The Anti-tumor Function And Mechanism Of MiR-550-1 In Acute Myeloid Leukemia

Part I Discovering the new tumor-suppressor miRNA, miR-550-1, analyzing its expression level and upstream regulatory mechanism in AMLPurpose:Screening new tumor suppressor microRNAs (miRNAs) and analyzing their expression level in AMLMethods:1.Exiqon miRCURY LNA arrays analyze the different expression level of miRNAs between 85 AML patients and 15 normal controls; 2.Confirming arrays’ results by TaqMan qPCR; 3.Using miR-22 promoters’CpG island as negative control and analyzing the methylation level of miR-550-1 promoters’CpG island in 194 AML patients’sample of TCGA database; 4.Detecting the expression level of miR-550-1 after treating AML cell with 0,0.5,1,2,4μM Decitabine for 96h.Results:1.According to Exiqon miRCURY LNA arrays,12 miRNAs which were down-regulation in AML patients compared to normal control were screened out, as follows: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＜0.0001, FDR<0.0001); 2.MiR-550-1 was selected as the research object. MiR-550-1 was down-regulation, which was further confirmed by Taqman qPCR (P=0.003) in AML patients; 3.By analyzing TCGA database, we found the methylation level of miR-550-1 promoter CpG island in 194 AML samples was positive compared with the promoter CpG island of miR-22; 4.After treating Kasumi-1 and MV4-11 with 0,0.5,1,2,4μM decitabine for 96h, the expression level of miR-550-1 could be increased 1.19,1.18,1.14,1.29 and 3.30,7.33,20.15,13.96 fold, respectively.Conclusion:1.miR-550-1 is down-regulation in AML patients compared with normal control; 2.The promoter’s CpG island of miR-550-1 is high methylation compared with negative control; 3.The demethylation reagent could induce up-regulation of miR-550-1 in AML cell lines.Part Ⅱ Detecting the impacts of miR-550-1 for cell activity, cell proliferation, cell apoptosis, cell cycle, colony formation in AML and the development of AML mice modelPurpose:Exploring the effect of exogenous overexpression of miR-550-1for biological function in AML cell lines and mouse myeloid progenitor cellMethods:1.Constructing the normal and mutant miR-550-1 overexpression retrovirus plasmid; 2.Constructing stable miR-550-1 overexpression cell line by retrovirus and confirming the overexpression effect by TaqMan qPCR; 3.Examining the influence of aberrant overexpression miR-550-1 for AML cell vability and proliferation by MTT and trypan blue counting; 4.Testing cell cycle and cell apoptosis by flow cytometry; 5.Checking cell growth, cell apoptosis and cell cycle relative signal pathways’changing by Western blot; 6.Testing m6A expression level by Dot blot method; 7.Detecting mouse myeloid progenitor cell’s colony ability by colony formation assay; 8.Checking cell morphology and differentiation status by Wright-Giemsa staining; 9.Testing impact of ectopic expression miR-550-1 for developing AML in mouse primary and secondary bone marrow transplant models.Results:1.After infecting Kasumi-1, MV4-11, mouse myeloid progenitor cell with virus retro virus, miR-550-1 was significantly increased (P<0.001); 2. Forced expression of miR-550-1 greatly inhibited cell viability and proliferation; 3.The G0/G1 cell ratio increased 10.4% after infecting 72h, and the proportion of apoptotic cells increased 7.76% after infecting 96h, PARP was active, p-AKT, CDK6 and m6A expression levels were inhibited; 4.Co-infection miR-550-1 and MLL-AF9, AE9a fusion gene into mouse myeloid progenitor cells could suppress the colony number and cell number compared to only infecting fusion gene (P<0.05) and lead to cell differentiation and maturation; 5. For mouse primary (median survival time 105 vs.77.5 days) and secondary (median survival time 33 vs.27 days) bone marrow transplant, ectopic miR-550-1 group could prolong the over survival time compared with MLL-AF9 group.Conclusion:1.Forced expression of miR-550-1 could inhibit cell growth, decrease cell viability, promote cell apoptosis, induce G0/G1 arrest and repress m6A expression level; 2.Ectopic expression of miR-550-1 repressed mouse myeloid progenitor cell colony formatting ability and caused cell differentiation; 3.Aberrant expression of miR-550-1 might postpone AML development, extend survival time, decrease proportion of blast cell, promote leukemia cell differentiation and reduce the tumor burden in mouse bone marrow transplant model.Part III Exploring the anti-tumor molecular biological mechanism of miR-550-1 in AMLPurpose:Finding the molecular mechanism of miR-550-1 for inhibiting AML in vitro and in vivo and selecting potential target geneMethods:1.Screening target gene by online target gene database; 2.Combining TCGA database, CALGB database and in-house database results, further identified special target gene; 3.Confirming the target gene by luciferase assays, qPCR and Western blot; 4.Construction of overexpression target gene lentivirus vector and performing rescue experiment; 5.Through IPA software analyzed interrelation between the target gene, and the impact on downstream signaling pathways.Results:1.We identified four potential target genes, as follows:WWTR1, FZD5, SOX 11 and PAX8 by analyzing target gene online database, TCGA database and CALGB database; 2.Luciferase assays, qPCR and Western blot confirmed that miR-550-1 transcriptionally repressed WWTR1, FZD5, SOX11 and PAX8 by interacting with the essential binding sequence located in 3’UTR (P<0.05); 3. For the rescue experiment, overexpression WWTR1 could decrease 9.1% Gl phase cell, increase cell viability and promote cell growth in Kasumi-1; 4.Wnt signal pathway was the most apparent pathway which was regulated by miR-550-1 according to IPA results.Conclusion:1.WWTR1, FZD5, SOX11 and PAX8 were target genes of miR-550-1; 2.Ectopic expression of WWTR1 partly reversed miR-550-1 anti-tumor’s function in Kasumi-1; 3.Wnt signal pathway was the most apparently regulated and the downstream of miR-550-1.