way mTOR signaling pathway N-Glycan biosynthesis NOD-like receptor signaling pathw. Oxidative phosphorylation Pentose phosphate pathway Phagosome Phosphatidylinositol signaling sys. PPAR signaling pathway Propanoate metabolism Proteasome Protein export Purine metabolism Pyrimidine metabolism Pyruvate metabolism Regulation of actin cytoskeleton RNA degradation RNA polymerase SNARE interactions in vesi. trans. Spliceosome Steroid biosynthesis Tight junction Toll-like receptor signaling 24900801 pathway Ubiquitin mediated proteolysis Valine, leucine and isoleucine deg. Vasopressin-regulated water reab. MiRNA Expression and Function in Pediatric AML Pathways with highest enrichment Pathway Name VEGF signaling pathway Wnt signaling pathway Ago1 KASUMI-1 NB4 SNB19 Pathways 11741928 with highest signal intensity KASUMI-1 Ago1 NB4 SNB19 Pathways were also compared to data from unrelated astrocytoma cell line SNB19 to determine AML specific pathways associated with miRNA regulatory machinery. Significance levels: p,0.05 = ; p,0.01 = ; p,0.001 = . doi:10.1371/journal.pone.0056334.t003 identified in Ago complexes in the KASUMI-1 cell line by our study, indicating that it may not be a true target in the KASUMI-1 cell context. We identified two other members of the MAP kinase pathway to be regulated by these miRNA clusters as complexed to the Ago2 and Ago3 proteins. TGF-receptor 2 and downstream apoptosis signal regulated kinase 1 of the p38 MAP kinase pathway are both complexed together and have a binding site for miR-23a/b, but not for the other two members of the primary transcript, miR-24 and miR-27. Overall, we extended previous profiling studies in pediatric AML identifying four different subgroups based upon miRNA expression and could identify previously described deregulated miRNAs between t- and t-positive samples as well as previously not recognized differentially expressed miRNAs. Moreover, we experimentally identified miRNAs and mRNAs associated with the Argonaute complexes using a modified PARCLIP-Array method. This is the first time, 
this technique has been applied to AML cells and provides insights into the complexity of regulation of AML-relevant pathways by concerted action of different Ago proteins. In addition, this work gives the scientific community a reliable experimental resource for future functional PF-04447943 custom synthesis testing of single miRNA-mRNA function in core-binding factor AML and promyelocytic AML, since we avoided artifacts by using native cell lines that do not over express tagged Argonaute proteins. selected AML-relevant pathways. Ago-associated mRNAs and miRNAs were mapped upon pathways from the KEGG database. The drawing was adapted from the KEGG database. Boxes delineate gene products of mRNAs identified from Ago-complexes in KASUMI-1, NB4 or both cell lines. Next to the gene products little squares indicate the amount of mRNAs associated with the Ago-proteins and identified in total RNA in the respective color as indicated in the figure. miRNAs with sequence similarity were summarized in sequence groups or families and miRNAs with binding sites to the respective mRNA are given. Sequence group names are preceded by a dot if miRNAs were identified as differentially expressed between t- samples and t samples compared to all others in our pediatric AML patients cohort. Supporting Information MLL-rearranged patient samples. The PAM algorithm selected significantly regulated miRNAs as class identifiers from patient samples with all cytogenetic subtypes
