本文選題：急性髓系白血病 + miR-550-1　； 參考：《浙江大學》2016年博士論文

【摘要】：第一部分急性髓系白血病中新的抑癌性microRNA——miR-550-1的發(fā)現(xiàn),分析其在AML患者中的表達水平,并探討其上游調控機制目的：篩選出急性髓系白血病(AML)中新的抑癌性microRNAs (miRNAs),并分析其表達情況。方法：1.Exiqon miRCURY LNA arrays分析85例AML患者與15例正常對照標本間差異表達的miRNAs; 2.TaqMan qPCR鑒定AML患者中特定miRNA的表達水平,驗證芯片結果；3.分析TCGA數據庫中194例AM,患者miR-550-1啟動子區(qū)CpG島甲基化程度,并利用miR-22的啟動子區(qū)CpG島甲基化程度作陰性對照；4.利用0、0.5、1、2、4μM不同濃度去甲基化藥物地西他濱處理AML細胞株96h,觀察處理后miR-550-1表達量改變的情況。結果：1. Exiqon miRCURY LNA arrays芯片篩選出了12個在AML患者中低表達而在正常對照中高表達的miRNAs,分別為：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,假發(fā)現(xiàn)率(false discovery rate, FDR)均小于0.0001)；2.從篩選的結果中我們挑選了miR-550-1作為研究對象,通過TaqmanqPCR檢測發(fā)現(xiàn)AML患者中miR-550-1的表達量較正常對照確實是顯著下調的(P=0.003)；3.通過分析TCGA數據庫,我們發(fā)現(xiàn)194例AML患者miR-550-1啟動子區(qū)CpG島較miR-22啟動子區(qū)CpG島的甲基化程度明顯升高；4.用0、0.5、1、2、4μM不同濃度地西他濱處理Kasumi-1和MV4-11細胞96h后,可誘導細胞內miR-550-1表達上調,相對表達水平分別為Kasumi-1 (1、1.19、1.18、1.14、1.29), MV4-11 (1、3.30、7.33、20.15、13.96).結論：1.與正常對照標本相比,AML患者中miR-550-1表達量顯著降低；2.miR-550-1啟動子區(qū)CpG島處于高度甲基化狀態(tài)；3.利用去甲基化藥物地西他濱可以促進AML細胞株中miR-550-1的表達。第二部分觀察miR-550-1對急性髓系白血病細胞活性、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發(fā)生發(fā)展的影響目的：探索外源性過表達miR-550-1后對AML細胞株及小鼠髓系祖細胞生物學功能的影響。方法：1.構建miR-550-1逆轉錄病毒過表達載體,PCR定點突變方法構建miR-550-1種子序列突變的變異體miR-550-1逆轉錄病毒載體；2.利用逆轉錄病毒感染AML細胞株和小鼠髓系祖細胞,構建miR-550-1穩(wěn)定過表達細胞株,其中小鼠髓系祖細胞還需共轉染MLL-AF9或AE9a融合基因,利用TaqMan qPCR驗證過表達miR-550-1是否成功；3.通過MTT、臺盼藍染色細胞計數檢測AML細胞活性和細胞增殖；4.流式細胞術檢測AML細胞株的細胞周期和細胞凋亡；5.Western blot方法檢測細胞增殖、周期、凋亡相關信號通路蛋白的變化；6.Dot blot方法檢測細胞內m6A表達水平變化；7.小鼠髓系祖細胞集落培養(yǎng)檢測細胞克隆形成能力；8.Wright-Giemsa染色檢測細胞形態(tài)及分化情況；9.小鼠骨髓移植模型檢測miR-550-1對一代、二代移植小鼠AML疾病發(fā)生發(fā)展的影響。結果：1.逆轉錄病毒感染細胞后可顯著升高Kasumi-1、MV4-11、小鼠髓系祖細胞內miR-550-1的表達水平(P0.001)；2.