Investigation of regulatory mechanism of homeostasis using functional nucleic acids.

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Osteopontin(OPN) is a member of extracellular matrix proteins which regulate a variety of cell functions including adhesion, migration, tumor cell invasion, and inflammatory responses. OPN binds to the various cell surface receptors, integrins and CD44. We have previously demonstrated that the neutralizing anti-OPN antibody could ameliorate rheumatoid arthritis and concanavalin A(ConA)-induced hepatitis in murine models and thus OPN could be a potential therapeutic target for various inflammatory diseases. Recently, a technique known as RNA interference (RNAi) has been successfully adapted to mammalian cells so that RNAi can be a powerful tool to silencing gene expression and function. Therefore, we determined whether small interfering RNA targeting OPN (OPN siRNA) can be useful for disease control. First, we demonstrated the silencing effect of OPN siRNA against exogeneous and endogeneous OPN expression. Next, we evaluated OPN siRNA's potential to treat or prevent OPN-mediated disease … More s. In Matrigel^<TM> assay, invasion of mouse Lewis lung carcinoma and HT1080 human fibrosarcoma cells was significantly inhibited by OPN siRNA. Using a ConA-induced hepatitis model that is a well-characterized murine model of T cell-mediated liver diseases, we have shown that OPN siRNA pretreatment could inhibit the liver tissue damage as reflected by the serum ALT levels. Importantly, serum ALT levels well correlated with the liver OPN mRNA expression levels. These findings suggest that silencing OPN expression with OPN siRNA may represent a new approach for the treatment of tumor invasion and inflammatory liver diseases. To improve the siRNA function targeting OPN mRNA, we constructed a series of novel siRNAs to which various chemical modifications were introduced. There was only one siRNA molecule, which showed the inhibitory effect comparable to the standard siRNA although most of the siRNA complexes containing the chemical modifications negatively functioned on the suppression effect. Furthermore, we carried out oligonucleotide array analysis to investigate comprehensive gene expression profile associated with siRNA treatment. It was found that the expression profiles of some genes specifically changed in response to the siRNA treatment. Less