All ETDs from UAB

Advisory Committee Chair

Mohammad Q Hassan

Advisory Committee Members

Amjad Javed

Heidi Erlandsen

Mary Macdougall

Document Type

Thesis

Date of Award

2013

Degree Name by School

Master of Science in Dentistry (MScD) School of Dentistry

Abstract

MicroRNAs are endogenous non-coding RNA (~22nt) that bind to the 3' untranslated region (UTR) of target messenger RNA (mRNA) and facilitate translational repression and degradation. Previous studies have demonstrated the essential role of miRNA in many biological processes, including skeletal development and tooth formation. Dlx3, a homeobox gene, is critical for craniofacial patterning and postnatal skeletal development. Mutations in the Dlx3 gene in humans result in two autosomal dominant phenotypes known as tricho-dento-osseous syndrome (TDO) and amelogenesis imperfecta with taurodontism (AIHHT). Dlx3 expression has been observed in cranial neural crest cells, endochondral osteoblasts, odontoblasts, ameloblasts, hypertrophic chondrocytes, and the developing limb. To date there are no studies showing the miRNA regulation of Dlx3 gene expression during dentinogenesis. The objective of this study was to test if miRNA(s) can control Dlx3 during odontoblast growth and differentiation leading to changes in dentin extracellular matrix protein gene expression. Our hypothesis is that Dlx3 expression is regulated during dentinogenesis by specific miRNAs. Through initial screenings using miRNA reporter assay and western blot analysis we identified and confirmed that Dlx3 is a potential target for miR-665. Three different odontoblast-like cell lines, MO6-G3, MDPC-23, and OD-21, were infected with control, miR-665, and anti-miR-665 Lentiviruses to study the regulation of Dlx3 and downstream dentin proteins during dentinogenesis by miR-665. The following key findings were observed: 1) MiR-665 directly targets the 3'UTR of Dlx3 mRNA and downregulates levels of Dlx3 mRNA and protein; 2) MiR-665 significantly downregulates dentin matrix proteins including DSPP, DMP1, OPN, FAM20C, KLK4, and MMP20 in the odontoblast cells; 3) Critical tooth transcription factors, Runx2 and Osterix, were significantly downregulated resulting in the inhibition of odontoblast differentiation; 4) The odontoblast potential of miRNA overexpressing OD-21 cells was inhibited significantly as measured by inhibition of alkaline phosphatase staining; 5) Silencing complex analysis showed a substantial amount of Dlx3 mRNA was bound to miR-665. This study demonstrates for the first time the critical role of miR-665 in regulating Dlx3 during dentinogenesis. Our results will help to identify molecular players involved in the process of dentinogenesis and will help to develop novel potent RNA therapeutics for dental diseases.

Included in

Dentistry Commons

Share

COinS