From microrna sequencing to target validation: An inquiry into one data set
MetadataShow full item record
In this study, we aim to characterize microRNA expression and predict and verify microRNA target genes. We begin by analyzing short RNA high-throughput sequencing data. The high-throughput data is sequenced from RNA that was isolated from cells with constitutively active or inactive NF-kB. We then create a candidate list of microRNA's that we hypothesize to be differentially expressed as a result of NF-kB activation based on sequencing reads, and we confirm differential expression using real-time PCR in three candidate miRNA's, one of which is hsa-miR-497. Using a luciferase reporter assay, we confirm IKK-Beta as a target of hsa-miR-497 at the level of mRNA regulation. To validate has-miR-497's direct effect and regulation of IKK-Beta, we repeat our luciferase reporter assay experiment after introducing a four base-pair mutation in the miRNA binding site of the IKK-Beta 3'UTR. The downregulation of IKK-Beta as a result of hsa-miR-497 expression in the cell is significantly attenuated with the introduction of a four base-pair mutation in the miRNA binding site. We also demonstrate the ability of a short-term NF-kB activation to significantly upregulate the expression of hsa-miR-497 using an IL-1B cytokine time-course study. Finally, we test for and confirm the biological existence of a novel unannotated miRNA from the high-throughput sequencing data that we have mapped to the Drosha Isoform 1 transcript in the human genome using reverse transcription and quantitative-PCR.