Document Type

Dissertation

Date of Degree

2012

Degree Name

PhD (Doctor of Philosophy)

Degree In

Genetics

First Advisor

Paul B. McCray

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that when mutated causes the disease cystic fibrosis (CF). Many obstacles hinder the understanding of CF disease pathogenesis, impeding advancements in understanding how mutations cause disease, and slowing the progress towards new treatments. To this end, we have profiled the transcriptome (mRNA and microRNA) of human and newborn pig CF and non-CF airway epithelia. We show that the use of cross-species transcriptomics allows the identification of genes differentially expressed owing to the loss of CFTR, and not due to confounding environmental or secondary disease progression influences. The identification of reduced OAS1 expression in CF samples is a case in point. We also demonstrate the utility of transcriptome profiling and longitudinal studies in pigs, providing greater understanding of the molecular mechanisms underlying CF disease progression.

MicroRNAs (miRNAs) comprise a large family of ~21-nt long non-coding RNAs that function as key post-transcriptional regulators of gene expression. Very little is known of how CFTR is regulated in the cell, both transcriptionally and post-transcriptionally. We discovered three miRNAs: miR-509-3p, miR-494 and miR-138 with possible CFTR regulatory functions. miR-509-3p or -494 directly target the CFTR mRNA, and decrease CFTR levels when over expressed; while inhibiting them had the opposite effect. Upon stimulating human airway epithelial cells with TNFα or IL-1β, we observed an increase in expression of both miRNAs mediated in part by the NF-κB transcription factor complex, with a concurrent decrease in CFTR expression. Gene ontology classification of predicted targets of miR-509-3p and/or miR-494 expressed in the airway epithelium revealed enrichment for genes in ion transport pathways. To our knowledge, this is the first suggestion of a possible role for miRNAs regulating a broad range of important epithelial electrolyte and fluid transport proteins.

The study of miR-138 mediated regulation of CFTR expression has led to novel discoveries in the field of CFTR transcriptional control. We discovered SIN3A to be a novel transcriptional repressor of CFTR, interacting with CTCF on the CFTR promoter at the -20.9 kb DHS. By validating SIN3A as a conserved target of miR-138, we also discovered miR-138 to be a novel transcriptional regulator/activator of CFTR.

The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and CF. Manipulating the miR-138/SIN3A regulatory network improved the biosynthesis of CFTR-ΔF508, restoring Cl- transport to human CF airway epithelia. To our knowledge, this is the first example of an individual miRNA having such broad regulatory functions. This discovery also provided novel targets for restoring CFTR function in cells affected by the most common CF mutation. To this end, we are utilizing the molecular signatures of miR-138 over-expression and SIN3A knockdown to identify candidate genes for RNA interference screens, and to identify candidate small molecule drugs that might mimic the effects of these two interventions. The goal of this approach is to develop a new therapeutic agent that restores anion transport to airway epithelia and other cell types and tissues affected by CF.

Keywords

CFTR, Cystic Fibrosis, Gene Therapy, microRNA, RNAi

Pages

xvi, 314 pages

Bibliography

Includes bibliographical references (pages 271-314).

Copyright

Copyright 2012 Shyam Ramachandran

Included in

Genetics Commons

Share

COinS