【作者】 袁媛；

【导师】 司维柯；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2011， 博士

【摘要】 目的:已有大量研究显示,Wnt家族成员Wnt5a与许多实体肿瘤的发生、发展密切相关,但其与白血病发生的关系,以及作用机制迄今不明。我们前期的研究发现,Wnt5a在髓系和淋巴系白血病病例、白血病细胞株中表达降低或缺失,但在治疗缓解的病例中显示Wnt5a表达恢复。并且在白血病细胞株K562、HL-60和U937中发现Wnt5a基因启动子甲基化,在多发性骨髓瘤、髓系和淋巴系白血病病例中,同样也存在甲基化的现象,但正常人和已经缓解的病例中却没有发现甲基化。此外,外源性Wnt5a可诱导K562细胞发生分化,并观察到个别向单核细胞分化的现象。以上均提示,Wnt5a表达缺失或降低可能是白血病发生的机制之一。本研究旨在前期研究的基础上,以本实验室已成功构建的稳定过表达Wnt5a的K562细胞株(K562-Wnt5a)和对照细胞株(K562-vector)为模型,更加深入的探讨Wnt5a在K562细胞中发挥其作用的机制。结果表明Wnt5a介导不同受体组合,可激活非经典通路并抑制经典Wnt信号通路;亦可激活经典Wnt信号通路。由此我们认为,这种不同信号通路的相互转化,可能导致Wnt5a在白血病的发生中起不同作用,为今后有望以Wnt5a为靶点的的白血病分子靶向治疗和诊断提供理论依据。方法:1.在K562细胞中Wnt5a调控Ror2、Frz4、LRP5受体表达及定位的研究。1.1 Western blotting、免疫荧光染色、免疫细胞化学染色、激光共聚焦三维重构的方法,分别检测在K562细胞中,Wnt5a对Ror2、Frz4、LRP5受体的表达和定位的影响。1.2免疫荧光染色检测Ror2分别在293、HepG2、L02细胞中的表达定位,并对比分析。1.3免疫电镜检测,在K562细胞中Wnt5a是否诱导Ror2受体内吞。2.在K562细胞中Wnt5a通过不同受体途径激活经典或非经典Wnt信号通路的研究。2.1在K562细胞中,Wnt5a通过Ror2/Frz4受体途径激活非经典通路,并抑制经典Wnt信号通路的检测及分析。2.1.1 Western blotting、免疫共沉淀的方法,检测Wnt5a是否通过Ror2/Frz4复合受体发挥作用。2.1.2 Western blotting、免疫荧光染色分别检测,β-catenin在K562细胞的总细胞、细胞核和细胞浆中的表达水平,及其在细胞内分布定位的情况。2.1.3 Western blotting、双荧光素酶报告基因,检测β-catenin磷酸化水平及β-catenin/TCF转录活性的变化,以及对下游靶基因cyclinD1表达的影响。2.2在K562细胞中,Wnt5a通过Frz4/LRP5受体途径,激活经典Wnt信号通路的检测及分析。2.2.1运用Ror2特异性抗体孵育K562细胞,以达到阻断非经典Wnt5a/Ror2/Frz4通路转导的目的。2.2.2 Western blotting、免疫共沉淀的方法,验证非经典Wnt5a/Ror2/Frz4信号通路的转导是否被阻断。2.2.3 Western blotting、免疫共沉淀检测,阻断非经典Wnt5a/Ror2/Frz4通路后,Wnt5a是否通过Frz4/LRP5复合受体发挥作用。2.2.4 Western blotting分别检测,阻断非经典Wnt5a/Ror2/Frz4通路后,β-catenin,磷酸化β-catenin,以及cyclinD1表达水平的变化。2.2.5阻断非经典Wnt5a/Ror2/Frz4通路后,细胞增殖试验、流式细胞术,分别检测K562细胞增殖及细胞周期的变化。结果:1.过表达Wnt5a导致Ror2、Frz4受体表达显著高于对照细胞,LRP5表达无明显变化。此外,发现Ror2受体聚集表达于细胞核凹陷的胞浆区域,而Frz4、LRP5受体则主要定位于细胞膜及细胞浆中。2.免疫电子显微镜进一步检测,在细胞超微结构下显示,Wnt5a-K562细胞中具有Ror2免疫原性的颗粒物质(表现为电子密度显著增高)通过细胞膜内陷进入细胞浆,并最终定位于细胞核凹陷处的胞浆区域。3.与对照细胞相比,在Wnt5a-K562细胞中,显示Wnt5a与Ror2共表达、且相互作用增强。此外,Ror2与Frz4受体的结合亦增强。推断Wnt5a可能通过Ror2/Frz4复合受体发挥作用。4.Wnt5a没有影响β-catenin在总细胞和细胞浆中的表达水平,但改变了其在细胞核中的分布,即减少了β-catenin入核。5.Wnt5a过表达导致β-catenin出现磷酸化,使β-catenin/TCF的转录活性显著降低,最终导致下游靶基因cyclinD1表达下调。推测Wnt5a通过Ror2/Frz4受体途径激活非经典通路,并抑制经典Wnt信号通路。6.Ror2抗体通过与细胞中Ror2受体的特异性结合,阻断了非经典Wnt5a/Ror2/Frz4信号通路的转导。显示Wnt5a诱导Frz4高表达,且Frz4与LRP5受体相互作用增强,推断在Ror2受体表达减少或缺失时,Wnt5a即通过Frz4/LRP5复合受体发挥作用。7.阻断非经典Wnt5a/Ror2/Frz4信号通路后,发现β-catenin、cyclinD1均表达上调,并且β-catenin未出现磷酸化。推断此时在K562细胞中,Wnt5a介导Frz4/LRP5受体途径激活了经典Wnt信号通路。8.激活了经典Wnt5a/Frz4/LRP5信号通路,使K562细胞的G1期下降,S期增高,变化明显,表明细胞处于增殖旺盛的状态,促进了细胞增殖,与细胞增殖试验的结果一致。结论:1.在K562细胞中,过表达Wnt5a诱导Ror2和Frz4受体表达增高,并在Frz4的协同下,介导Ror2受体内吞,激活非经典通路并抑制经典Wnt信号通路:从而抑制了β-catenin的核转移,促进其磷酸化,使β-catenin/TCF转录活性降低,下调下游靶基因cyclinD1的表达。此时Wnt5a可能起抑癌因子的作用。2.在K562细胞中,当Ror2表达减少或缺失时,Wnt5a介导Frz4/LRP5受体途径激活经典Wnt信号通路,上调β-catenin及cyclinD1的表达,并促进K562细胞增殖。其中Frz4分别与Ror2或LRP5受体结合成为不同的复合受体,协同Wnt5a转导经典或非经典Wnt信号通路。由此可见Frz4受体在此过程中起重要的枢纽作用。3.Wnt5a发挥抑癌基因或是癌基因的作用可能与细胞的种类,即细胞表面所表达的受体有关。由于Wnt5a可通过不同的受体激活不同的信号通路,转导活化不同的下游靶基因表达,从而产生不同的调节作用。由此可见,Wnt5a在白血病中发挥的作用也与其相关分子的复杂性密切相关。更多还原

