Monoallelic loss of RB1 Enhances Osteogenic Differentiation and Delays DNA Repair Without Inducing Tumorigenicity

Abstract

The Retinoblastoma (RB1) gene plays a pivotal role in osteogenic differentiation. Our previous study, employing temporal gene expression analysis using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), revealed the deregulation of osteogenic differentiation in patient-derived heterozygous RB1 mutant orbital adipose-derived mesenchymal stem cells (OAMSCs). The study revealed increased Alizarin Red staining, suggesting heightened mineralization without a corresponding increase in osteogenic lineage-specific gene expression. In this study, we performed high-throughput RNA sequencing on RB1+/+ and RB1+/- patient-derived OAMSCs differentiated towards the osteogenic lineage to investigate the pathways and molecular mechanisms. The pathway analysis revealed significant differences in cell proliferation, DNA repair, osteoblast differentiation, and cancer-related pathways in RB1+/- OAMSC-derived osteocytes. These findings were subsequently validated through functional assays. The study revealed that osteogenic differentiation is increased in RB1+/- cells, along with enhanced proliferation of the osteocytes. There were delayed but persistent DNA repair mechanisms in RB1+/- osteocytes, which were sufficient to maintain genomic integrity, thereby preventing or delaying the onset of tumors. This contrasts with our earlier observation of increased mineralization without corresponding gene expression changes, emphasizing the importance of high-throughput analysis over preselected gene set analysis in comprehending functional assay results.