OSCAR Regulates The Proliferation And Migration Of BMSCs By Orchestrating Wnt Signaling Pathway

Objective:Osteoclast-associated receptor（OSCAR）is an activating receptor for collagen,which is highly expressed in osteoclasts and is involved in osteoclast differentiation.However,its role for osteoblast-spectrum cells is unclear.In this study,we investigated the potential role of OSCAR in regulating the proliferation and migration of bone marrow mesenchymal stem cells（BMSCs）and its possible molecular mechanism by constructing an Oscar knockout mouse model(Oscar^-/-).Methods:We generated Oscar^-/-mice by CRISPR/Cas9 technology.BMSCs from 4-8 weeks old wild-type（WT）and Oscar knockout（KO）mice were extracted by amended whole bone marrow culture method.Wound-Healing assay and transwell migration assay were performed to examine the migration ability of BMSCs from different genotypes of mice.The Cell Counting Kit-8（CCK-8）assay and flow cytometry were designed to explore the effect of OSCAR on the proliferation of BMSCs.After osteogenic induction of different genotypes of BMSCs,we used RNA-sequence technology to analyze differentially expressed genes and performed signaling pathway analysis by bioinformatics methods to identify the signaling pathways enriched with differentially expressed genes.Quantitative real-time polymerase chain reaction（q RT-PCR）verified the expression of related genes.Meanwhile,cellular proteins were extracted for Western Blot（WB）to detect the expression of molecules associated with proliferation and migration.Immunofluorescence（IF）were performed to determine the cellular localization ofβ-catenin molecules in different genotypes of BMSCs.Results:In this study,we observed diminished proliferation and migration of BMSCs in Oscar^-/-mice compared to WT mice.The results of RNA-sequence and bioinformatics analysis showed that the genes expression of cell adhesion molecules,ECM-receptor interaction,transforming growth factor-β（TGF-β）and Wnt signaling pathway were significantly decreased in BMSCs of Oscar^-/-mice compared to WT mice.The vast majority of genes with reduced expression were associated with bone development,bone formation,and osteogenic differentiation.And q RT-PCR results further confirmed the results of RNA-sequence analysis.The WB results showed that compared with WT BMSCs,Oscar^-/-BMSCs showed no significant difference in the expression ofβ-catenin and its phosphorylation,but the expression of Wnt1-induced secreted protein 1（WISP1）and Myc was decreased.IF results showed thatβ-catenin content in the cytoplasm was increased in BMSCs after Oscar knockout.Conclusion:Oscar promotes the proliferation and migration of BMSCs by activating the canonical Wnt signaling pathway and regulating the expression of downstream target genes Wisp1 and Myc.Oscar knockout reduced the proliferation and migration of BMSCs.