【Abstract】 Objective:Wnt5a, one member of the Wnt family, has been found to be correlated with tumorigenesis, but the specific relations and the mechanism are still not clear, especially concerning with leukemia. Our previous studies reported that Wnt5a was doesn’t expressed or down-regulated in myeloid and lymphoid leukemia, and myeloid leukemia cell lines. However, Wnt5a was found to be expressed in leukemias cases of complete remission. Wnt5a gene promoter region methylation in K562, HL-60 and U937 cell lines, also in multiple myeloma, myeloid and lymphoid leukemia cases, but was not found in normal and patients with complete remission. In addition, we have demonstrated that exogenous Wnt5a inhibits the proliferation of K562 cells and induces differentiation. We hypothesis that Wnt5a may be associated with leukemia. The K562 cells that stably overexpress Wnt5a protein were constructed successfully, and our current study shows that Wnt5a activate noncanonical or canonical Wnt signaling pathways in K562 cells through specific coupling of different receptor. Thus, we think that Wnt5a may play different role in the progression of leukemia.This study established the foundation for wnt5a as a tumor marker and a gene therapy target for leukemia.Methods:1. Wnt5a regulation Ror2, Frz4, LRP5 receptor expression.1.1 Western blotting, immunofluorescence, immunocytochemistry were used for detecting Wnt5a regulating Ror2, Frz4, LRP5 receptor expression and localization in K562 cells.1.2 K562、293、HepG2、L02 cells were analyzed for the localization of Ror2 by immunofluorescence. 1.3 Wnt5a induction Ror2 receptor internalization was observed by Immune electron microscopy in K562 cells.2. Wnt5a activates noncanonical or canonical Wnt signaling pathways through specific coupling of related coreceptors.2.1 Wnt5a mediated Ror2/Frz4 co-receptor to activate noncanonical pathway and inhibi classical Wnt signaling pathway.2.1.1 Western blotting, immunoprecipitation were used to observe that Wnt5a carrying out diverse roles via Ror2/Frz4 co-receptor.2.1.2 Western blotting, immunofluorescence were used for analyzing the expression and distribution ofβ-catenin in total cells, nuclei and cytoplasm of K562 cells.2.1.3 Western blotting were used for observing the expression ofβ-catenin phosphorylation and cyclinD1, andβ-catenin transcriptional activity was used for detecting by dual-luciferase assay.2.2 Wnt5a activates classical Wnt signaling pathway through Frz4/LRP5 co-receptor.2.2.1 Ror2 receptor leads to blocking the Wnt5a/Ror2/Frz4 nonclassical pathway through specific coupling of Ror2 antibody in K562 cells.2.2.2 To make it clear whether the Wnt5a/Ror2/Frz4 signal blocked was evaluated by Western blotting, immunoprecipitation.2.2.3 We assessed whether Wnt5a carrying out its diverse roles via Frz4/LRP5 co-receptor was detected by Western blotting, immunoprecipitation.2.2.4 Western blotting was used for detecting changes ofβ-catenin, p-β-catenin