Objective. To elucidate the role of microRNA (miRNA) in the pathogenesis of rheumatoid arthritis (RA), we analyzed synoviocytes from RA patients for their miRNA expression.Methods. Synoviocytes derived from surgical specimens obtained from RA patients were compared with those obtained from osteoarthritis (OA) patients for their expression of a panel of 156 miRNA with quantitative stem-loop reverse transcription-polymerase chain reaction. The miRNA whose expression decreased or increased in RA synoviocytes as compared with OA synoviocytes were identified, and their target genes were predicted by computer analysis. We used an in vitro system of enhancing the expression of specific miRNA by transfection of precursors into synoviocytes, and then we performed proliferation, cell cycle, and apoptosis assays, as well as enzyme-linked immunosorbent assays for cytokine production. The effects of transfection on predicted target protein and messenger RNA (mRNA) were then examined by Western blot analysis and luciferase reporter assay.Results. We found that miR-124a levels significantly decreased in RA synoviocytes as compared with OA synoviocytes. Transfection of precursor miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G 1 phase. We identified a putative consensus site for miR-124a binding in the 3 -untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA. Induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. Luciferase reporter assay demonstrated that miR-124a specifically suppressed the reporter activity driven by the 3 -untranslated regions of CDK-2 and MCP-1 mRNA.Conclusion. The results of this study suggest that miR-124a is a key miRNA in the posttranscriptional regulatory mechanisms of RA synoviocytes.MicroRNA (miRNA) are a well-established class of small (ϳ22 nucleotides) endogenous noncoding RNAs that influence the stability and translation of messenger RNA (mRNA) (1). Using various computational and experimental approaches, hundreds of miRNA have been identified in numerous animal species. The miRNA genes are transcribed by RNA polymerase II as primary miRNA (pri-miRNA) (2,3). The RNase III enzyme Drosha then processes the nuclear pri-miRNA, yielding a ϳ70-nucleotide molecule known as precursor miRNA (pre-miRNA) (4), which is exported from the nucleus. Maturation of the pre-miRNA into miRNA is then mediated by the cytoplasmic enzyme Dicer (5), after which the mature miRNA is loaded into the RNA-induced silencing complex (RISC)
It is the first report that PGE(2) can induce articular chondrocyte apoptosis in vitro. It is also suggest that apoptosis of chondrocytes by PGE(2) is linked with cAMP-dependent pathway.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease that is characterized by muscle dystrophin deficiency. We report that intravenous (IV) infusion of an antisense oligonucleotide created an in-frame dystrophin mRNA from an outof-frame DMD mutation (via exon skipping) which led to muscle dystrophin expression. A 10-year-old DMD patient possessing an out-of-frame, exon 20 deletion of the dystrophin gene received a 0.5 mg/kg IV infusion of an antisense 31-mer phosphorothioate oligonucleotide against the splicing enhancer sequence of exon 19. This antisense construct was administered at one-week intervals for 4 wk. No side effects attributable to infusion were observed. Exon 19 skipping appeared in a portion of the dystrophin mRNA in peripheral lymphocytes after the infusion. In a muscle biopsy one week after the final infusion, the novel in-frame mRNA lacking both exons 19 and 20 was identified and found to represent approximately 6% of the total reverse transcription PCR product. Dystrophin was identified histochemically in the sarcolemma of muscle cells after oligonucleotide treatment. These findings demonstrate that phosphorothioate oligonucleotides may be administered safely to children with DMD, and that a simple IV infusion is an effective delivery mechanism for oligonucleotides that lead to exon skipping in DMD skeletal muscles.
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