supplementary materials: Key genes and regulatory network of hypoxic preconditioning on osteoblasts

Hypoxia preconditioning has been studied more than 40 years but the exact regulation mechanism of it on osteoblast is still unclear. The purpose of our study was to clarify the key genes and pathways involved in hypoxia preconditioning on osteoblasts. Rat osteoblast (ROS1728) was used and divided to three groups. Cells of hypoxia preconditioning group were received 10 min hypoxia cultivation/10 min normoxic cultivation 3 times before received 4 hours hypoxia cultivation and long-time hypoxia group cells were received 4 hours hypoxia cultivation. Control group cells were received 4 hours normoxic cultivation. We analyzed the different expressed miRNA in each two groups by high throughput sequencing. MiRwalk 3.0 was used to predicate the target genes of miRNAs and make Gene Set Enrichment Analysis (GSEA). Besides, STRING 11.0 database and Cytoscape software were used to make PPI networks and select the hub genes. Finally, CCK-8, ALP, IL6, IL10 and VEGF were detected in each group. Hypoxia preconditioning upregulated 37 miRNAs and downregulated 105 miRNAs compared with long-time hypoxia. 21496 target genes were involved and enriched to 90 Gene ontology (GO) terms with 123 pathways. 268 genes were enriched in biological process of protein phosphorylation and 698 genes involved in metabolic pathways (p<0.001) by GESA. Vav2, LOC502894 and Oxtr were the hub genes. In addition, hypoxia preconditioning improved cell survival by CCK-8 test (p<0.001), and enhanced ALP, IL10 and VEGF expression and inhibited IL6 expression (p<0.001). The mechanism of hypoxia preconditioning on osteoblasts is related with protein phosphorylation. The key pathway was metabolic pathways and the hub genes were Vav2, LOC502894 and Oxtr.