ABSTRACT
OBJECTIVE: To explore the effect of TRIB3 on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs) and reveal the potential role of TRIB3 in bone regeneration. METHODS: TRIB3-knockdown and TRIB3-overexpression hASCs were used to explore the effect of TRIB3 on osteogenic differentiation by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, quantitative real-time polymerase chain reaction (qRT-PCR) and heterotopic bone formation. The regulation of miR-24-3p on TRIB3 was detected by qRT-PCR and western blot. Ribonucleic acid (RNA) sequencing was performed to investigate the downstream regulatory network of TRIB3. RESULTS: TRIB3 promoted the osteogenic differentiation of hASCs both in vitro and in vivo. This process was regulated epigenetically by the post-transcriptional regulation of miR-24-3p, which could bind directly to the three prime untranslated region (3'UTR) of TRIB3 and inhibit TRIB3 expression. The downstream regulatory network of TRIB3-mediated osteogenic differentiation was related to calcium ion binding and cell metabolism, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and nuclear factor-κB (NF-κB) signalling pathways. CONCLUSION: TRIB3 is a promising therapeutic target for hASC-based bone tissue engineering and the epigenetic regulation of TRIB3 through miR-24-3p permits regulatory controllability, thus promoting osteogenesis through an important metabolic target while obtaining a safe and controllable effect via post-transcriptional epigenetic regulation.
Subject(s)
Cell Cycle Proteins/genetics , Mesenchymal Stem Cells , MicroRNAs , Protein Serine-Threonine Kinases/genetics , Repressor Proteins/genetics , Cell Differentiation , Cells, Cultured , Epigenesis, Genetic , Humans , MicroRNAs/genetics , Osteogenesis/genetics , RNA Processing, Post-TranscriptionalABSTRACT
OBJECTIVE: To explore graphene's effects on the gene expression profile of mesenchymal stem cells, and to reveal the mechanisms of graphene-guided osteogenic differentiation. METHODS: Human adipose-derived mesenchymal stem cells (hASCs) were cultured on single-layer graphene-coated titanium disks or titanium disks in proliferation medium (control) or osteoinduction medium for 7 days before RNA extraction. After library construction and RNA sequencing, identification of differentially expressed genes was performed through Limma package of R platform, with a cut-off value of log fold change (logFC) > = |1|. Pathway and Gene ontology (GO) analyses were conducted on DAVID Bioinformatics Resources 6.8 (NIAID/NIH). Network analyses were performed by the Ingenuity Pathways Analysis (IPA). RESULTS: Signalling pathway analysis revealed the top five pathways - cytokine-cytokine receptor interaction, neuroactive-ligand receptor interaction, calcium signalling pathway, PI3K-Akt signalling pathway and cell adhesion molecules. GO analyses demonstrated significant changes on cell adhesion, calcium signalling, and epigenetic regulation. IPA network analyses demonstrated that inflammation-related pathways were influenced by graphene, while the downstream factors of histone H3 and H4 were also altered especially under the existence of osteoinduction medium. CONCLUSION: Graphene promotes osteogenic differentiation of hASCs mainly by influencing cell adhesion, cytokine-cytokine receptor interactions, inflammatory responses, and potentially influences histone H3 and H4 through epigenetic regulation.
