14例,完全缓解组32例)和35例健康对照者共151例骨髓单个核细胞中CIP2A的表达。结果显示,在AML初治组中,CIP2A mRNA的阳性率为77.14%(54/70)；在复发组中阳性率为70.86%(11/14),均较完全缓解组及正常对照组有显著性差异(完全缓解组CIP2A mRNA的阳性率为6.25%,正常对照组为2.86%)(P<0.001)；而初治组与复发组、完全缓解组与正常对照组之间CIP2A mRNA阳性率的差异均无统计学意义(P>0.05)。应用Western blot的方法检测了相应标本的CIP2A在蛋白水平的表达,初治组和复发组标本的CIP2A蛋白也呈现高表达状态,与RT-PCR结果一致。2.CIP2A对HL60细胞增殖、分化及凋亡的影响(1)CIP2A对HL60细胞增殖的影响经过72h的血清饥饿,HL60细胞中CIP2A的表达水平明显下降。沉默CIP2A的表达后,HL60细胞生长速度减慢,克隆形成能力下降,较对照组有显著性差异。(2)CIP2A对HL60细胞分化的影响收集转染CIP2A siRNA后72h的HL60细胞,瑞氏染液染色后油镜下观察细胞形态,显示约1/3的细胞出现了晚期早幼粒细胞的形态特征；转染CIP2A siRNA的HL60细胞CDllb的表达率为11.25±2.20%,较转染control siRNA的HL60细胞明显增加,P<0.05。(3) CIP2A对HL60细胞凋亡的影响分别收集转染CIP2A siRNA或control siRNA 72h后的HL60细胞,检测细胞凋亡情况,转染CIP2A siRNA组凋亡率为5.1±0.87%；转染control siRNA组凋亡率为0.80±0.30%,二者比较P<0.01。3. CIP2A对c-Myc表达和PP2A活性的影响沉默CIP2A表达后,c-Myc蛋白的水平随之下降,PP2A的活性明显恢复。结论：1.CIP2A在初治及复发的AML患者中高表达,而在完全缓解的患者及正常对照组的表达很低,提示CIP2A可能作为AML的肿瘤标志。2.CIP2A在HL60细胞中的高表达可以促进细胞增殖、抑制细胞分化和凋亡。3.CIP2A的生物学功能与其高表达引起PP2A活性下降、c-Myc蛋白的表达升高有关。4.抑制CIP2A的表达是AML细胞中PP2A活性的恢复和c-Myc的降解的新途径,因此干预CIP2A的表达有可能作为AML新的靶向治疗策略。研究目的：慢性髓细胞白血病(chronic myelocytic leukemia, CML)是一种获得性造血干细胞的恶性克隆性疾病,主要涉及髓系。表现为外周血粒细胞显著增多并有成熟障碍、脾脏肿大。目前认为Ph染色体的出现和BCR/ABL融合基因的形成是CML的主要发病机制,而有关CML急变的机制仍不明确。CML病程发展缓慢,分为慢性期(chronic phase, CP)、加速期(accelerated phase, AP)和急变期(blastic phase, BP)三个时期。急变期为CML的终末期,CML一旦急变,预后极差,患者往往在数月内死亡因此,寻找新的CML急变的相关基因对于揭示CML急变的分子机制及发现治疗的新靶点具有重要的意义。已有研究发现,CML在发生及急变的过程中均存在PP2A功能的失活,因此认为PP2A抑癌活性的丧失对于CML的发生甚至急性变都有着至关重要的作用。CIP2A是新近发现的一种PP2A的内源性抑制剂,主要存在于人类恶性肿瘤组织中,可以直接和c-Myc的氨基末端结合,保护c-Myc第62位丝氨酸(serine 62, S62)免受脱磷酸作用,阻止c-Myc蛋白的降解,维持其稳定,从而抑制c-Myc相关的PP2A活性。已经证实,CIP2A在一些实体瘤如头颈部鳞状细胞癌、结肠癌、胃癌和乳腺癌中过表达,而CIP2A在CML中的表达及其作用尚未见报道。本实验旨在通过研究CIP2A在不同分期CML患者中的表达和对K562增殖、分化、凋亡的影响以及与BCR/ABL的调控关系,探讨CIP2A在CML发生及急变中的作用及其机制。材料与方法：1.用RT-PCR、QRT-PCR及Western-Blot法检测CML患者中CIP2A的表达收集不同分期的CML患者及健康志愿者的骨髓标本,分离骨髓单个核细胞,提取RNA及蛋白,采用RT-PCR及Western blot的方法分别检测CIP2A在mRNA和蛋白水平的表达。用QRT-PCR法检测CIP2A mRNA在CML不同分期表达水平的差异。2.用RNA干扰、血清饥饿实验、细胞生长曲线、克隆形成实验、瑞氏染色和流式细胞术研究CML中CIP2A的作用以CML急变细胞系K562为研究对象,采用CIP2A siRNA干扰的方法,沉默CIP2A的表达,检测K562细胞增殖、分化与凋亡的相关指标,从而探讨CIP2A在CML发病中的作用。(1)通过绘制细胞生长曲线、软琼脂克隆形成实验及血清饥饿实验研究CIP2A对K562细胞增殖的影响。(2)通过瑞氏染色观察转染CIP2A siRNA后K562细胞形态的变化,探讨CIP2A对K562细胞分化的影响。(3)通过Annexin V与PI双染法流式细胞仪检测K562细胞凋亡率的变化,观察CIP2A对K562细胞凋亡的影响。3.用RNA干扰和伊马替尼干预研究CIP2A作用的机制(1)检测K562细胞转染CIP2A siRNA后c-Myc表达水平及PP2A活性的变化。(2)BCR/ABL对CIP2A的调节作用使用酪氨酸激酶抑制剂甲磺酸伊马替尼(imatinib mesylate,IM)作用于K562细胞,检测CIP2A表达的变化。(3)CIP2A对BCR/ABL的调节作用分别转染CIP2A siRNA和CIP2A质粒使CIP2A表达下调或上调,检测BCR/ABL和SHP-1表达的相应变化。结果：1.CIP2A在CML患者中的表达情况检测71例CML患者(CP 58例,AP/BP13例)和35例健康对照者骨髓单个核细胞中CIP2A的表达。CML-CP组58例标本中44例表达CIP2A转录产物,阳性率为75.86%(44/58)；CML-AP/BP组13例标本中CIP2A阳性者为10例,阳性率为76.93%(10/13)；正常对照组CIP2A表达的阳性率仅为2.86%(1/35)。CML-CP组和CML-AP/BP组与正常对照组的CIP2AmRNA的表达水平均有显著性差异(P<0.001)。应用Western blot的方法检测了相应标本的CIP2A在蛋白水平的表达,CML-CP组和CML-AP/BP组标本的CIP2A蛋白也呈现高表达现象,与RT-PCR结果一致。CML-AP/BP期CIP2A mRNA的表达水平较CML-CP组明显增高(P<0.001),约是CML-CP组的4倍。CIP2A mRNA在CML患者骨髓单个核细胞中的表达与BCR/ABL融合基因的表达具有一致性2.CIP2A对K562细胞增殖、分化和凋亡的影响经过72h的血清饥饿,CIP2A水平明显下降。CIP2A siRNA沉默CIP2A的表达后,K562细胞生长速度减慢,克隆形成能力下降,72h时细胞形态未发生明显变化,细胞凋亡增多。3.CIP2A作用机制的研究(1) CIP2A siRNA沉默CIP2A的表达后,c-Myc蛋白的水平下降,PP2A的活性得到了显著恢复。(2) BCR/ABL对CIP2A的调节作用IM作用于K562细胞后,BCR/ABL表达被抑制的同时,CIP2A的表达亦明显下降。