The regulation of microRNAs in cancer through novel transcriptional and post-transcriptional mechanisms

Thesis (Ph.D.)--University of Washington, 2012MicroRNAs are small non-coding RNA molecules that serve as important regulators of gene expression. While understanding of the functional roles of miRNAs in both normal physiology and disease has rapidly expanded, the regulation of these molecules remains a largely open question. We have investigated the regulation of mRNAs at multiple steps of their biogenesis. We began by studying the transcriptional regulation of miRNAs in ovarian cancer, and developed a computational pipeline by which to identify putative transcription factor: miRNA interactions. We found that the miR-200 family of miRNAs is regulated by p63 and p73, two members of the p53 transcription factor family. The miR-200 miRNAs serve as potent regulators of the epithelial-mesenchymal transitions that influence tumor invasion; therefore, identifying positive regulators may facilitate the future modulation of these miRNAs for therapeutic applications. We have also examined the transcriptional regulation of miR-210, which is potently induced under low oxygen conditions. We found that under normoxic conditions miR-210 is rapidly activated by HIF-1 alpha in response to cell density, demonstrating the importance of a cell's microenvironment in shaping its miRNA expression patterns. We next examined the downstream regulation of miRNAs at the post-transcriptional level. We discovered that miRNAs frequently show 3' non-templated nucleotide additions, forming a large number of miRNA isomiRs that expand the diversity of the miRNA transcriptome. We identified multiple nucleotidyl transferase enzymes that are responsible for these modifications in a miRNA-specific fashion, including three enzymes--TUT1, MTPAP, and ZCCHC6--not previously known to modify miRNAs. Finally, we have investigated the functional effects these 3' additions and their nucleotidyl transferase regulators exert on miRNA activity. We found 3' additions are neither universally stabilizing nor destabilizing; instead, certain nucleotide additions, such as increased 3' uridylation, are associated with reduced miRNA abundance. Additionally, we identified two nucleotidyl transferases, TUT1 and PAPD4, which broadly maintain the expression of miRNAs. Taken together, our work reveals several novel mechanisms of regulation for miRNAs that are influential in tumor initiation and spread. While some factors control the expression of many miRNAs, other proteins alter the abundance of a subset of miRNAs to enable a precise regulatory response