miR-550-1在急性髓系白血病中的抑癌作用及其机制研究

发布时间：2018-06-06 03:29

本文选题：急性髓系白血病 + miR-550-1　；参考：《浙江大学》2016年博士论文

【摘要】：第一部分急性髓系白血病中新的抑癌性microRNA——miR-550-1的发现,分析其在AML患者中的表达水平,并探讨其上游调控机制目的：筛选出急性髓系白血病(AML)中新的抑癌性microRNAs (miRNAs),并分析其表达情况。方法：1.Exiqon miRCURY LNA arrays分析85例AML患者与15例正常对照标本间差异表达的miRNAs; 2.TaqMan qPCR鉴定AML患者中特定miRNA的表达水平,验证芯片结果；3.分析TCGA数据库中194例AM,患者miR-550-1启动子区CpG岛甲基化程度,并利用miR-22的启动子区CpG岛甲基化程度作阴性对照；4.利用0、0.5、1、2、4μM不同浓度去甲基化药物地西他滨处理AML细胞株96h,观察处理后miR-550-1表达量改变的情况。结果：1. Exiqon miRCURY LNA arrays芯片筛选出了12个在AML患者中低表达而在正常对照中高表达的miRNAs,分别为：miR-150、miR-148a、miR-29a、miR-29b、 miR-184、miR-22、miR-342-3p、miR-423-5p、miR-550-1、miR-660、miR-768-3p、 miR-768-5p (P值均小于0.0001,假发现率(false discovery rate, FDR)均小于0.0001)；2.从筛选的结果中我们挑选了miR-550-1作为研究对象,通过TaqmanqPCR检测发现AML患者中miR-550-1的表达量较正常对照确实是显著下调的(P=0.003)；3.通过分析TCGA数据库,我们发现194例AML患者miR-550-1启动子区CpG岛较miR-22启动子区CpG岛的甲基化程度明显升高；4.用0、0.5、1、2、4μM不同浓度地西他滨处理Kasumi-1和MV4-11细胞96h后,可诱导细胞内miR-550-1表达上调,相对表达水平分别为Kasumi-1 (1、1.19、1.18、1.14、1.29), MV4-11 (1、3.30、7.33、20.15、13.96).结论：1.与正常对照标本相比,AML患者中miR-550-1表达量显著降低；2.miR-550-1启动子区CpG岛处于高度甲基化状态；3.利用去甲基化药物地西他滨可以促进AML细胞株中miR-550-1的表达。第二部分观察miR-550-1对急性髓系白血病细胞活性、增殖、凋亡、周期、克隆形成能力以及小鼠急性髓系白血病骨髓移植模型发生发展的影响目的：探索外源性过表达miR-550-1后对AML细胞株及小鼠髓系祖细胞生物学功能的影响。方法：1.构建miR-550-1逆转录病毒过表达载体,PCR定点突变方法构建miR-550-1种子序列突变的变异体miR-550-1逆转录病毒载体；2.利用逆转录病毒感染AML细胞株和小鼠髓系祖细胞,构建miR-550-1稳定过表达细胞株,其中小鼠髓系祖细胞还需共转染MLL-AF9或AE9a融合基因,利用TaqMan qPCR验证过表达miR-550-1是否成功；3.通过MTT、台盼蓝染色细胞计数检测AML细胞活性和细胞增殖；4.流式细胞术检测AML细胞株的细胞周期和细胞凋亡；5.Western blot方法检测细胞增殖、周期、凋亡相关信号通路蛋白的变化；6.Dot blot方法检测细胞内m6A表达水平变化；7.小鼠髓系祖细胞集落培养检测细胞克隆形成能力；8.Wright-Giemsa染色检测细胞形态及分化情况；9.小鼠骨髓移植模型检测miR-550-1对一代、二代移植小鼠AML疾病发生发展的影响。结果：1.逆转录病毒感染细胞后可显著升高Kasumi-1、MV4-11、小鼠髓系祖细胞内miR-550-1的表达水平(P0.001)；2.在Kasumi-1、MV4-11、小鼠髓系祖细胞中miR-550-1过表达后可以明显抑制细胞活性和细胞增殖能力；3Kasum i-1细胞过表达miR-550-1,72h后G0/G1细胞比例增加10.4%,96h凋亡细胞比例增加7.76%,PARP蛋白激活,p-AKT和CDK6蛋白表达被抑制,m6A表达水平被抑制；4.共感染miR-550-1和MLL-AF9、AE9a融合基因的小鼠髓系祖细胞较单独转染融合基因的细胞每一代的克隆形成数要少(P0.05),克隆中细胞总量也显著降低(P0.05),分化成熟的细胞逐代增加；5.在小鼠骨髓移植中,与对照组相比较,miR-550-1过表达组小鼠一代移植(中位生存时间分别为77.5天和105天)和二代移植(中位生存时间分别为27天和33天)的总体生存时间均显著延长(P值分别为0.035和0.010)。结论：1.过表达miR-550-1后可抑制AML细胞生长,降低细胞活性,促进细胞凋亡,诱导细胞发生G0/G1期阻滞,抑制RNA上m6A表达；2.过表达miR-550-1后可抑制共感染MLL-AF9或AE9a融合基因的小鼠髓系祖细胞每代克隆形成能力,并诱导细胞分化；3.过表达miR-550-1可抑制小鼠骨髓移植模型AML的发生发展,延长小鼠总体生存时间,降低恶性原始细胞比例,促进小鼠细胞分化,减轻小鼠肿瘤负荷。第三部分探索miR-550-1的靶基因和影响急性髓系白血病发生发展的分子机制目的：探索miR-550-1在体内体外影响急性髓系白血病发生发展的分子机制,即通过调控哪些潜在靶基因而实现其生物学功能,并通过rescue实验验证靶基因功能。方法：1.结合在线数据库预测miR-550-1的潜在靶基因；2.通过TCGA、CALGB和已检测mRNA表达谱芯片结果确定miR-550-1潜在靶基因,并分析在AML患者中各靶基因的表达量与miR-550-1表达水平间的相关性；3.构建潜在靶基因3'UTR的luciferase荧光双报告载体,对靶基因与miR-550-1的结合序列进行鉴定；4.利用qPCR、Western blot方法对miR-550-1过表达细胞株中靶基因在RNA和蛋白质水平上的表达情况进行验证；5.克隆与miR-550-1表达负性相关靶基因的CDS区,构建其慢病毒过表达载体；6.利用靶基因的慢病毒载体在miR-550-1过表达细胞株中进行rescue实验；7.通过IPA软件分析靶基因间的相互联系,以及对下游信号通路的影响。结果：1.结合生物信息学网站靶基因预测结果、TCGA和CALGB数据库以及前期mRNA表达谱芯片结果,识别miR-550-1的四个潜在下游靶基因,分别为WWTR1、FZD5、SOX 11、PAX8;2.双荧光素酶报告实验发现较对照或突变序列而言,只有含WWTR1、FZD5、SOX11、PAX8正常3'UTR序列的质粒与miR-550-1共转后可降低荧光比值(P均0.05),通过Western blot. qPCR结果发现,过表达miR-550-1后可以降低VWTR1、FZD5、SOX11、PAX8的蛋白水平和mRNA水平；3.在过表达miR-550-1的Kasumi-1稳定细胞株中,过表达WWTR1靶基因后G0/G1期细胞比例较未过表达WWTR1组减少了9.1%,细胞活性增加,生长增快；4.IPA软件分析显示miR-550-1对下游Wnt信号通路的影响最为明显。结论：1. WWTR1、FZD5、SOX11、PAX8均为miR-550-1下游靶基因；2.miR-550-1通过抑制WWTR1、FZD5、SOX11、PAX8,可调控AML细胞株中Wnt信号通路的活性；3.过表达靶基因WWTR1可以部分逆转miR-550-1对AML细胞株生长抑制、周期阻滞的作用。