and cyclinD1 expression.2.2.5 MTT cell proliferation assay, flow cytometry were used for observing the proliferation and growth cycle of K562 cells.Results:1.Ror2 and Frz4 were found strongly expresseing more strongly in K562-Wnt5a than in control cells, but no change of the expression of LRP5 occurred. Furthermore, Ror2 localized in foci of the nuclear invagination area in K562-Wnt5a. Frz4, LRP5 localized in the cell membrane and cytoplasm.2. Immune electron microscopy showed that immunoreactive particulate matter occurred on the plasmalemma of K562-Wnt5a cells, which was internalized into the cytoplasm, and localized in the nuclear invagination cytoplasmic area.3. Compared with the control cells, Wnt5a and Ror2 co-expressed, and enhanced binding of each other in K562-Wnt5a cells. Wnt5a also enhanced binding to Frz4. Wnt5a played its roles via Ror2/Frz4 co-receptor.4. Wnt5a has no effect on total and cytoplasmicβ-catenin expression, but can decrease expression ofβ-catenin in the nucleus.5. Phosphotyrosine ofβ-catenin was detected in K562-Wnt5a cells, and TOPflash reporter assay demonstrated significant down-regulation ofβ-catenin transcriptional activity and leaded to downregulation cyclinD1.6. Ror2 receptor blocked the Wnt5a/Ror2/Frz4 nonclassical pathway through specific coupling of Ror2 antibody. Subsequently, Wnt5a induced increased expression of Frz4, and enhanced interaction between Frz4 and LRP5 proteins. We think that Wnt5a play a role through Frz4/LRP5 co-receptors when Ror2 expression was absenced or reduced.7. Wnt5a induced Increased expression ofβ-catenin and cyclin D1, and P-β-catenin was not detected in K562-Wnt5a cells. We think that Wnt5a carry out its roles by classical Wnt signaling pathway via Frz4/LRP5 co-receptor.8. MTT and flow cytometry assay results indicate that compared with control, overexpression Wnt5a promoted the proliferation of K562 cells.Conclusion:1. Overexpression Wnt5a resulted in increasing expression of Ror2 and Frz4. Wnt5a activates nonclassical signaling and inhibit the classical Wnt pathway by Frz4 synergistic through Ror2 internalization. Tyrosine phosphorylation ofβ-catenin was detected in Wnt5a-K562 cells. Results show thatβ-catenin transcriptional activity was negatively regulated by Wnt5a through decreasing the localization and expression ofβ-catenin in the nucleus, and leaded to decreasing cyclinD1 expression levels.2. Wnt5a activated canonical signaling pathways through Ror2/Frz4 co-receptor when Ror2 expression was absence or reduced. We think that Frz4 is required for Wnt5a coupling Frz4 to LRP5 or Ror2 co-receptors, to induce noncanonical or canonical Wnt signaling pathways. 3. Wnt5a proteins can lead to different outcomes in tumorigenesis. Wnt5a carry out diverse roles by different signaling pathways via specific to coupling different receptor in different cells. which is also the mechanism for Wnt5a in leukemia genesis.更多还原