MicroRNA (miRNA) is an important regulator of cellular proliferation, differentiation and death. Leukemia-specific signature of miRNAs suggests that epigenetic dysregulation of miRNAs is important for leukemogenesis. We focused on the role of DNA methylation of miR-203 which targets BCR-ABL1 mRNA. The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment. Imatinib induced the demethylation of the miR-203 promoter region, resulting in low expression of targeted BCR-ABL1 gene, and loss of proliferation of leukemic cells. In conclusion, demethylation of miR-203 is one of the molecular mechanisms of imatinib-induced inhibition of BCR-ABL1-positive leukemic cells.

Background: MicroRNAs (miRNA) are expected as useful biomarkers for various diseases. We studied the pre-analytical factors causing variation in the analysis of miRNA.Material and Methods: Blood samples were collected from 25 healthy subjects. Plasma and serum were obtained from the same samples. The levels of miR-451, -16, -126, and -223 were analyzed using RT-qPCR. Cel-miR-39 was added as a spiked-in control in each sample.Results: With the exception of miR-451, the levels of the miRNAs in plasma were higher than in serum. After high-speed centrifugation, the levels of miRNAs were almost equal between plasma and serum except for miR-451. Membrane filtration with 0.45 µm pore size reduced the levels of plasma miRNAs. The coagulation accelerators for serum processing did not affect the analysis of miRNA. The use of fraction containing particles of > 0.45 µm in size showed the inhibitory effect on the analysis of plasma miR-451. The RNase inhibitor was effective for protecting against the degradation of miRNAs.Conclusion: Plasma contains factors modifying miRNA profiles. The immediate processing of plasma with membrane filtration and RNase inhibitor may be a relevant method for achieving the stable analysis of miRNA

No abstract

4042 Poster Board III-977 Background MicroRNA (miRNA) is noncoding short RNA which plays important roles for cell death, proliferation, development and differentiation. MiRNA binds to 3' UTR region of target messenger RNA(mRNA), and negatively regulates gene expressions by inducing instability of mRNAs or translation block. This function of miRNA depends on the presence of cytoplasmic protein complex, RNA induced silencing complex(RISC). A main component of RISC is argonoute2(Ago2), of which deficiency or knockout results in severe cellular dysfunction. We and several groups reported erythroid-specific expressions of miRNAs(Masaki et al., BBRC 346:509-514, 2007). Among those miRNAs, miR-451 is especially specific to erythroid cells. Knockout of miR-451 results in reduction of red blood cell(RBC) production, and a high amount of miR-451 is contained in RBCs which are enucleated, and previously seem to be genetically inactive. Roles of miRNAs in mature RBCs for the biology of RBC are entirely unclear. In this study, we analyzed amounts of miRNA-Ago2 complexes in normal human RBCs. Materials & Methods Circulating blood cells were obtained from 5 normal subjects, and fractionated using density centrifugation and ultracentrifugation methods. RNA was isolated directly from each cell fraction, or isolated after immunoprecipitation by anti-Ago2 antibody(Wako, Osaka, Japan). MiRNA was measured by reverse transcriptase-based quantitative real time polymerase chain reaction(RT-qPCR), using microRNA assay kits(ABI). Results Packed RBCs contained miR-451 at the concentrations of 10,000 folds higher than granulocytes and miR-16 at the same level, whereas miR-155, -152, -221, -223 were contained at very low levels. After ultracentrifugation of circulating RBCs, packed RBCs were fractionated into 4 fractions:buffy coat cells(BFC), upper, middle, lower fractions of packed RBC(BFC, UF, MF, LF, respectively). Numbers of leukocytes were 21.9 and 0.88 per 104 RBCs in BFC and UF, respectively. They were low in MF and LF, 0.20 and 0.10. Coexisting reticulocytes in each fractions were 15.3, 5.50, 3.75 and 1.75 per 106 RBCs in BFC, UF, MF, LF, respectively. We used let-7a as an internal control for RT-qPCR assays, since expression of let-7a was relatively constant between 4 fractions. Analysis of total RNA isolated directly from each fraction showed constantly high expression of miR-16 and -451 in RBCs from each fraction. Analysis of immunoprecipitated miRNAs showed that the almost same amounts of miR-16 and -451 were present as miRNA-Ago2 complex in RBCs from the lower fraction(mature RBCs). Discussion & Conclusion We report, for the first time, the presence of miRNA-RISC complex in mature RBCs. Ratio of miRNA bound to Ago2 in total miRNAs is depend on species of miRNA. Our observation showed that almost all miRNA in mature RBCs is present as a miRNA-RISC complex. This finding clearly suggests that miRNAs in mature RBCs are active molecules, and also suggests that enucleated mature RBCs are still using epigenetic controlling system for some biological purpose. The well-known target gene of miR-16 is BCL2. There is a possibility that miR-16 negatively regulates expression of immortalize gene, BCL2, resulting in suppression of proliferation, and in keeping a differentiated state. On the other hand, the target gene of miR-451 is not well determined. Experiments using Zebra fishes showed knockout of miR-451 induced severe reduction of RBC production, suggesting that miR-451 is one of key molecules for erythropoiesis. In conclusion, our observations suggest that miRNA-Ago2 complexes in mature RBCs may be playing roles for homeostasis of normal erythropoiesis, and the analysis of miRNAs-Ago2 complex in RBCs is relevant for understanding normal and pathological erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

