Functional Studies On MiRs Related To Acute Myeloid Leukemia

IntroductionHematopoietic stem cells (HSCs) have been studied for many years, which give rise to progenitors that become progressively restricted to several or single blood cell lineages. These progenitors are devoted to the production of mature blood cells, including red blood cells, megakaryocytes, granulocytes, monocyte/macrophage cells and lymphocytes. It is a very hot reseach field with potential applications that may change our lives. It's very important for haematopoiesis to develop and maintain life. There are two aspects on regulation of haematopoiesis:one regulation is self-renewal of hematopoietic stem cells, the other is how to control the differentiation and proliferation of hematopoietic stem cells. It's a very precise and complicated regulation involved in several genes "opening or closing", abnormal regulation will result in leukaemogenesis in general. Recently, many studies on miRNAs have shown that miRNA play a key role in haematopoiesis, including transformation of development stage, cell proliferation, differentiation, apoptosis and so on. It is related significantly with leukaemogenesis and metastasis.More and more scientists have engaged themselves in functional study of miRNAs involved in different differentiation stages, which are helpful for revealing such key projects as transforming normal stem cell to leukaemic cell, pathogenesis of leukemia and diagnosis and treatment of leukemia. In AML, the accumulation of leukaemic cells (also referred to as blasts) arises from a failure of myeloid progenitors to mature, and therefore AML are classified in subtypes based on the stage at which normal differentiation is blocked in the leukaemic cells. To provide insights into the complex genetic alterations in leukemogenesis, we performed a genome-wide bead-based miRNA expression analysis and selected minimal "interesting miRNAs" (for example, miR-17-92, miR-196b, miR-9) after statistical analysis. Potential targets of these "interesting miRNAs" were predicted by bioinformatics website (prediction programs) and verified by luciferase reportor assay. Proliferative and differentiative cell model were established in vitro. We performed forced expression of miR-17-92, miR-196b, miR-9 to examine the function about proliferation and differentiation.Objectives1. To investigate the miRNA expression profiles in bone marrow mononuclear cell from AML sample and normal control, and identify the specific miRNAs in AML2. To verify targets of the miRNAs in leukemogenesis, and investigate the roles of miRNA in the regulation of proliferation, differentiation and apoptosis in mouse progenitor cell and AML cell line, which can provide a basic for discussing the roles of miRNAs in the pathogenesis of AML.Methods1. Bead-based miRNA expression profiling assay (Exiqon 10.0) and gene array were performed, and TIGR Mutiple Array Views software package (TMeV version 4.0) was used to perform data analysis to identify the miRNAs and genes correlated with leukemogenesis of AML. Combined with data for analysis of gene array, targets of differential expression miRNAs in AML were predicted by prediction program (TargetScan, PITA,et al) 2. Cloning mir genes by PCR assay and constructing in retroviral expression vector.3. Luciferase reportor assay was performed to validate the targets of "interesting miRNAs" (miR-17-92, miR-196b, miR-9).4. Colony-forming/replating assays was conducted to identify colony forming capacity of mouse normal bone marrow progenitor cells and proliferation5. To observe the effect of proliferation and apoptosis on Hela,293T cell and mouse progenitor cell after forced expression of mir-17-92, mir-196b, mir-9 or their combination.6. Establishment of THP1 cell model on differentiation induced by PMA, expression rate of CD11b, CD 14 marker by FACs and Morphological changes of THP-1 cells were used for evaluating differentiation of THP1 cell7. Detection of relative expression of miR-17-92, miR-196b and miR-9 in THP1 cell differentiation model and to observe the effect on differentiation of THP1 cell by overexpression of these three mir genes at time point of 48h (adding PMA)8. Based on the potential target genes of mir-17-92, mir-196b, mir-9, analysis of GO processes and pathways were conducted with GeneGo MetaCore software.Results1.112 miRNAs abnormally expressed in AML were selected by bead-based miRNA expression profiling assay,3 miRNAs (miR-17-92,miR-196b,miR-9)expressed in bone marrow mononuclear of AML sample were selected and further studied after minimizing number of "interesting miRNA " by data analysis of TIGR Mutiple Array Views software package (TMeV version 4.0) and ANOVA analysis. Potential targets of miR-17-92, miR-196b and miR-9 were BIM (BCL2L11) and PLK2, CDKN1B or TGFBI by luciferase reportor assay, respectively2.