Effect of knockdown of lncRNA MRF on gene expression during osteogenic induction of human BMSCs
Mesenchymal stem cells (MSCs), known for their ability to differentiate into osteoblasts, play a pivotal role in bone metabolism. Prior to this study, we identified a novel lncRNA called MCP1 regulatory factor (MRF), which exhibits significant involvement in immune regulation of BMSCs. Moreover, we observed noticeable expression changes of MRF during the osteogenic differentiation of BMSCs. However, the exact role and underlying mechanism of MRF in the osteogenic differentiation of BMSCs remain elusive. QRT-PCR analysis was used to assess the expression of MRF. RNA interference and overexpression plasmids were employed to modulate MRF expression and observe changes in the osteogenic differentiation capacity of BMSCs. Downstream pathways involved in MRF-mediated regulation of BMSCs' osteogenic differentiation were predicted using transcriptome sequencing. The functionality of MRF in vivo was validated through a mouse tibial drilling defect model. In patients with osteoporosis, there is a conspicuous escalation in the expression of MRF within BMSCs. During the bone differentiation process of BMSCs, the MRF gradually diminishes over time. The knockdown of MRF amplifies the osteogenic differentiation of BMSCs, promoting an increased expression of bone-related proteins such as RUNX2, ALP, and COL1A1. Transcriptome sequencing and western blot indicated that cAMP/PKA/CREB signaling pathway was significantly activated after lncRNA-MRF knockdown. The mouse tibial drilling defect model demonstrates that the knockdown of MRF conspicuously advances ossification in vivo. MRF modulates the cAMP/PKA/CREB signaling axis through FSHR, thereby adjusting the ossification differentiation of BMSCs. Our research suggests that MRF may a potential target for bone related disorders. Overall design: To investigate the regulatory role of lncRNA MRF in the osteogenic differentiation of BMSCs, we collected BMSCs from three different patient sources and used third-generation BMSCs for experiments. Each group of BMSCs was divided into two groups, which were subjected to lncRNA MRF knockdown sirna treatment and negative control sirna treatment were performed, followed by osteogenic induction for three days. TRIzol was used to extract the total RNA from each group of samples.