Supplementary Materials1. APL individual specimens. RNAi-mediated attenuation of miR-181a/b expression in NB4 cells was sufficient to reduce colony forming capacity, proliferation and survival. Mechanistic investigations revealed that miR-181a/b targets the ATRA-regulated tumor suppressor gene RASSF1A by direct binding to its 3UTR. Enforced expression of miR-181a/b or RNAi-mediated attenuation of RASSF1A inhibited ATRA-induced granulocytic differentiation via regulation of the cell cycle regulator cyclin D1. Conversely, RASSF1A overexpression enhanced apoptosis. Lastly, RASSF1A levels were reduced in PML/RAR knock-in mice and APL patient samples. Taken together, our results define miR-181a and miR-181b as oncomiRs in PML/RAR-associated APL, and they reveal RASSF1A as a pivotal element in the granulocytic differentiation program induced by ATRA in APL. Introduction Acute promyelocytic leukemia (APL) is usually characterized by specific chromosomal translocations involving the retinoic acid receptor TP-434 ic50 (RAR), (1,2). The most frequent translocation fuses the RAR with the promyelocytic leukemia protein (PML) gene (3). At physiological levels of retinoids, the PML/RAR Smoc2 fusion protein causes block of differentiation and neoplastic transformation by disrupting the function of PML and repressing transcription TP-434 ic50 of genes regulated by RAR (2,4,5). Pharmalogical doses of retinoids can overcome this block, lead to the expression of granulocytic specific transcription factors like C/EBP (6) and thereby induce terminal differentiation of APL blasts and (1,2). Recent studies recognized a group of small molecules that are involved in posttranscriptional regulation of gene expression. MicroRNAs (miRNAs) are endogenous, non-protein coding small RNAs which play crucial functions in the post-transcriptional regulation of target genes by direct targeting of mRNAs for cleavage, translational repression or destabilization (7). A selected quantity of miRNAs has been shown to play important functions in hematopoietic differentiation (8) as well as in the formation and maintenance of leukemia (9). We as well as others already showed that miR-223, miR-34a and miR-30c are important factors in myeloid differentiation (10-13). While some miRNAs like miR-223 have been implied in TP-434 ic50 APL differentiation (14) and tumorigenesis, there is still a lack of knowledge about the expression and function of other miRNAs. In this study, we showed that this genomic clustered miR-181a and miR-181b (miR-181a/b) are highly expressed in APL and downregulated during ATRA-induced differentiation (14-16). By analyzing APL and AML patient samples as well as PML/RAR knock-in mice, we exhibited that miR-181a and miR-181b display a very specific PML/RAR-dependency test to determine statistical significance of experimental results. A and and (Fig. 1D,K,L). Furthermore, we demonstrate that cytostatics and arsenic trioxide which are typically used in APL therapy and predominately inducers of apoptosis does not impact miR-181a/b expression (Physique 1I,J). These results expand and confirm previous observations (10,14-16) and suggest a specific role for the miR-181 family in the response to ATRA in APL. Diverse publications illustrate the expression pattern and define multiple functions for miR-181a and miR-181b in hematopoiesis and leukemia, whereas miR-181c and miR-181d are less explained (8,27-31). The fact that ATRA prospects to the degradation of PML/RAR and thereby changes gene expression let presume that miR-181a/b expression is dependent on PML/RAR (1). We followed miR-181a/b expression upon ATRA-treatment of the non-APL cell lines U937 and HL60. Both cell lines respond to ATRA, but show no significant switch in miR-181a/b TP-434 ic50 expression (Fig. 1E,G,F,H). This observation substantiates the proposed PML/RAR-dependency of miR-181a/b expression. The miR-181a/b-cluster has been shown TP-434 ic50 to be upregulated in AML patients with C/EBP-mutations which have a favorable prognosis and to be associated with favorable outcome in patients with cytogenetically normal AML and cytogenetically abnormal AML (32-34). Combining these data, high expression of miR-181a and miR-181b occurs in combination with a favorable end result of AML. In APL, a combination of ATRA and arsenic trioxide therapy generates a complete remission rate (CR) of over 90% (35). Our observation that this miR-181a/b-cluster is highly expressed in APL and significantly downregulated upon ATRA-treatment points to a role for the microRNA cluster as prognostic marker in t(15;17). Beside its function as transcriptional repressor (2), PML/RAR is also able to induce transcription, whereas this effect seems to be indirect due the sequestration of corepressors (36). In this study, we demonstrate the.