â‘ Forced expression of mir-17-92, mir-196b or mir-9 alone could result in a significant number (>700) of colonies, the number of colonies in every group was more than twice as many as the one in empty group (about 300). It showed these three miRNAs could enhance the colony forming capacity of mouse normal bone marrow progenitor cells and proliferation/development (P<0.05). Most of colonies were typeâ…¢. Morphological change showed a lot of immature cells were observed.â‘¡The number of colonies was sharply decreased after 2nd rounds of plating, but the decreasing tendency in the group of miR-9 or miR-9+miR196b was lower than others, and morphological change also showed these two groups still have some of immature cells.â‘¢he number of colonies in group of miR-17-92+miR196b or miR-9+miR196b was less than that in group of miR-17-92 or miR-9 alone (P<0.05).â‘£ith increasing of time and rounds of plating, the number of colonies decreased significantly (P<0.05) and came to zero at the end of 3rd round of plating.3.â‘ The relative fluorescence units(RFU) for viability or apoptosis in group of forced expression of the mir-17-92 or mir-196b are significantly higher than that in the control groups in Hela (a human cervical cancer cell line) and 293T (a variant of human embryonic kidney 293 cell line) cells (p<0.05).â‘¡Forced expression of mir-17-92 or mir-9 or mir-17-92 plus mir-9 in mouse normal bone marrow progenitor cells showed the similar results as in Hela and 293T cell.â‘¢after forced expression of mir-196b+mir-9/mir-17-92, inhibition of proliferation and anti-apoptosis for mouse progenitor cells were observed4. In general, expression rate of CD11b, CD 14 on THP1 cell increased with the time increased (F=10.084, P=0.003).FACs analysis revealed that expression rate of CD11b, CD 14 at the time point 48h,72h,96h was significantly higher than that in negative control(no PMA+THP1) (p<0.05),especially for CD11b. Morphological evidence indicated that the size of cell and nuclear became smaller, nuclear-cytoplasmic ratio (N/C ratio) decreased, nuclear depressed, presented bandformnucleus or leaflike shape.5. RNA at different time point appeared good band after RNA electrophoresis was performed and value of A260/A280 was between 1.9 to 2.1. With time increased, relative expression in group of miR-196b, miR-9 and miR-17-92 were all significantly decreased, comparing with that in group of negative control (no PMA) (p<0.05).Every group at time point 48h and 72h were the most significant.6.â‘ After forced expression of mir-9,mir-17-92 or mir196b for 48h, expression rate of CD11b, CD 14 in group mir-9,mir-17-92 or mir 196b were lower than that in negative groups(NO PMA or empty vector transfection).â‘¡orphological change in the groups of mir-9,mir-17-92 or mir196b was not more significant than that of negative control. The cells that had been found less in number were bandformnucleus or leaflike, and smaller in size, smaller nuclear, decreased nuclear-cytoplasmic ratio (N/C ratio) and depressed nuclear.Conclusions1. Expression of mir-17-92, mir-196b and mir-9 gene in AML were obviously up-regulated, suggesting these three miRs may involve in the pathogenesis of AML.2. Cloning a series of aberrant mir genes including mir-17-92, mir-196b and mir-9 and Construction of Recombinant Retroviral Vector (pMSCVneo/pMSCVPIG) Containing these mir gene (pre-miRNA), which provide a basis for functional study.3. Forced expression of mir-17-92, mir-196b and mir-9 alone can significantly increase colony forming capacity of mouse normal bone marrow progenitor cells and proliferation. However, Forced expression of mir-196b can inhibit significantly the enhancement of colony forming capacity of mouse normal bone marrow progenitor cells for overexpression of mir-17-92 or mir-9 gene.4. Forced expression of the mir-17-92 or mir-196b significantly inhibited apoptosis while significantly increasing viability of Hela and 293T cells(No data was provided for miR-9). Forced expression of mir-17-92 or mir-9 or mir-17-92 plus mir-9 in mouse normal bone marrow progenitor cells showed the similar results as in Hela and 293T cell. miR-196b cannot inhibited apoptosis and increase viability. However, miR-196b can inhibit the ability of proliferation and anti-apoptosis for mir-17-92 or mir-9.5. A differentiation model of THP1 cell induced by PMA was established successfully.6. miR-196b, miR-9 and miR-17-92 were all negatively correlated with differentiation of AML cell7. CDKN1B gene is the direct target of miR-196b, and miR-196b may play a role in cell cycle and leukemogenesis through negatively regulating CDKN1B involved pathway of cell proliferation or differentiation.