Investigating the functional role of lncRNA CASC20 in heterotopic bone formation

Heterotopic Ossification (HO) is a lamellar bone formation in the soft tissues and usually follows injury, trauma or joint replacement. A genome wide association study of HO patients after total hip arthroplasty identified lncRNA Cancer Susceptibility 20 (CASC20) as a gene that is strongly associated with HO severity. Previous findings from my MRes demonstrated an unreported upregulation of CASC20 during BMP2-induced osteodifferentiation of hMSCs. Based on these studies, I hypothesised that CASC20 is a novel regulator of bone formation. This doctoral thesis investigated the role of CASC20 in regulating osteogenic and chondrogenic differentiation processes. Using a diverse range of experimental models, including hMADs, ASC52teloSOX9, and P512MSCs, this research explored CASC20's impact on key osteogenic and chondrogenic markers. Techniques such as in silico miR prediction, CASC20 lentiviral expression, RT-qPCR, and miR-Seq were used to assess the effect of CASC20 on microRNAs (miRs), genes and biological pathways. Here I found that CASC20 consistently played a pivotal role in the early stages of osteogenesis and chondrogenesis. CASC20's modulation of miRs emerged as a consistent regulatory mechanism throughout the experiments. The study identified 13 genes commonly targeted by the putative CASC20-interacting miRs, including well-known osteogenic and chondrogenic players such as MAPK1. To confirm the findings, future steps include genotyping human mesenchymal stem cells and conducting comprehensive experiments to validate the observed effects. The creation of human CRISPR CASC20 KO models will further elucidate CASC20's role. In conclusion, this doctoral thesis provides valuable insights into the intricate mechanisms governed by CASC20 in osteogenesis and chondrogenesis. Further research and exploration are needed to comprehensively elucidate the extent of CASC20's influence in these critical biological processes.