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MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression by inducing RNA cleavage or translational inhibition. Most human miRNAs are intragenic and are transcribed as part of their hosting transcription units. We hypothesized that the expression profiles of miRNA host genes and of their targets are inversely correlated and devised a novel procedure, HOCTAR (host gene oppositely correlated targets), which ranks predicted miRNA target genes based on their anti-correlated expression behavior relative to their respective miRNA host genes. HOCTAR is the first tool for systematic miRNA target prediction that utilizes the same set of microarray experiments to monitor the expression of both miRNAs (through their host genes) and candidate targets. We applied the procedure to 178 human intragenic miRNAs and found that it performs better than currently available prediction softwares in pinpointing previously validated miRNA targets. The high-scoring HOCTAR predicted targets were enriched in Gene Ontology categories, which were consistent with previously published data, as in the case of miR-106b and miR-93. By means of overexpression and loss-of-function assays, we also demonstrated that HOCTAR is efficient in predicting novel miRNA targets and we identified, by microarray and qRT-PCR procedures, 34 and 28 novel targets for miR-26b and miR-98, respectively. Overall, we believe that the use of HOCTAR significantly reduces the number of candidate miRNA targets to be tested compared to the procedures based solely on target sequence recognition. Finally, our data further confirm that miRNAs have a significant impact on the mRNA levels of most of their targets.

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

A kindred with hereditary spherocytosis and beta-thalassaemia trait was identified. Detailed studies of the red cell membrane proteins on polyacrylamide gels with sodium dodecyl sulphate (SDS-PAGE) demonstrated the presence of band 3 (anion transporter) deficiency in all HS subjects (20-25% reduction) whereas spectrin content was in the normal range. The molecular defect of beta thalassaemia in this kindred was due to a beta(0) codon 39 (C-T) mutation, as assessed by beta globin gene amplification and ASO-probe hybridization. Seven subjects of this family were studied: two were normal, two had HS alone, two co-inherited HS and beta-thalassaemia trait, and one had beta-thalassaemia trait only. The two subjects with HS alone had a typical clinical form of spherocytosis with anaemia, reticulocytosis and increased red cell osmotic fragility. The two with both HS and beta-thalassaemia trait were not anaemic and showed a small, well-compensated haemolysis. Hence the finding of red cells with abnormalities of both HS and beta-thalassaemia indicates that beta-thalassaemic trait 'silences' HS caused by band 3 deficiency.

Background The BIO14.6 hamster is an excellent animal model for inherited cardiomyopathy, because of its lethal and well-documented course, due to a spontaneous deletion of delta-sarcoglycan gene promoter and first exon. The muscle disease is progressive and average lifespan is 11 months, because heart slowly dilates towards heart failure. Methodology/Principal Findings Based on the ability of adeno-associated viral (AAV) vectors to transduce heart together with skeletal muscle following systemic administration, we delivered human delta-sarcoglycan cDNA into male BIO14.6 hamsters by testing different ages of injection, routes of administration and AAV serotypes. Body-wide restoration of delta-SG expression was associated with functional reconstitution of the sarcoglycan complex and with significant lowering of centralized nuclei and fibrosis in skeletal muscle. Motor ability and cardiac functions were completely rescued. However, BIO14.6 hamsters having less than 70% of fibers recovering sarcoglycan developed cardiomyopathy, even if the total rescued protein was normal. When we used serotype 2/8 in combination with serotype 2/1, lifespan was extended up to 22 months with sustained heart function improvement. Conclusions/Significance Our data support multiple systemic administrations of AAV as a general therapeutic strategy for clinical trials in cardiomyopathies and muscle disorders.