NPM1突变蛋白介导核蛋白PML异常定位及稳定性促进白血病细胞的自噬和增殖能力

发布时间：2018-06-20 23:30

本文选题：NPM1 + 基因突变　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:急性髓系白血病(acute myeloid leukemia,AML)是一组临床上细胞遗传学和分子生物学具有高度异质性的恶性血液病。核仁磷酸蛋白(nucleophosmin,NPM1)基因突变是AML最常见的一种基因突变。已有的文献报道NPM1基因突变是白血病发生的始动因素。然而,NPM1突变基因促进AML恶性转化的分子机制仍尚未完全阐明。自噬是真核细胞中比较保守的一种代谢途径,通过溶酶体途径降解胞内折叠错误的蛋白质或受损的细胞器以维持细胞稳态。越来越多的证据表明自噬与肿瘤的发生发展和治疗疗效密切相关。此外,最新临床研究显示,NPM1突变阳性AML样本中存在肿瘤抑制因子早幼粒细胞白血病蛋白PML异常的亚细胞定位。本文主要探讨NPM1基因突变对白血病细胞自噬的调控作用,进一步观察自噬水平的改变对NPM1突变白血病细胞体外增殖的影响,并初步探讨异常表达的PML蛋白在NPM1突变介导的白血病细胞自噬中的作用。方法:首先采用过表达和慢病毒介导的RNA干扰方法,观察A型NPM1基因突变(NPM1-mA)对白血病细胞自噬水平的调控作用,进一步观察自噬诱导剂rapamycin或抑制剂3-methyladenine(3-ma)处理对npm1-ma介导的细胞体外增殖能力的影响。qrt-pcr和westernblot检测髓系白血病细胞株和临床aml样本中pml的表达水平;免疫组化和免疫荧光检测pml蛋白在髓系白血病细胞中的分布情况。免疫共沉淀检测npm1突变蛋白与pml蛋白之间的相互作用。过表达或者干扰npm1-ma后观察pml蛋白水平的变化。cycloheximide和mg132处理进一步观察npm1-ma对pml蛋白稳定性的调控。接下来,利用慢病毒介导的rna干扰技术下调pml水平,探讨干扰pml对天然携带npm1突变基因的oci-aml3白血病细胞株的自噬、体外增殖能力以及akt信号分子的改变。最后,通过rescue实验观察过表达pml对npm1-maknockdown的oci-aml3细胞株自噬和体外增殖能力的影响。结果:首先,过表达npm1-ma能够增强thp-1白血病细胞株自噬水平,3-ma处理能够减弱npm1-ma介导的细胞体外增殖能力;相反,干扰npm1-ma则抑制oci-aml3细胞自噬水平,而rapamycin处理能逆转npm1-maknockdown介导的细胞体外增殖能力。其次,在npm1-ma阳性的aml样本和oci-aml3细胞株中证实了pml的表达和蛋白的异常胞质定位。免疫共沉淀验证了npm1-ma蛋白与pml蛋白的相互作用。此外,过表达npm1-ma能上调pml蛋白表达水平,干扰npm1-ma则下调pml蛋白表达水平。cycloheximide和mg132处理进一步验证了npm1突变蛋白是通过抑制蛋白酶体途径来增强pml蛋白的稳定性。接着,干扰pml导致oci-aml3体外增殖能力和克隆形成能力减弱。另外,干扰PML可抑制细胞自噬活性进而影响细胞增殖能力,这可能与AKT信号分子的改变有关。最后,rescue实验表明,过表达PML能够逆转NPM1-mA下调对白血病细胞自噬和增殖能力的抑制作用。结论:NPM1突变蛋白可促进白血病细胞的自噬和增殖能力。机制上,NPM1-mA与PML蛋白相互作用介导PML的异常胞质定位及稳定性;PML可能通过AKT信号分子调控白血病细胞的自噬活性,进而增强NPM1突变白血病细胞的体外增殖能力。本研究提示抑制自噬或者靶向PML蛋白可作为临床上治疗NPM1突变白血病的一种新策略。
[Abstract]:Objective: acute myeloid leukemia (AML) is a group of malignant hematopathy with high heterogeneity in clinical cytogenetics and molecular biology. The mutation of nucleolar phosphoric acid (nucleophosmin, NPM1) gene is the most common gene mutation in AML. It has been reported that the mutation of NPM1 gene is the beginning of the occurrence of leukemia. Factors. However, the molecular mechanism of NPM1 mutation to promote the malignant transformation of AML is still not fully elucidated. Autophagy is a relatively conservative metabolic pathway in eukaryotic cells to degrade wrong proteins or damaged organelles through the lysosome pathway to maintain cell homeostasis. The more evidences indicate the occurrence of autophagy and the occurrence of tumors Development and therapeutic efficacy are closely related. In addition, the latest clinical study shows that the subcellular localization of the tumor suppressor promyelocytic leukemia protein PML abnormality exists in the NPM1 mutation positive AML sample. This paper mainly discusses the regulation of NPM1 mutation on autophagy in leukemic cells and further observation of the change of autophagy to NPM1 mutation The effect of the proliferation of leukemic cells in vitro, and the role of abnormal expression of PML protein in autophagy mediated by NPM1 mutation in leukemia cells. Methods: first, using overexpression and lentivirus mediated RNA interference to observe the regulation of A NPM1 gene