(3) CIP2A对BCR/ABL的调节作用下调CIP2A的表达后,BCR/ABL的水平也呈下降趋势,而SHP-1的表达则呈现上升趋势；相反的,上调CIP2A的表达可导致BCR/ABL水平升高,SHP-1的表达下降。结论：1.CIP2A在CML患者中高表达,加速期和急变期患者CIP2A-mRNA的表达水平明显高于慢性期。提示CIP2A不仅与CML的发病有关,而且在CML急变的过程中发挥了一定的作用。2.CIP2A在K562细胞中的高表达可以促进细胞增殖,并抑制其凋亡。3.CIP2A的生物学作用与其高表达所致的PP2A活性下降、c-Myc蛋白的表达升高有关。4.IM可通过抑制BCR/ABL下调CIP2A的表达,丰富了IM的作用途径。5.CIP2A与BCR/ABL之间存在着正反馈相互调节作用,一方面,CIP2A可抑制c-Myc相关的PP2A活性,PP2A的失活可以通过SHP-1的介导上调BCR/ABL的表达；另一方面,BCR/ABL也可诱导CIP2A表达,进而抑制PP2A的活性,这种正反馈作用参与了CML急性变。通过BCR/ABL诱导CIP2A表达效应产生的PP2A活性的抑制是CIP2A参与CML发病与急变的可能机制之一。6.干预CIP2A的表达可能成为CML急变的靶向治疗新策略。更多还原

【Abstract】 Introduction:Acute myeloid leukemia (AML) is characterized by the clonal expansion of myeloid hematopoietic cells with uncontrolled proliferation, differentiation arrest and decreased apoptosis. AML is the most common type of leukemia in adults. Despite the fact that improvements in chemotherapy, strengthened supportive treatment and the wild spread use of molecular targeted therapy and hematopoietic stem cell transplantation improve the efficacy of treatment, but the five-year survival rate in patients under the age of 60 years old is only 30-40%, and while in patients over 60 years old is less than 10%. Therefore, it is vital to identify the fundamental pathogenesis of AML, which may lead to the development of novel treatments. CIP2A (Cancerous Inhibitor of PP2A, CIP2A), also called KIAA1524, p90, is a newly identified endogenous inhibitor of protein phosphatase 2A (PP2A). CIP2A interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage independent cell growth and in vivo tumor formation. Thus CIP2A is identified as a human oncoprotein. Although CIP2A is over-expressed in some human solid tumors, such as head and neck squamous cell carcinoma, colon cancer, gastric cancer, and breast cancer, its expression and roles in AML is still unknown. Methods:We collected the bone marrow samples from patients with AML and healthy controls, and then isolated mononuclear cells by centrifugation over Ficoll-Hypaque gradients, extracted total RNAs and protein. CIP2A mRNA and protein expressions were determined in bone marrow mononuclear cells of both patients with AML and healthy controls using reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. We used siRNA to knock-down CIP2A expression in HL60 cells and then examined its potential roles during the pathological progression of AML.1. Cell proliferation study was conducted by drawing cell growth curve, soft agar colony formation assay and serum starvation test.2. Cell differentiation was determined by both microscopic morphology analysis following Wright’s staining and immunophenotypic feature of CD11b expression analysed by flow cytometry.3. Cell apoptosis rate was detected by flow cytometry through AnnexinⅤand PI staining.4. After silencing the expression of CIP2A, we detected the changes of c-Myc level and PP2A activity.Results:A total of 151 bone marrow samples from 116 patients with AML [70 patients with newly diagnosed AML,14 patients with relapsed AML and 32 patients with AML in complete remission (AML-CR)] and 35 healthy controls were collected for this