Date of Completion

7-24-2020

Embargo Period

7-24-2023

Keywords

microRNA, miR-29-3p, RISC, osteoclast, osteoblast, skeleton, bone

Major Advisor

Anne Delany

Co-Major Advisor

Sun-Kyeong Lee

Associate Advisor

Archana Sanjay

Associate Advisor

Peter Maye

Associate Advisor

Yi Wu

Associate Advisor

Mina Mina

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Osteoclasts are large multinucleated bone cells of the myeloid lineage. Osteoclasts secrete proteases and create an acidic environment, causing bone resorption. Osteoclast differentiation, driven by MCSF and RANKL, is tightly regulated by numerous factors, including microRNAs (miRNAs, miRs). miRNAs are short non-coding RNAs that post-transcriptionally regulate gene expression by binding to complementary mRNA sequences, leading to mRNA degradation or destabilization. The aim of these studies was to investigate the function of the miRNA-29-3p family in osteoclasts both in vivo and in vitro.

We generated mice which globally express a miR-29-3p tough decoy which competitively inhibits miR-29-3p activity. The examination of both two-month-old and six-month-old animals allowed for determination of function in growing and skeletally mature animals. Histomorphometry failed to demonstrate an impact of the decoy on the osteoclast or bone marrow adipocyte lineages at either age. However, in two-month-old animals, we observed a significant decrease in bone formation rate and expression of osteoblastic genes in calvaria. By six-months, bone formation rate had normalized in the decoy mice, however, bone volume and trabecular number were decreased. Together, these studies indicate that global miR-29 inhibition more strongly affected osteoblast function.

We also studied the impact of miR-29-3p inhibition in these cell lineages in vitro. Despite our observations in vivo, miR-29 decoy did not affect the osteoblast lineage in vitro, suggesting the phenotype in vivo may be the result of extra-skeletal effects of global miR-29 inhibition. In the adipocyte lineage, marker gene expression was modestly increased by the miR-29 decoy, suggesting miR-29-3p inhibition may promote adipogenesis. Lastly, osteoclast differentiation was inhibited by the decoy in vitro, without changes in osteoclast function.

To better understand the mechanism by miR-29-3p regulates osteoclastogenesis, we performed both candidate gene studies and an RNA-seq study to identify potential miR-29-3p targets and affected pathways in the monocytic lineage. We identified novel targets in the E-cadherin/CDH1 signaling complex, as well as targets associated with macrophage polarization. These data suggest miR-29-3p may promote osteoclast differentiation, in part, by pushing osteoclast precursors away from the M2 lineage, towards the pro-inflammatory M1 macrophage lineage, which shares similar characteristics to bone resorbing osteoclasts.

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