[Abstract]:The first part of the acute myeloid leukemia, the discovery of a new tumor suppressor microRNA - miR-550-1, analyzed its expression in AML patients and explored the aim of its upstream regulatory mechanism: to screen out the new tumor suppressor microRNAs (miRNAs) in acute myeloid leukemia (AML) and to analyze its expression. Methods: 1.Exiqon miRCURY LNA arrays points. The differential expression of miRNAs between 85 AML patients and 15 normal controls was analyzed; 2.TaqMan qPCR was used to identify the expression level of specific miRNA in AML patients and to verify the results of the chip; 3. analysis of the 194 AM in the TCGA database, the degree of CpG Isle methylation in the miR-550-1 promoter region of the patients and the negative control of the CpG island methylation degree in the miR-22 promoter region. 4. the AML cell line 96h was treated with 0,0.5,1,2,4 mu M with different concentration of demethylation drugs, and the changes of miR-550-1 expression after treatment were observed. Results: 1. Exiqon miRCURY LNA arrays chips were used to screen 12 low expressions in AML patients and high expression in normal controls. -29b, miR-184, miR-22, miR-342-3p, miR-423-5p, miR-550-1, miR-660, miR-768-3p, miR-768-5p (P values are less than 0.0001, false discovery rate is less than 0.0001). 2. from the screening results we selected as the research pair of images. The control was significantly down-regulated (P=0.003); 3. by analyzing the TCGA database, we found that the degree of methylation of CpG island in the miR-550-1 promoter region of the AML patients was significantly higher than that of CpG island in the miR-22 promoter region; 4. with 0,0.5,1,2,4 u M and different concentrations of the Kasumi-1 and MV4-11 cell 96h, the expression of the intracellular expression could be induced. The relative expression level was Kasumi-1 (1,1.19,1.18,1.14,1.29) and MV4-11 (1,3.30,7.33,20.15,13.96). Conclusion: 1. compared with normal control specimens, miR-550-1 expression in AML patients decreased significantly; CpG island in 2.miR-550-1 promoter region was in high methylation state; 3. using demethylation drug, citripara could promote AML fine. The second part observed the effect of miR-550-1 on the activity, proliferation, apoptosis, cycle, clone formation, and development of acute myeloid leukemia bone marrow transplantation model in acute myeloid leukemia: To explore the biological work of AML cell lines and medullary progenitor cells after miR-550-1 overexpression of miR-550-1 Methods: 1. construction of miR-550-1 retrovirus