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Genes can be silenced with short-interfering RNA molecules (siRNA). siRNAs are widely used to identify gene functions and have high potential for therapeutic treatments. It is critical that the siRNA specifically targets the expression of the gene of interest but has no off-target effects on other genes. Although siRNAs were initially considered to be exclusively active on mature mRNAs in the cytoplasm, additional studies have shown that siRNAs are present in the nucleus as well, suggesting that pre-mRNA sequences containing introns and other untranslated regions can also be targeted. In this study, we investigated the extent to which off-targets may occur in Drosophila melanogaster by looking at mature mRNA sequences and pre-mature RNA sequences separately. First, an in silico approach revealed that, based on sequence similarity, numerous off-targets are predicted to occur in RNAi experiments. Second, existing microarray data were used to investigate a possible effect of the predicted off-targets based on analysis of in vitro data. We found that the occurrence of off-targets in both mature and pre-mature RNA sequences in RNAi experiments can be extensive and significant. Possibilities are discussed how to minimize off-target effects.

Pantothenate kinase–associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines. Ten to thirty percent of the gene transcripts from the genome were enriched in the Ago2-IP fraction of untreated cells, representing the HL miRNA-targetome. In silico analysis indicated that similar to 40% of these gene transcripts represent targets of the abundantly co-expressed miRNAs. To identify targets of miR-17/20/93/106, RIP-Chip with anti-miR-17/20/93/106 treated cells was performed and 1189 gene transcripts were identified. These genes were analyzed for miR-17/20/93/106 target sites in the 5'-UTRs, coding regions and 3'-UTRs. Fifty-one percent of them had miR-17/20/93/106 target sites in the 3'-UTR while 19% of them were predicted miR-17/20/93/106 targets by TargetScan. Luciferase reporter assay confirmed targeting of miR-17/20/93/106 to the 3'-UTRs of 8 out of 10 genes. In conclusion, we report a method which can establish the miRNA-targetome in untreated human cells and identify miRNA specific targets in a high throughput manner. This approach is applicable to identify miRNA targets in any human tissue sample or purified cell population in an unbiased and physiologically relevant manner.

Abstract 265 In Hodgkin Lymphoma (HL), the Hodgkin Reed-Sternberg (HRS) cells are a minority of large mono- or multi-nucleated B cells characterized by a loss of B cell phenotype, constitutive NF-kB activation, a disturbed cell cycle and anti-apoptotic features. In this study we investigated the role of deregulated miRNA expression in the pathogenesis of HL. MiRNA in situ hybridization (ISH) in HL tissue was performed to determine expression of miRNAs previously reported to be highly abundant in HL cell lines, in HRS cells. Next we identified the miRNA-targetome of two HL cell lines by immunoprecipitation of RISC in untransfected and transfected cell lines. miRNA ISH confirmed expression of miR-17-5p, miR-24, miR-106a, miR-146a, miR-150, miR-155, miR-181b and miR-210 in HRS cells. Ago2-immunoprecipitation followed by microarray analysis of the co-immunoprecipitated mRNA revealed that the miRNA-targetome of HL comprises of about 2,500 genes. Inhibition of the anti-miR-17 seed family revealed that about 500 of these genes are regulated by miRNAs of the miR-17 seed family. Gene ontology (GO) analysis for the total miRNA-targetome of HL showed a significant enrichment of genes involved in the regulation of cell cycle, apoptosis, immune system development and NF-kB cascade. The miRNA-targetome of HL contained several genes known to be mutated in HRS cells, including A20, FAS, NFKB1A, NFKB1E, PERP and SOCS1. Also, using previously reported gene expression data, we defined a set of genes downregulated in HL cell lines (L428 and L1236) compared to germinal center B cells (GCB) and compared them to the miRNA-targetome of the same cell lines. This resulted in the identification of 149 genes in L428 and 183 genes in L1236 that were subjected to miRNA mediated repression. Unexpectedly, only a few of all the reported inactivated genes in HRS cells that might contribute to loss of B cell phenotype (MYBL1 and CXCR4) were found to be regulated by miRNAs in HL. In conclusion, we confirmed the expression of miRNAs in the HRS cells of HL tissue and identified miRNA repressed genes in HL. Our data indicated that aberrant miRNA expression contributes to the deregulation of apoptosis, cell cycle, and NF-kB pathways but not loss of B cell phenotype in HL. Disclosures: No relevant conflicts of interest to declare.

MiRNAs are small regulatory RNAs which control gene expression at the post-transcriptional level. Tumor cells of Hodgkin lymphoma (HL), the so-called Hodgkin Reed-Sternberg (HRS) cells, originate from defective germinal center B cells. HRS cells are characterized by their loss of B cell phenotype, large multi-nucleated cell size, a disturbed cell cycle and resistance to undergo apoptosis. The aim of our study is to investigate the role of deregulated miRNA expression to the pathogenesis of HL. To identify genes which are regulated by miRNAs in a high throughput manner, an approach called Ribonucleoprotein ImmunoPrecipitation – microarray (RIP-CHIP) was applied. As a first step, the AGO2 protein, which is part of the RISC complex, is immunoprecipitated (IP) with an AGO2 specific antibody. RNA isolation of the IP fraction followed by microarray analysis thus leads to the identification of miRNA targets from the whole transcriptome. With this approach, 1255 genes were found to be commonly regulated by miRNAs in both L1236 and L428 HL cell lines. Genes known to be absent or down regulated in HRS cells, like CDKN1A, CDKN1B, and PRDM1, were included in this gene list. Gene ontology analysis of these candidate miRNA target genes using Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed a significant enrichment in KEGG pathways termed p53 signaling pathway and cell cycle. TargetScan prediction and seed sequence search at the 3′UTRs of these genes both indicated that about half of these genes were targets of the highly abundant miRNAs found in Hodgkin lymphoma cell lines. 10 randomly selected target genes were analyzed using luciferase reporter assays. For all genes, targeting by the predicted miRNA was confirmed using specific antisense Locked Nucleic Acids (LNA) inhibitors. In conclusion, we demonstrated an approach for large scale identification of endogenous miRNA targets without prior manipulation of the cells. HL specific target genes that may contribute to the characteristic phenotype of HRS cells and to the malignant transformation of germinal center B cells were identified.