在Kasumi-1、MV4-11、小鼠髓系祖細胞中miR-550-1過表達后可以明顯抑制細胞活性和細胞增殖能力；3Kasum i-1細胞過表達miR-550-1,72h后G0/G1細胞比例增加10.4%,96h凋亡細胞比例增加7.76%,PARP蛋白激活,p-AKT和CDK6蛋白表達被抑制,m6A表達水平被抑制；4.共感染miR-550-1和MLL-AF9、AE9a融合基因的小鼠髓系祖細胞較單獨轉染融合基因的細胞每一代的克隆形成數要少(P0.05),克隆中細胞總量也顯著降低(P0.05),分化成熟的細胞逐代增加；5.在小鼠骨髓移植中,與對照組相比較,miR-550-1過表達組小鼠一代移植(中位生存時間分別為77.5天和105天)和二代移植(中位生存時間分別為27天和33天)的總體生存時間均顯著延長(P值分別為0.035和0.010)。結論：1.過表達miR-550-1后可抑制AML細胞生長,降低細胞活性,促進細胞凋亡,誘導細胞發(fā)生G0/G1期阻滯,抑制RNA上m6A表達；2.過表達miR-550-1后可抑制共感染MLL-AF9或AE9a融合基因的小鼠髓系祖細胞每代克隆形成能力,并誘導細胞分化；3.過表達miR-550-1可抑制小鼠骨髓移植模型AML的發(fā)生發(fā)展,延長小鼠總體生存時間,降低惡性原始細胞比例,促進小鼠細胞分化,減輕小鼠腫瘤負荷。第三部分探索miR-550-1的靶基因和影響急性髓系白血病發(fā)生發(fā)展的分子機制目的：探索miR-550-1在體內體外影響急性髓系白血病發(fā)生發(fā)展的分子機制,即通過調控哪些潛在靶基因而實現(xiàn)其生物學功能,并通過rescue實驗驗證靶基因功能。方法：1.結合在線數據庫預測miR-550-1的潛在靶基因；2.通過TCGA、CALGB和已檢測mRNA表達譜芯片結果確定miR-550-1潛在靶基因,并分析在AML患者中各靶基因的表達量與miR-550-1表達水平間的相關性；3.構建潛在靶基因3'UTR的luciferase熒光雙報告載體,對靶基因與miR-550-1的結合序列進行鑒定；4.利用qPCR、Western blot方法對miR-550-1過表達細胞株中靶基因在RNA和蛋白質水平上的表達情況進行驗證；5.克隆與miR-550-1表達負性相關靶基因的CDS區(qū),構建其慢病毒過表達載體；6.利用靶基因的慢病毒載體在miR-550-1過表達細胞株中進行rescue實驗；7.通過IPA軟件分析靶基因間的相互聯(lián)系,以及對下游信號通路的影響。結果：1.結合生物信息學網站靶基因預測結果、TCGA和CALGB數據庫以及前期mRNA表達譜芯片結果,識別miR-550-1的四個潛在下游靶基因,分別為WWTR1、FZD5、SOX 11、PAX8;2.雙熒光素酶報告實驗發(fā)現(xiàn)較對照或突變序列而言,只有含WWTR1、FZD5、SOX11、PAX8正常3'UTR序列的質粒與miR-550-1共轉后可降低熒光比值(P均0.05),通過Western blot. qPCR結果發(fā)現(xiàn),過表達miR-550-1后可以降低VWTR1、FZD5、SOX11、PAX8的蛋白水平和mRNA水平；3.在過表達miR-550-1的Kasumi-1穩(wěn)定細胞株中,過表達WWTR1靶基因后G0/G1期細胞比例較未過表達WWTR1組減少了9.1%,細胞活性增加,生長增快；4.IPA軟件分析顯示miR-550-1對下游Wnt信號通路的影響最為明顯。結論：1. WWTR1、FZD5、SOX11、PAX8均為miR-550-1下游靶基因；2.miR-550-1通過抑制WWTR1、FZD5、SOX11、PAX8,可調控AML細胞株中Wnt信號通路的活性；3.過表達靶基因WWTR1可以部分逆轉miR-550-1對AML細胞株生長抑制、周期阻滯的作用。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R733.71

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