All ETDs from UAB

Advisory Committee Chair

Quamarul Hassan

Advisory Committee Members

Hope Amm

Louis Patrick

Document Type

Thesis

Date of Award

2022

Degree Name by School

Master of Science (MS) Heersink School of Medicine

Abstract

The differentiation of mesenchymal stem cells to pre-osteoblasts, lies upon various transcription factors and physiological signaling, but is also governed by the micro-RNA-23a cluster. The miR-23a cluster has miR-23a, miR-27a and miR-24-2 transcribed from a single promoter as a sole RNA primary transcript. Its effects have been observed in embryonic stem cells, mesenchymal stem cells (MSCs), and in cancer (1-2). It regulates the lineage commitment of MSCs. (3). With respect to bone formation, miR-23a cluster is involved MSCs commitment to and osteoblast maturation (4). Here, through in vivo and in vitro experiments, we report that knock down of microRNA-23a cluster using CRISPR gene editing, caused increased expression of early (Alp, Runx2, Sp7 (Osx) and Dlx3) and late (Ocn) bone essential genes involved in osteoblast maturation and differentiation. We employed the CRISPR/Cas9 complex after designing 3 single guide RNA (sgRNA), targeting the miR-23a cluster to be repressed using the KRAB motif attached to dCAS9 protein. In the calvarial defect model, bone regeneration was elevated in the tissue-specific miR-23a cluster knockdown mice when compared with the control group. Here we induced a defect on the calvaria of the mice. We studied the defect on day 0 and day 28 for bone regeneration for control and experiment. Collectively, our findings indicate that inhibition of the miR-23a cluster positively regulates osteoblast differentiation and, as a result, shows increased osteogenesis.

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