mutation (NPM1-mA) on the autophagy level of leukemic cells and further observe autophagy. Effects of inducer rapamycin or inhibitor 3-methyladenine (3-mA) treatment on the proliferation of npm1-ma mediated cells in vitro:.Qrt-pcr and Westernblot detection of PML expression in myeloid leukemia cell lines and clinical AML samples; immunohistochemical and immunofluorescence detection of the distribution of PML protein in myeloid leukemia cells. Immunofulcation Interaction between NPM1 mutant protein and PML protein. Over expression or disturbance of npm1-ma after npm1-ma observation of PML protein level change.Cycloheximide and MG132 treatment further observe the regulation of PML protein stability by npm1-ma. Next, use the lentivirus mediated RNA interference technique to adjust PML level, explore the interference PML on the natural carrying NPM Autophagy, in vitro proliferation ability and changes in Akt signal molecules of the 1 mutant gene oci-aml3 leukemia cells. Finally, the effects of PML on the autophagy and in vitro proliferation of npm1-maknockdown oci-aml3 cell lines were observed by rescue experiments. Results: first, overexpression of npm1-ma could enhance the autophagy level of THP-1 leukemia cell lines, 3 -ma treatment could weaken the proliferation ability of npm1-ma mediated cells in vitro; on the contrary, interference with npm1-ma inhibited the autophagy level of oci-aml3 cells, and rapamycin treatment could reverse the proliferation ability of npm1-maknockdown mediated cells in vitro. Secondly, the expression of PML and the abnormal cytoplasm of the protein were confirmed in the npm1-ma positive AML samples and oci-aml3 cell lines. The interaction between npm1-ma protein and PML protein was confirmed by immunoprecipitation. In addition, overexpression of npm1-ma could increase the expression of PML protein, interfering npm1-ma and decreasing PML protein expression level.Cycloheximide and MG132 treatment further verified that NPM1 mutation protein enhanced the stability of PML protein by inhibiting the egg white enzyme body pathway. In addition, interfering with PML in vitro proliferation and clonogenic ability of oci-aml3 decreased. In addition, interfering PML could inhibit cell autophagy and affect cell proliferation, which may be related to the change of AKT signal molecules. Finally, rescue experiment showed that overexpression of PML could reverse the inhibition of autophagy and proliferation of leukemic cells by NPM1-mA down regulation. Conclusion: NPM1 mutant protein can promote the autophagy and proliferation of leukemia cells. The interaction of NPM1-mA and PML protein mediates the abnormal cytoplasmic localization and stability of PML; PML may regulate the autophagy activity of leukemic cells through AKT signal molecules, and then enhance the proliferation ability of NPM1 mutant leukemia cells in vitro. These findings suggest that inhibition of autophagy or targeting of PML protein can be used as a new strategy for clinical treatment of NPM1 mutant leukemia.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71

【相似文献】