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

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.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96159/1/jandro_1.pd

Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/1/embr201285-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/2/embr201285.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102078/3/embr201285.reviewer_comments.pd

MicroRNA-21 and Dicer are Dispensable for Hepatic Stellate Cell Activation and the Development of Liver Fibrosis

Fibrosis and cancer represent two major complications of chronic liver disease. MicroRNAs have been implicated in the development of fibrosis and cancer, thus constituting potential therapeutic targets. Here, we investigated the role of miR-21, a microRNA that has been implicated in the development of fibrosis in multiple organs and also been suggested to act as "oncomir". Accordingly, miR-21 was the microRNA that showed the strongest upregulation in activated hepatic stellate cells (HSC) in multiple models of fibrogenesis, with an 8- to 24-fold induction compared to quiescent HSC. However, miR-21 antisense inhibition did not suppress the activation of murine or human HSC in culture or in liver slices. Moreover, antisense inhibition or genetic deletion of miR-21 in two independently generated knockout mice did not alter HSC activation or liver fibrosis in models of toxic and biliary liver injury. Despite a strong upregulation of miR-21 in injury-associated hepatocellular carcinoma and in cholangiocarcinoma, miR-21 deletion or antisense inhibition did not reduce the development of liver tumors. As inhibition of the most upregulated microRNA did not affect HSC activation, liver fibrosis and fibrosis-associated liver cancer, we additionally tested the role of microRNAs in HSC by HSC-specific Dicer deletion. Although Dicer deletion decreased microRNA expression in HSC and altered the expression of select genes, it only exerted negligible effects on HSC activation and liver fibrosis. In conclusion, genetic and pharmacologic manipulation of miR-21 does not inhibit the development of liver fibrosis and liver cancer. Moreover, suppression of microRNA synthesis does not significantly affect HSC phenotype and activation. This article is protected by copyright. All rights reserved