Background: Aucubin (AU), an iridoid glucoside isolated from many traditional herbal medicines, has anti-osteoporosis and anti-apoptosis bioactivities. However, the effect of AU on the treatment of bone-fracture remains unknown. In the present study, the aims were to investigate the roles and mechanisms of AU not only on osteoblastogenesis of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) and anti-oxidative stress injury in vitro, but also on bone-fracture regeneration by a rat tibial fracture model in vivo. Methods: CCK-8 assay was used to assess the effect of AU on the viability and proliferation of hBM-MSCs. The expression of specific genes and proteins on osteogenesis, apoptosis and signaling pathways was measured by qRT-PCR, western blotting and immunofluorescence analysis. ALP staining and quantitative analysis were performed to evaluate ALP activity. ARS and quantitative analysis were performed to evaluate calcium deposition. DCFH-DA staining was used to assess the level of reactive oxygen species (ROS). A rat tibial fracture model was established to validate the therapeutic effect of AU in vivo. Micro-CT with quantitative analysis and histological evaluation were used to assess the therapeutic effect of AU locally injection at the fracture site. Results: Our results revealed that AU did not affect the viability and proliferation of hBM-MSCs. Compared with control group, western blotting, PCR, ALP activity and calcium deposition proved that AU-treated groups promoted osteogenesis of hBM-MSCs. The ratio of phospho-Smad1/5/9 to total Smad also significantly increased after treatment of AU. AU-induced expression of BMP2 signaling target genes BMP2 and p-Smad1/5/9 as well as of osteogenic markers COL1A1 and RUNX2 was downregulated after treating with noggin and LDN193189. Furthermore, AU promoted the translocation of Nrf2 from cytoplasm to nucleus and the expression level of HO1 and NQO1 after oxidative damage. In a rat tibial fracture model, local injection of AU promoted bone regeneration. Conclusions: Our study demonstrates the dual effects of AU in not only promoting bone-fracture healing by regulating osteogenesis of hBM-MSCs partly via canonical BMP2/Smads signaling pathway but also suppressing oxidative stress damage partly via Nrf2/HO1 signaling pathway.

Increasing evidence suggests the immunomodulatory potential of genes in oncology. But the identification of immune attributes of genes is costly and time-consuming, which leads to an urgent demand to develop a prediction model. We developed a deep learning-based model to predict the immune properties of genes. This model is trained in 70% of samples and evaluated in 30% of samples. Furthermore, it uncovers 60 new immune-related genes. We analyzed the expression perturbation and prognostic value of these genes in gastric cancer. Finally, we validated these genes in immunotherapy-related datasets to check the predictive potential of immunotherapeutic sensitivity. This model classifies genes as immune-promoted or immune-inhibited based on the human PPI network and it achieves an accuracy of 0.68 on the test set. It uncovers 60 new immune-related genes, most of which are validated in the published literature. These genes are found to be downregulated in gastric cancer and significantly associated with the immune microenvironment in gastric cancer. Analysis of immunotherapy shows that these genes can discriminate between responder and non-responder. This model can facilitate the identification of immune properties of genes, decoding tumor-immune interactions for precision immunotherapy in oncology.

Background: The available therapeutic options of bone defects, fracture nonunion, and osteoporosis remain limited, which are closely related to the osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs). Thus, there remains an urgent demand to develop a prediction method to infer osteogenic differentiation–related genes in BMSCs.Method: We performed differential expression analysis between hBMSCs and osteogenically induced samples. Association analysis, co-expression analysis, and PPI analysis are then carried out to identify potential osteogenesis-related regulators. GO enrichment analysis and GSEA are performed to identify significantly enriched pathways associated with AOX1. qRT-PCR and Western blotting were employed to investigate the expression of genes on osteogenic differentiation, and plasmid transfection was used to overexpress the gene AOX1 in hBMSCs.Result: We identified 25 upregulated genes and 17 downregulated genes. Association analysis and PPI network analysis among these differentially expressed genes show that AOX1 is a potential regulator of osteogenic differentiation. GO enrichment analysis and GSEA show that AOX1 is significantly associated with osteoblast-related pathways. The experiments revealed that AOX1 level was higher and increased gradually in differentiated BMSCs compared with undifferentiated BMSCs, and AOX1 overexpression significantly increased the expression of osteo-specific genes, thereby clearly indicating that AOX1 plays an important role in osteogenic differentiation. Moreover, our method has ability in discriminating genes with osteogenic differentiation properties and can facilitate the process of discovery of new osteogenic differentiation–related genes.Conclusion: These findings collectively demonstrate that AOX1 is an osteogenic differentiation-relevant gene and provide a novel method established with a good performance for osteogenic differentiation-relevant genes prediction.

Background: As a powerful tool, bioinformatics analysis is playing an increasingly important role in many fields. Osteogenic differentiation is a complex biological process involving the fine regulation of numerous genes and signaling pathways. Method: Osteogenic differentiation-related genes are collected from the online databases. Then, we proposed two indexes Jaccard similarity and Sorensen-Dice similarity to measure the topological relevance of genes in the human PPI network. Furthermore, we selected three pathways involving osteoblast-related transcription factors, osteoblast differentiation, and RUNX2 regulation of osteoblast differentiation for investigation. Subsequently, we performed functional a enrichment analysis of these top-ranked genes to check whether these candidate genes identified by similarity-based metrics are enriched in some specific biological functions and states. we performed a permutation test to investigate the similarity score with four well-known osteogenic differentiation-related pathways including hedgehog signaling pathway, BMP signaling, ERK pathway, and Wnt signaling pathway to check whether these osteogenic differentiation-related pathways can be regulated by FOXA1. Lentiviral transfection was used to knockdown and overexpress gene FOXA1 in human bone mesenchymal stem cells (hBMSCs). Alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were employed to investigate osteogenic differentiation of hBMSCs. Result: After data collection, human PPI network involving 19,344 genes is included in our analysis. After simplifying, we used Jaccard and Sorensen-Dice similarity to identify osteogenic differentiation-related genes and integrated into a final similarity matrix. Furthermore, we calculated the sum of similarity scores with these osteogenic differentiation-related genes for each gene and found 337 osteogenic differentiation-related genes are involved in our analysis. We selected three pathways involving osteoblast-related transcription factors, osteoblast differentiation, and RUNX2 regulation of osteoblast differentiation for investigation and performed functional enrichment analysis of these top-ranked 50 genes. The results collectively demonstrate that these candidate genes can indeed capture osteogenic differentiation-related features of hBSMCs. According to the novel analyzing method, we found that these four pathways have significantly higher similarity with FOXA1 than random noise. Moreover, knockdown FOXA1 significantly increased the ALP activity and mineral deposits. Furthermore, overexpression of FOXA1 dramatically decreased the ALP activity and mineral deposits. Conclusion: In summary, this study showed that FOXA1 is a novel significant osteogenic differentiation-related transcription factor. Moreover, our study has tightly integrated bioinformatics analysis with biological knowledge, and developed a novel method for analyzing the osteogenic differentiation regulatory network.