Characterizing miRNA mediated regulation of proliferation

Cell proliferation is a fundamental biological process, and the ability of human cells to transition from a quiescent to proliferative state is essential for tissue homeostasis. Most cells in eukaryotic organisms are in a quiescent state, but on appropriate physiological or pathological stimuli, many types of somatic cells may exit quiescence, re-enter the cell cycle and begin to proliferate. The ability of cells to remain viable while quiescent, exit quiescence and re-enter into the cell cycle is the basis for varied physiological processes such as wound healing, lymphocyte activation and hepatocyte regeneration, but is also a hallmark of cancer. The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. Our understanding of miRNA biology has significantly improved, but the miRNA regulatory networks that govern cell proliferation are still largely unknown. We characterized a miR-22 Myc network that mediates proliferation through regulation of the interferon response and multiple cell cycle arrest genes. We identified several cell cycle arrest genes that regulate the effects of the tumor suppressor p53 as direct targets of miR-22, and discovered that miR-22 suppresses interferon gene expression. We go on to show that miR-22 is activated by the transcription factor Myc as quiescent cells enter proliferation, and that miR-22 represses the Myc transcriptional repressor MXD4, mediating a feed forward loop to elevate Myc expression levels. To more effectively determine miRNA targets, we utilized a combination of RNA-induced silencing complex immunoprecipitations and gene expression profiling. Using this approach for miR-191, we constructed an extensive transcriptome wide miR-191 target set. We show that miR-191 regulates proliferation, and targets multiple proto-oncogenes, including CDK9, NOTCH2, and RPS6KA3. Recent advances in determining miRNA targetomes have revealed widespread non-canonical miRNA-target pairing. We experimentally identified the transcriptome wide targets of miR-503, miR-103, and miR-494, and observed evidence of non-canonical target pairing for these miRNAs. We went on to confirm that miR-503 requires pairing outside of the canonical 5' seed region to directly target the oncogene DDHD2. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis.