2008 Rustbelt RNA Meeting
RRM

 

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Talk on Friday 06:10-06:30pm submitted by Zhongxin Lu

MicroRNAs in the NF-kappaB Pathway

Zhongxin Lu (Department of Biochemistry and Molecular Biology), Apana Takwi (Department of Biochemistry and Molecular Biology), Yanglong Zhu (Department of Biochemistry and Molecular Biology), Yong Li (Department of Biochemistry and Molecular Biology)

Abstract:
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-B 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