MicroRNAs (miRNAs) constitute a class of small noncodingRNAs that play important roles in a variety of biological processes including development, apoptosis, proliferation, and differentiation. Here we show that the expression of miR-199a and miR-199a* (miR-199a/a*), which are processed from the same precursor, is confined to fibroblast cells among cultured cell lines. The fibroblast-specific expression pattern correlated well with methylation patterns: gene loci on chromosome 1 and 19 were fully methylated in all examined cell lines but unmethylated in fibroblasts. Transfection of miR-199a and/or -199a* mimetics into several cancer cell lines caused prominent apoptosis with miR-199a* being more pro-apoptotic. The mechanism underlying apoptosis induced by miR-199a was caspasedependent, whereas a caspase-independent pathway was involved in apoptosis induced by miR-199a* in A549 cells. By employing microarray and immunoblotting analyses, we identified the MET proto-oncogene as a target of miR-199a*. Studies with a luciferase reporter fused to the 3-untranslated region of the MET gene demonstrated miR-199a*-mediated down-regulation of luciferase activity through a binding site of miR-199a*. Interestingly, extracellular signal-regulated kinase 2 (ERK2) was also down-regulated by miR-199a*. Coordinated down-regulation of both MET and its downstream effector ERK2 by miR199a* may be effective in inhibiting not only cell proliferation but also motility and invasive capabilities of tumor cells.

In order to develop an anti-NF-kappaB siRNA as a novel class of anti-inflammatory drug, we have isolated a highly efficient siRNA targeting the p65 (RelA) subunit of NF-kappaB, hereafter named REL1096. To determine whether down-regulation of p65 by REL1096 can block the induction of inflammatory cytokines after treatment with tumor necrosis factor-alpha (TNF-alpha), human primary fibroblast-like synoviocytes were isolated from patients with rheumatoid arthritis. When treated with REL1096, the TNF-mediated induction of downstream target genes such as inflammatory cytokines, chemokines, and anti-apoptosis genes was drastically inhibited. To enhance the inhibitory effect of REL1096, cells were treated with siRNA targeting the p50 subunit of NF-kappaB together with REL1096. In addition to effective downregulation of inflammatory cytokines, knockdown of both p65 and p50 resulted in much more extensive apoptosis when compared to cells treated with either REL1096 or p50-siRNA alone. Thus, our results provide evidence for the potential use of siRNA targeting NF-kappaB as an effective means to treat rheumatoid arthritis. In addition to effective amelioration of synovial inflammation by downregulation of inflammatory cytokines, increased apoptosis by dual knockdown of p65 and p50 may prove advantageous in preventing invasiveness and destructiveness of hyperplastic synoviocytes.