2008 Rustbelt RNA Meeting
MicroRNAs (miRNA) are short 20-25 nucleotide RNA molecules that negatively regulate gene expression in animals and plants. We have developed a vector-based genetic library including minigenes for most known human miRNAs. This library provides a widely applicable resource for gain-of-function studies for miRNA and will significantly facilitate our studies to identify novel miRNA function. In this work, we have designed a dual-reporter method to investigate miRNA functions in the NF-ƒÛB signaling pathway in conjunction with our genetic library. A firefly luciferase gene (luc) under the control of a minimal CMV (mCMV) promoter, which is downstream of transcription response elements (TRE), is integrated into the chromosomes of a host cell. The mCMV promoter is only activated when a specific transcription factor (TF) (for example NF-kappaB) binds to the TRE. We use lentiviral-based TF vector to integrate the first reporter into the host chromosomes. The second reporter Renilla luciferase (Rluc) upstream of the 3'-UTR sequence from the TF gene is constitutively expressed. With miRNAs directly targeting the TF gene, both reporters will be down-regulated. However, if miRNA indirectly down-regulates the TF, only the first reporter expression will be affected. Similarly, miRNAs targeting any gene that modulates the TF function can be screened with its 3'-UTR downstream of the second reporter. Coupled with our vector-based lentiviral miRNA genetic library, we are able to investigate whether any miRNA is relevant to a signaling pathway rather than whether one miRNA down-regulates one particular gene. We have identified one miRNA from the above assay that upregulated NF-kappaB-dependent reporter expression the most. We then studied the target of this miRNA, which inhibits NF-kappaB activation. Moreover, the transcription of this miRNA is controlled by NF-kappaB. Disclosing this miRNA in the NF-kappaB pathway would present a gene regulatory mechanism for chronic or persistent NF-kappaB activation by TNF-alpha, in which the miRNA mediates cellular NF-kappaB signaling through a positive feedback loop. These results may also offer clues to NF-kappaB activation in some tumors and provide novel biomarkers for cancer diagnosis.
Keywords: microRNA, NF-kappaB, signal pathway