overexpression vector, PCR fixed-point mutation method to construct variant miR-550-1 retrovirus vector of miR-550-1 seed sequence; 2. using retrovirus infected AML cell line and mouse medullary progenitor cells to construct a miR-550-1 stable overexpressed cell line, in which the medullary lineage of mice is fine. MLL-AF9 or AE9a fusion gene was co transfected, and TaqMan qPCR was used to verify the success of miR-550-1 over expression of miR-550-1; 3. through MTT, trypan blue staining cells were used to detect AML cell activity and cell proliferation, and 4. flow cytometry was used to detect the cell cycle and apoptosis of AML cell lines, and 5.Western blot method was used to detect cell proliferation, cycle and apoptosis. The change of related signaling pathway protein; 6.Dot blot method to detect the change of m6A expression in cell; 7. mouse myeloid progenitor cell culture to detect cell clone formation ability; 8.Wright-Giemsa staining to detect cell morphology and differentiation; 9. mouse bone marrow transplantation model was used to detect miR-550-1 to the first generation, and the two generation transplanted mice AML disease hair. Results: the expression level of miR-550-1 in Kasumi-1, MV4-11, mouse medullary progenitor cells (P0.001) was significantly increased after 1. retroviral infection cells, and 2. in Kasumi-1, MV4-11, mouse myeloid progenitor cells could obviously inhibit cell viability and cell proliferation ability; 3Kasum I-1 cells overexpressed miR-. After 550-1,72h, the proportion of G0/G1 cells increased by 10.4%, the proportion of 96h apoptotic cells increased by 7.76%, PARP protein was activated, the expression of p-AKT and CDK6 protein was inhibited, and the expression level of m6A was suppressed; 4. co infected mouse medullary progenitors of miR-550-1 and AE9a gene were less than those of each generation of cells transfected with the fusion gene alone (P0.05). The total number of cells in the clones decreased significantly (P0.05) and the differentiated mature cells increased by generation. 5. in mice bone marrow transplantation, the total survival time of the miR-550-1 overexpressed group (median survival time of 77.5 and 105 days respectively) and the two generation (27 days and 33 days respectively) in the mice was compared with the control group. Lengthening (P values are 0.035 and 0.010 respectively). Conclusion: 1. overexpression of miR-550-1 can inhibit the growth of AML cells, reduce cell activity, promote cell apoptosis, induce G0/G1 arrest and inhibit m6A expression on RNA, and 2. overexpression of miR-550-1 can inhibit the formation of each generation of mouse progenitor cells co infected with MLL-AF9 or AE9a fusion genes. 