CASC20 encodes a long noncoding RNA (lncRNA) and is hypothesised to be a novel regulator of bone formation through promotion of osteogenesis and a reduction in chondrogenesis.
Understanding the role CASC20 plays could help uncover novel therapeutic approaches for treatment and/or prevention of diseases such as heterotopic ossification (HO), where GWAS has shown CASC20 is associated with susceptibility. This thesis describes the work done to investigate the role of CASC20 in musculoskeletal development and its mechanism of action. lncRNAs can act via decoy, scaffold or guide mechanisms and evidence was gathered to determine which of these is responsible for the actions of CASC20.
An RNA-sequencing analysis of murine cells expressing CAS20 during osteogenic differentiation was performed to observe changes in gene expression and identify CASC20 micro (mi)RNA targets. This work was then repeated in primary, adipose-derived human mesenchymal stromal cells (hMSCs), isolated from the stromal vascular fraction (SVF) in knee fat pads obtained from patients undergoing joint replacement surgery. The hMSCs were transduced with a CASC20 overexpressing (OE) or control (CTRL) lentivirus and differentiated to observe the effects of CASC20 on osteogenesis and chondrogenesis. However, whilst these cells were able to differentiate, the process of lentiviral transduction with CASC20 caused them to lose their differentiative capacity. Pilots with adenoviral transduction demonstrated this was a viable alternative to be used going forward for CASC20 overexpression in primary hMSCs.
Future work is ongoing to investigate whether the action of CASC20 is through BMP2, a neighbouring gene critical to osteogenesis, by clustered regularly interspaced palindromic repeats (CRISPR) deletion of CASC20 regions. This work alongside that described in this thesis helps provide further insight into the action of CASC20 and its role as a potential novel regulator of bone formation.
