Title The essential role of miR-27a in craniofacial and body skeletons Abstract MicroRNAs are ~22 nucleotides noncoding RNAs which control gene expression at post-transcriptional levels. Owing to its ability to simultaneously modulate a vast amount of genes, microRNA has been postulated to work as a master regulator for cell type-specific development and function. Although gain-of-function analysis with microRNA overexpression can affect these processes in transgenic mice, loss-of-function study often fails to detect phenotypic alterations and is unable to faithfully determine their role in development and disease. Moreover, results obtained from in vitro analysis sometimes are not supported by functional analyses in vivo. Studies of Dicer, an enzyme essential for biosynthesis of microRNAs, have implicated their importance in bone remodeling. However, the exact miRNA(s) involved has not been identified. The cluster of miR-23a~27a~24-2 consists of three miRNAs generated from a single transcript. Dysregulation of miR-23a and miR-27a has been shown in osteoporosis patients but their regulatory role remains elusive. In cell culture study, high levels of miR-23a or miR-27a inhibits OB differentiation, suggesting that they are negative regulators for bone formation. In mice, OB-specific expression of miR-23a~27a~24-2 cluster reveals its effects on osteocyte but not OB differentiation. The animal study does not support the cell culture analysis. There is an urgent need to develop loss-of-function models to definitively assess the function of these microRNAs in vivo. To fill our knowledge gaps, we created a mouse model deficient for miR-27a using CRSPR-Cas9 gene editing. The loss of this single miRNA causes severe osteoporosis in the craniofacial and body skeletons, suggesting the function of miR-27a cannot be substituted. The bone loss phenotype also becomes more prominent with age. New genetic evidence, indicating miR-27a as a positive regulator in bone remodeling, strongly argues against the previous cell culture study. In this application, we will characterize craniofacial and skeletal defects in miR-27a deficient mice. We will examine osteoblast and osteoclast abnormalities associated with the mutation to cause an imbalance in skeletal remodeling. To further elucidate the mechanism by which miR-27a regulates bone formation and resorption, we will identify its direct targets using unbiased screening. The completion of this proposal has outstanding potential to advance our knowledge base of epigenetic regulation in bone metabolism, leading to novel strategies for prevention and treatment of craniofacial and skeletal disorders.
This proposal investigates basic epigenetic elements controlling the formation and maintenance of healthy craniofacial and body skeletons. Using CRISPR-Cas9 gene editing, we have created mouse strains ideally suited for assessing the function of microRNA and elucidating the mechanism underlying skeletal remodeling. The results obtained are highly relevant to human health, e.g. osteoporosis, and has potentials to gain insights into therapeutic strategies for human disorders.
