Diversity in Intracellular MicroRNA Regulatory Networks: MicroRNA-21 and Beyond.

Abstract

microRNAs (miRNAs) comprise a broad class of short non-coding RNA that regulate gene expression by guiding the RNA induced silencing complex (RISC) to mRNAs containing complementary ‘seed sites’. General principles of miRNA action in mammals have emerged over the last decade from research using cultured cancer cell models; however, it is unclear whether these principles apply in vivo, where few functional studies have been performed in healthy tissue. Furthermore, it is uncertain whether there are mechanistic or functional heterogeneities between the hundreds of miRNAs conserved across mammals. The primary aim of this thesis was to compare the repressive activities of three highly abundant miRNAs— miR-21, miR-122, and let-7— in healthy liver tissue, and to contrast them with measured or previously reported activities in cancer cell lines. miRNA activities were measured based on (i) array profiling following pharmacological inhibition, and (ii) binding to polysome-associated target mRNAs. It was found that miR-21, compared to miR-122 and let-7, has surprisingly little impact on regulation of canonical seed-matched mRNAs. Moreover, miR-21 showed greatly reduced binding to polysome-associated target mRNAs, possibly due to reduced thermodynamic stability of seed pairing. Significantly, these trends are reversed in human cervical carcinoma (HeLa) cells, where miR-21 and other miRNAs showed enhanced target binding within polysomes and miR-21 triggers strong degradative activity toward target mRNAs. Taken together, these results suggest that certain miRNA activities can be highly context dependent and miRNA pathways are overactive under pathological conditions. Additionally, bioinformatic analysis of sequence features in miR-21 and miR-122 responsive targets revealed low to moderate correspondence with previously established targeting trends derived from HeLa, exposing the complexity of in vivo target selection and suggesting cross-talk with other regulatory networks. As an additional aim, a single molecule method was developed for directly observing the kinetic diversity in miRNA processes. The method combines particle tracking with step-wise photobleaching to probe dynamics and stoichiometry in complex assembly. Pilot studies indicate that fluorophore labeled let-7 miRNAs, detectable as single molecules, undergo a biphasic kinetic assembly when microinjected into live HeLa cells.