MicroRNA Involvement In The Cisplatin-selected Pleiotropic Multidrug-resistant Phenotype
Pouliot, Lynn Margaret
Gottesman, Michael M
Drug resistance remains a major obstacle to the successful treatment of cancer. Cancer cells acquire a pleiotropic resistance phenotype to cisplatin (CisPt), conferring cross-resistance to other xenobiotics. This pleiotropic resistance occurs in a single-step reducing CisPt accumulation and altering cellular phenotypes. miRNAs are involved in differentiating and maintaining cell phenotype, and therefore we have investigated their influence on cisplatin resistance. This was accomplished by silencing the miRNA biosynthesis pathway, preventing the maturation of new miRNAs in response to treatment with CisPt and reversing CisPt resistance. Next we assessed miRNA expression differences between parental and resistant cell lines using a microarray analysis, and identified the miR-181 family as the most overexpressed in cisplatin-resistant (CP-r) cells. Functional assays were performed to determine if family members caused an increase or decrease in viability when treated with CisPt. When expression was increased, miR-181a*/c/c* increased cell viability while miR-181a/d decreased viability. To identify other miRNAs that mediate CisPt-resistance, we conducted parallel high-throughput screens for CisPt-sensitizing miRNA mimics, and for RNAi-silenced kinases that decreased viability in the same cells. We validated the top hits from our screen, and showed that the miR-15/16/195/424/497 family mediates CisPt-resistance, by targeting CHK1 and WEE1 mRNA. Lastly, we explored the involvement of GCF2, a negative transcription factor, in silencing RHOA, leading to disorganization of the cytoskeleton, and decreased CisPt accumulation. These studies demonstrate 1) the critical involvement of the miRNA biosynthesis pathway in maintaining the CP-r phenotype 2) functional assays are critical for determining the influence of a specific miRNA on a phenotype or pathology. In addition this dissertation identified two miRNA-cell cycle kinase (WEE1, CHK1) mRNA pairings, and a pathway involving the transcriptional repressor GCF2 and RHOA in acquired CisPt resistance.
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