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Fabbri E.,Section of Biochemistry and Molecular Biology | Borgatti M.,Section of Biochemistry and Molecular Biology | Montagner G.,Section of Biochemistry and Molecular Biology | Bianchi N.,Section of Biochemistry and Molecular Biology | And 7 more authors.
American Journal of Respiratory Cell and Molecular Biology | Year: 2014

In this study we analyzed the microRNA profile of cystic fibrosis (CF) bronchial epithelial IB3-1 cells infected with Pseudomonas aeruginosa by microarray and quantitative RT-PCR, demonstrating that microRNA 93 (miR-93), which is highly expressed in basal conditions, decreases during infection in parallel with increased expression of the IL-8 gene. The down-regulation of miR-93 after P. aeruginosa infection was confirmed in other bronchial cell lines derived from subjects with and without CF, namely CuFi-1 and NuLi-1 cells. Sequence analysis shows that the 3′-UTR region of IL-8 mRNA is a potential target of miR-93 and that the consensus sequence is highly conserved throughout molecular evolution. The possible involvement of miR-93 in IL-8 gene regulation was validated using three luciferase vectors, including one carrying the complete 3′-UTR region of the IL-8m RNA and one carrying the same region with a mutated miR-93 site. Up-modulation of IL-8 after P. aeruginosa infection was counteracted in IB3-1, CuFi-1, and NuLi-1 cells by pre-miR-93 transfection. In addition, IL-8 was up-regulated in uninfected cells treated with antagomiR-93. Our results support the concept of a possible link between microRNA expression and IL-8 induction in bronchial epithelial cells infected with P. aeruginosa. Specifically, the data presented here indicate that, in addition to NF-κB-dependent up-regulation of IL-8 gene transcription, IL-8 protein expression is posttranscriptionally regulated by interactions of the IL-8 mRNA with the inhibitory miR-93. Copyright © 2014 by the American Thoracic Society.

PubMed | Hebrew University of Jerusalem, University of Ferrara, Roadboud University Medical Center, University of Cape Town and 6 more.
Type: Journal Article | Journal: Human molecular genetics | Year: 2015

Congenital myopathies are genetically and clinically heterogeneous conditions causing severe muscle weakness, and mutations in the ryanodine receptor gene (RYR1) represent the most frequent cause of these conditions. A common feature of diseases caused by recessive RYR1 mutations is a decrease of ryanodine receptor 1 protein content in muscle. The aim of the present investigation was to gain mechanistic insight into the causes of this reduced ryanodine receptor 1. We found that muscle biopsies of patients with recessive RYR1 mutations exhibit decreased expression of muscle-specific microRNAs, increased DNA methylation and increased expression of class II histone deacetylases. Transgenic mouse muscle fibres over-expressing HDAC-4/HDAC-5 exhibited decreased expression of RYR1 and of muscle-specific miRNAs, whereas acute knock-down of RYR1 in mouse muscle fibres by siRNA caused up-regulation of HDAC-4/HDAC-5. Intriguingly, increased class II HDAC expression and decreased ryanodine receptor protein and miRNAs expression were also observed in muscles of patients with nemaline myopathy, another congenital neuromuscular disorder. Our results indicate that a common pathophysiological pathway caused by epigenetic changes is activated in some forms of congenital neuromuscular disorders.

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