Multi-omics identification of key targets for the osteogenic differentiation of human bone marrow mesenchymal stromal cells under oxidative stress
Background: Human bone marrow mesenchymal stromal cells (hBMSCs) are multipotent stromal cells capable of osteogenic differentiation, making them a promising cell source for bone tissue engineering and regenerative medicine. Identifying key factors that regulate hBMSCs osteogenic differentiation is crucial for enhancing bone regeneration strategies. Objective: This study aims to identify target genes underlying impaired osteogenic differentiation of hBMSCs under oxidative stress (OS) through integrated transcriptomic and proteomic approaches, and delineate the role of proenkephalin (PENK) in this process. Methods: OS model and impaired osteogenic differentiation model were established in hBMSCs using hydrogen peroxide (H2O2). Cellular oxidative stress levels were assessed using Dihydroethidium (DHE) fluorescent probes and JC-1 staining. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin Red staining (ARS). Key genes and proteins were predicted via integrated transcriptomic and proteomic analyses. The role of PENK in osteogenic differentiation was validated using lentiviral transfection. Results: This study established 400 µM H2O2 as the optimal concentration for inducing impaired osteogenic differentiation in hBMSCs. Integrated transcriptomic and proteomic analysis identified 18 pivotal regulatory genes that orchestrate impaired osteogenic differentiation of hBMSCs under OS. Among these, PENK was identified as a potential therapeutic target involved in regulating oxidative stress-impaired osteogenic differentiation of hBMSCs. Functional validation confirmed that PENK overexpression promotes osteogenic differentiation in hBMSCs. Conclusion: OS contributes to impaired osteogenic differentiation in hBMSCs. PENK regulates osteogenic differentiation of hBMSCs under OS and holds promise as a novel therapeutic target for bone regeneration and repair. Overall design: This study aims to elucidate the transcriptomic landscape underlying the impaired osteogenic differentiation of human bone marrow mesenchymal stromal cells (hBMSCs) under oxidative stress (OS). A multi-group comparison design was employed: Experimental Groups: Control: hBMSCs in standard growth medium. OS: hBMSCs treated with 400 µM H2O2. Osteogenic Differentiation (OD): hBMSCs in osteogenic induction medium. OS+OD (Key Model): hBMSCs treated with 400 µM H2O2 and cultured in osteogenic induction medium (model of impaired osteogenesis). PENK OE + OS+OD (Functional Validation): PENK-overexpressing hBMSCs subjected to the OS+OD condition. Primary Comparison: The core analysis compares the OS+OD group versus the OD group to directly pinpoint genes specifically dysregulated during osteogenesis under OS conditions. Replication: Each group was analyzed with 3 biological replicates to ensure statistical robustness. Validation: Key candidate genes identified from transcriptomic analysis (prioritizing PENK) were functionally validated using lentiviral overexpression.