MicroRNAs (miRNAs) are 18-24 nucleotide RNA molecules that regulate the stability or translational efficiency of complementary target mRNAs. miRNAs participate in a diverse array of cellular processes including proliferation, apoptosis, and differentiation. Moreover, globally abnormal miRNA expression patterns appear to be a ubiquitous feature of human cancer cells. Work from our laboratory and others has established that miRNAs function as critical downstream components of several important oncogenic and tumor suppressor pathways. Furthermore, some miRNAs themselves act as novel oncogenes and tumor suppressors. These findings suggest a causative role for miRNA dysregulation in cancer pathogenesis. We have recently classified miRNA expression patterns in pancreatic cancer cells and identified a cohort of miRNAs that are frequently abnormally expressed compared to normal cells. Based on these findings, we now propose a series of experiments designed to test the following central hypothesis: Key miRNAs that are controlled by critical signaling pathways and are abnormally expressed in pancreatic cancer cells act as novel oncogenes and tumor suppressors. We will test these hypotheses by pursuing three specific aims. In Aim 1, we will functionally evaluate the consequences of miRNA dysregulation in vitro by examining neoplastic cellular phenotypes following enforced expression or inhibition of selected miRNAs. We will also employ somatic cell gene knockout methodologies to examine the consequences of complete loss-of-function of specific miRNAs. Finally, we will initiate mechanistic studies of the role of miRNAs in pancreatic cancer by characterizing an important miRNA:target mRNA interaction. In Aim 2, we will take advantage a novel zebrafish model of pancreatic cancer developed by our co- investigator Dr. Leach. By expressing miRNAs in this model specifically in the developing pancreas, we will be able to assess the contribution of miRNAs to pancreatic cancer pathogenesis in vivo in a high-throughput manner. Finally, in Aim 3, we will characterize a novel mouse model that we have generated with loss of function of a miRNA with anti-tumorigenic activity that we have shown to be frequently absent in pancreatic cancer cells. We will also breed these mice to a well-established model of pancreatic cancer to specifically assess the contribution of loss of this miRNA to disease progression. We envision that these studies will contribute significantly to our understanding of the molecular basis of pancreatic cancer and may reveal novel pathways that are amenable to therapeutic intervention. RELEVANCE (See instructions): Pancreatic ductal adenocarcinoma (pancreatic cancer) is the fourth most common cause of cancer-related death, accounting for approximately 33,000 lives each year in the United States. The five year survival rate of <5% underscores the ineffectiveness of current therapies and the profound direness of this disease. We propose to study the role of a recently-described class of molecules called microRNAs in pancreatic cancer biology, with the hope of revealing new insights that could lead to novel therapeutic strategies.