Background MicroRNAs (miRNAs) are short noncoding RNAs regulating a variety of biological processes by post-transcriptionally silencing via targeting mRNA. Recently there are many reports demonstrating that epigenetic alterations correlate to the characteristics of tumor cells, and that miRNAs were also reported to be regulated by methylation of CpG islands within the promoter region. MiR-203 is epigenetically silenced in human BCR-ABL1-positive leukemic cell lines and primary chronic myelogenous leukemia (CML) cells by the methylation of promoter region. In this study, we analyzed the effect of imatinib, a tyrosine-kinase inhibitor specific to BCR-ABL1 protein, on the expression of miRNA in BCR-ABL1-positive cells. Materials & Methods Two CML cell lines (K562 and KU812) and one AML cell line (HL60) were treated with imatinib for 72 hours. Microarray analysis of miRNAs was conducted by 3D-Gene (TORAY) in K562 cells with/without imatinib. Methylation specific PCR and bisulfite direct sequencing was performed to evaluate methylation status of promoter region of miR-203. Validation of expressions of miRNAs, including miR-203, and mRNAs was analyzed by RT-qPCR. The expression of BCR-ABL protein was confirmed by Western blotting. The function of miR-203 for cell survival was evaluated by the transfection of anti-miRNA. Results The microarray analysis showed that 48 miRNAs of CpG-rich 212 miRNAs were upregulated over 2-fold after imatinib treatment, in K562 cells. The demethylated state of the promoter region of miR-203, one of 48 miRNAs, was confirmed by bisulfite direct sequencing. The expression of BCR-ABL mRNA, which is one of the target of miR-203, was inhibited with imatinib to 52% and 26% of the level in control cultures in K562 cells and KU812 cells, respectively. The expression of BCR-ABL protein was also inhibited. The addition of anti-miR-203 increased the expression level of BCR-ABL protein to 68.1% in the K562 cell culture with imatinib treatment. The expression of DNA methyltransferase (DNMT) mRNA was analyzed, and the expressions of DNMT1 and DNMT3B were significantly decreased after imatinib treatments in CML cell lines, whereas the expression of DNMT3A was not changed. Discussion & Conclusion We report, for the first time, that imatinib up-regulated miR-203 by inducing demethylation of the promoter region of miR-203 in CML cells. MiR-203 is the important miRNA to inhibit ABL1 and BCR-ABL1 mRNA, and imatinib-induced demethylation of miR-203 is the possible mechanism to suppress growth of BCR-ABL1-positive leukemic cells. It was suggested that the demethylation was partially caused by down regulation of DNMT1 and DNMT3B after imatinib treatments in CML cell lines. In conclusion, imatinib not only inhibits the activity of tyrosine kinase but also induces DNA demethylation of miR-203 in CML cells. Disclosures: No relevant conflicts of interest to declare.