study. CIP2A mRNA was presented in 54 of 70 (77.14%) patients with newly diagnosed AML and in 11 of 14 (70.86%) patients with relapsed AML, which was significantly higher than AML-CR specimens (2/32,6.25%) and healthy controls (1/35,2.86%) (P<0.001). However, in terms of CIP2A mRNA expression, the AML-CR group was not statistically significant different from normal controls (P>0.05). Similarly, there was no statistically significant difference between the newly diagnosed and relapsed AML group. In addition, over-expression of CIP2A protein was also verified by Western blot in corresponding specimens. After 72h of serum starvation, CIP2A level was significantly decreased. Knock-down of CIP2A in HL60 cells slowed down cell proliferation, decreased clonogenic activity, promoted cell differentiation and inceased cell apoptosis. Upon the knock-down of CIP2A, the level of c-Myc protein was consequently reduced, and the activity of PP2A recovered dramatically.Conclusions:Our findings suggest that CIP2A is over-expressed in patients with newly diagnosed/relapsed AML, indicating that CIP2A may serve as a novel tumor marker for AML. The high expression of CIP2A in HL60 cells may be related to active cell proliferation, arrest cell differentiation and inhibit cell apoptosis through the inactivation of PP2A and the increase of c-Myc protein. This study may shed light on the molecular function of CIP2A in myeloid leukemogenesis and support the potential development of CIP2A-based targeted therapy for the treatment of AML. Introduction:Chronic myelocytic leukemia (CML) is an acquired clonal hematopoietic stem cell malignant disease, mainly involving myeloid, caused by the BCR/ABL fusion protein which arises from the translocation of chromosomes 9 and 22. Usually CML experiences three phases:chronic phase (CP), accelerated phase (AP) and blastic phase (BP). Once the disease progresses into BP, the survival is usually less than 1 year. Until now, precise mechanisms responsible for transition of CML chronic phase into blast crisis are still unclear. It has been reported that the tumor suppressor PP2A is functionally inactivated in occurrence and blast crisis of CML. So it is believed that inactivation of PP2A is essential for the pathogenesis even blast crisis of CML. CIP2A is a newly identified endogenous inhibitor of PP2A. Therefore the research of CIP2A in CML is very important to disclose possible mechanisms of CML progression and look for new therapy targets. Although CIP2A is over-expressed in some human solid tumors, such as head and neck squamous cell carcinoma, colon cancer, gastric cancer, and breast cancer, its expression and roles in CML is still unknown.Methods:We collected the bone marrow samples from patients with CML and healthy controls, and then isolated mononuclear cells by centrifugation over Ficoll-Hypaque gradients, extracted total RNAs and protein. CIP2A mRNA and protein expressions were determined in bone marrow mononuclear cells of both patients with CML and healthy controls using RT-PCR and Western blot, respectively. The difference between patients with CML-CP and