3. overexpression of miR-550-1 could inhibit the occurrence and development of mouse bone marrow transplantation model AML, prolong the total survival time of mice, reduce the proportion of malignant primitive cells, promote the differentiation of mouse cells and reduce the load of tumor in mice. The third part explores the target gene of miR-550-1 and affects the development of acute myeloid leukemia. Molecular mechanism Objective: To explore the molecular mechanism of miR-550-1 in the development of acute myeloid leukemia in vitro and in vivo, namely, to realize its biological function by regulating which potential target genes to realize its biological function, and to verify the target gene function through rescue experiment. Method: 1. the potential target gene of miR-550-1 is predicted by online database; 2. through TCGA, CALGB The potential target gene of miR-550-1 was determined with the results of the detected mRNA expression chip, and the correlation between the expression of target genes and the level of miR-550-1 expression in AML patients was analyzed. 3. the luciferase fluorescent double reporting vector of the potential target gene 3'UTR was constructed, and the binding sequence of the target gene and miR-550-1 was identified, and 4. using qPCR, Western blo. T was used to verify the expression of target gene at the level of RNA and protein in miR-550-1 overexpressed cell lines; 5. clone and miR-550-1 expressed the CDS region of the negative related target gene, construct its lentivirus overexpression vector, and 6. use the lentivirus vector of the target gene to carry out rescue experiment in the miR-550-1 over the cell line, and 7. through IPA. The software analyses the interconnections between the target genes and the influence on the downstream signal pathways. Results: 1. the results of the target gene prediction of the bioinformatics website, the TCGA and CALGB database and the earlier mRNA expression chip results identify the four potential downstream target genes of miR-550-1, including WWTR1, FZD5, SOX 11, PAX8; 2. double Luciferase Report. It was found that only WWTR1, FZD5, SOX11, PAX8 normal 3'UTR sequences could reduce the ratio of fluorescence (P 0.05), and the results of Western blot. qPCR showed that the protein level and the level of PAX8 were reduced by Western blot. qPCR. In the Kasumi-1 stable cell line, after overexpressing WWTR1 target gene, the proportion of G0/G1 phase cells decreased by 9.1%, cell activity increased and growth increased faster than that of the unoverexpressed WWTR1 group; 4.IPA software analysis showed that miR-550-1 had the most obvious influence on the downstream Wnt signaling pathway. Conclusion: 1. WWTR1, FZD5, SOX11, PAX8 are the downstream target genes of miR-550-1. 0-1 by inhibiting WWTR1, FZD5, SOX11 and PAX8, the activity of Wnt signaling pathway in AML cell lines can be regulated, and 3. over expression of target gene WWTR1 can partly reverse the effect of miR-550-1 on the growth inhibition and periodic block of AML cell lines.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R733.71

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