MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate growth and differentiation. miRNAs are frequently located at cancer-specific fragile sites in the human genome, such as chromosome 7q. The nuclear oncogene SKI is up-regulated in acute myeloid leukemia (AML) with −7/del7q. Here we asked whether loss of miRNAs on chromosome 7q may explain this up-regulation. miR-29a expression was found to be down-regulated in AML with −7/del7q. Forced expression of miR-29a down-regulated Ski and its target gene, Nr-CAM, whereas miR-29a inhibition induced Ski expression. Luciferase assays validated a functional binding site for miR-29a in the 3′ untranslated region of SKI. Finally, in samples of AML patients, we observed an inverse correlation of Ski and miR-29a expression, respectively. In conclusion, up-regulation of Ski in AML with −7/del7q is caused by loss of miR-29a. miR-29a may therefore function as an important tumor suppressor in AML by restraining expression of the SKI oncogene.

1968 Poster Board I-991 Introduction: AML patients with deletion of chromosome 7 (−7) or deletion of 7q (−7q) have a poor prognosis. We have found that the nuclear oncogene SKI is overexpressed in AML, especially in AML with −7/−7q. SKI acts in AML as a repressor of retinoic acid induced myeloid differentiation (Ritter et al., (2006) Leukemia). As we found SKI up regulated in AML, we asked how SKI expression may be regulated. The aim of our study was to find a molecular background for increased SKI level. On chromosome 7 is a cluster of micro-RNAs (miRNAs) localized particularly around the fragile site 7q32 (Calin et al., (2003) PNAS). Therefore we investigated whether there exists a link between expression of miRNAs localized on chromosome 7 and up regulation of SKI expression in AML. Methods: We used micro RNA profiling analysis, FACS, Western blot, RQ-PCR and luciferase assays to determine the role of miRNA29a in regulating SKI expression. Results: We found that the expression of miRNA25, miRNA29a, miRNA183 and miRNA335 was downregulated in AML patients with -7/-7q. Transfection studies with these four miRNAs in HL60 cells revealed in FACS that miRNA29a inhibits SKI expression (60,4%) compared to nonsense control (100%) and other miRNAs (miRNA25: 91%, miRNA183: 101%, miRNA335: 93%). Western blot experiments confirmed that miRNA29a reduces SKI level in HL60 cells. In keeping, miRNA29a also represses expression of the SKI target gene Nr-CaM in IFB melanoma cells. Knock down of miRNA29a using miRNA29a inhibitor molecules induces SKI expression in the high miRNA29a and low SKI expressing cell line NW1539. Luciferase assays in NW1539 and HeLa transfected with 3′UTR-constructs and HeLa cells cotransfected with miRNA29a demonstrated that miRNA29a binds to 3′UTR of SKI in vitro. Furthermore, comparison of SKI and miRNA29a expression of AML patient samples indicates that miRNA29a expression is associated with low SKI level in vivo. Conclusion: Our data show that miRNA29a which is located on 7q32 regulates expression of the oncogene SKI in vitro and in vivo. We suggest the deletion of miRNA29a as mechanism for up regulation of SKI in AML with -7/-7q and thus propose that in AML, this effect may contribute to the tumor suppressive function of miRNA29a. Disclosures: No relevant conflicts of interest to declare.