CML-AP/BP was analyzed by QRT-PCR. We used siRNA to knock-down CIP2A expression in K562 cells and then examined its potential roles during the pathological progression of CML.1. Cell proliferation study was conducted by drawing cell growth curve, soft agar colony formation assay and serum starvation test.2. Cell differentiation was determined by microscopic morphology analysis following Wright’s staining.3. Cell apoptosis rate was detected by flow cytometry through Annexin V and PI staining. We also detected the expression of c-Myc and the activity of PP2A after knock-down of CIP2A. In addition, we inhibited the expression of BCR/ABL by imatinib mesylate (IM), and detected the variation of CIP2A. On the other hand, we checked out the change of BCR/ABL and SHP-1 after CIP2A was knocked down or up-regulated.Results:Bone marrow samples from 71 patients with CML (58 patients with CML-CP,13 patients with CML-AP/BP) and 35 healthy controls were collected for this study. CIP2A mRNA was presented in 44 of 58 (75.86%) patients with CML-CP and in 10 of 13 (76.93%) patients with CML-AP/BP, which was significantly higher than healthy controls (1/35,2.86%) (P<0.001). Furthermore, in terms of CIP2A mRNA expression, the CML-AP/BP group was significantly higher than CML-CP (P<0.001), and almost four times the CP. In addition, over-expression of CIP2A protein was also verified by Western blot in corresponding specimens. After 72h of serum starvation, the level of CIP2A was significantly decreased. Knock-down of CIP2A in K562 cells slowed down cell proliferation, decreased clonogenic activity, promoted cell apoptosis. Upon scilencing of CIP2A, the level of c-Myc protein was consequently reduced, and the activity of PP2A recovered dramatically. CIP2A mRNA expression in bone marrow mononuclear cells in CML patients was in accordance with the expression of BCR/ABL fusion gene. After the expression BCR/ABL was suppressed by IM, expression of CIP2A also significantly decreased. Followed with down-regulation of CIP2A expression, expression of BCR/ABL also decreased, while the levels of SHP-1 showed a rise trend. On the contrary, up-regulation of CIP2A expression led to the increase of BCR/ABL and decrease of SHP-1. Conclusions:Our findings suggest that CIP2A is over-expressed in patients with CML and the expression of CIP2A mRNA in CML-AP/BP group was significantly higher than that in CML-CP. This shows that CIP2A not only participates in the pathogenesis of CML, but also plays an important role in the progression of CML. The high expression of CIP2A in K562 cells may promote cell proliferation, inhibit cell apoptosis through the inactivation of PP2A and the increase of c-Myc protein. CIP2A and BCR/ABL can regulate each other forming a positive feedback loop. Functional inactivation of PP2A tumour suppressor activity caused by the effect BCR/ABL on CIP2A expression may be one of the possible mechanisms of blast crisis CML. This study may shed light on the molecular function of CIP2A in CML and interfering in CIP2A’expression will be a potential targeted therapy in patients with CML blast crisis.更多还原