Characterization of microRNA Regulation and Function in Hypoxia Biology

microRNAs are small ∼22 nucleotide non-coding RNA molecules that are responsible for the translational regulation of an estimated 60% of all protein-coding transcripts. As such, their own transcriptional regulation is tightly controlled in normal tissues. Unfortunately, this regulation is often altered in cancer to promote aberrant gene expression and facilitate cell survival. One of the most common features of the tumor microenvironment is hypoxia. Hypoxic tumor cells display increased genomic instability and are prone to therapy resistance, characteristics that can be ascribed to molecular gene expression changes. Arguably, the main effector molecule of hypoxia is HIF-1, a hypoxia-stabilized transcription factor, which drives irregular expression of many target genes. It has been shown that HIF-1 is capable of regulating microRNA expression. Several microRNAs have been implicated in tumorigenesis including miR-155 and miR-128, which both have altered hypoxic expression. miR-155 will be the main focus of this dissertation, while some preliminary work will be discussed for miR-128.;miR-155 has been described as an oncogenic microRNA or oncomiR, with over-expression observed in many human malignancies including lung cancer and lymphoma. We have found that miR-155 expression is induced by hypoxia in a HIF-1-dependent manner. We have shown that this induction of miR-155 expression promotes radiation resistance in hypoxic lung cancer cells, likely through the suppression of FOXO3a, a pro-apoptotic transcription factor. Additionally, targeting miR-155 with an antagomiR in hypoxia sensitizes these cells to radiation therapy. This work suggests the possibility of using anti-miR-155 molecules as an adjuvant to radiation therapy to improve tumor cell sensitivity to therapy.;In addition to radiation resistance, we have also found that miR-155 plays a critical role in maintaining genome integrity. Upon over-expression of miR-155, we observed an increase in mutation frequency demonstrated by a mutation reporter assay. Corresponding decreases in established miR-155 targets associated with DNA repair were observed by Western blot, including RAD51 and MLH1. Using microarray analysis to look for changes in gene expression in other DNA repair pathways, we found that miR-155 over-expression resulted in a decrease in mRNA expression of all four subunits of polymerase delta. While it is unlikely that miR-155 directly targets all four of these subunits, it is possible that a transcription factor responsible for their regulation might be targeted by miR-155. In fact, we have identified sites within each promoter where the transcription factor, FOXO3a, could putatively bind. We demonstrated that miR-155 over-expression leads to a decrease in POLD1, the catalytic subunit of polymerase delta, and FOXO3a at the protein level.;We have also shown that miR-155 over-expression results in a decrease in homologous recombination (HR) activity and a corresponding increase in non-homologous end joining (NHEJ). This increase in an error-prone double-strand break repair pathway may contribute to the observed increase in mutation frequency in the presence of miR-155 over-expression. Over-expression of miR-155 results in a baseline decrease in the number of double-strand breaks, which correlates with an increase in NHEJ. Taken together with the fact that miR-155 results in a suppression of polymerase delta, it is tempting to speculate that miR-155 facilitates a decrease in HR by suppressing polymerase delta activity, and enabling a compensatory increase in NHEJ or the more mutagenic MMEJ, which is catalyzed by polymerase theta.;Interestingly, we have also found that loss of miR-155 leads to an increase in baseline double-strand breaks and a decrease in NHEJ. Importantly, we did not find a significant shift in the mutation frequency suggesting that although miR-155 loss seems to delay DNA repair or promote DSB formation, this does not appear to affect the integrity of the genome.;Similar to radiation resistance, we have also shown that miR-155 over-expression confers gefitinib resistance in EGFR-mutant lung cancer cells. Normally, gefitinib, an EGFR inhibitor, enables the accumulation of BIM, a pro-apoptotic factor, forcing EGFR-mutant cells to undergo apoptosis. Resistance often occurs with a secondary mutation in the EGFR protein itself, rendering gefitinib inactive against EGFR signaling. Upon over-expression of miR-155, we show that BIM protein expression is reduced. Although not predicted by target prediction algorithms, it is possible that miR-155 directly targets BIM in one of three putative 6- or 7-mer binding sites in the BIM 3' UTR. Further work is necessary to understand the mechanism behind miR-155-driven resistance to gefitinib.;Taken together, this dissertation identifies an important role for miR-155 in maintaining genomic integrity and mediating therapy response. Future work will help determine the potential use of anti-miR-155 as an adjuvant therapy to improve clinical outcome.