Abstract: BACKGROUND: Osteoporosis can be attributed to an imbalance between osteoblastic bone formation and osteoclastic bone resorption. Osteoblasts are the basis of bone formation and are essential for bone growth and maintenance. Disorders of osteoblast function can lead to dysplasia of bone growth and osteoporosis. However, the key genes involved in osteoblast differentiation are still unknown.  
OBJECTIVE: Bioinformatics was used to identify key genes and upstream miRNAs during osteogenic differentiation of MC3T3-E1 cells and verify their effects on osteogenic differentiation and proliferation of MC3T3-E1 cells.
METHODS:  The osteogenic induction data of gene expression profile GSE46400 were downloaded from the Gene Expression Omnibus. Differentially expressed genes for osteogenesis differentiation were obtained with limma package. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes were analyzed using DAVID database. The STRING website was used to establish a protein-protein interaction network and CytoHubba plug-in was used to screen the key modules in the network to identify the key genes. MC3T3-E1 cells were cultured and transfected with siRNA and miRNA, separately. The change of prostaglandin-endoperoxide synthase 2 (Ptgs2) and ossification-related gene level was detected by real-time quantitative PCR 72 hours after transfection. The change of PTGS2 protein level was detected by western blot assay 72 hours after transfection. Alkaline phosphatase was quantified 14 days after transfection. Alizarin red staining and quantitative detection were performed 21 days after transfection.  
RESULTS AND CONCLUSION: (1) 229 differentially expressed genes were screened, of which 114 were up-regulated and 115 were down-regulated. Five genes enriched in the biological process of bone mineralization, including Mmp13, Ibsp, Gpnmb, Ptgs2 and Aspn, were highly expressed. Furthermore, CytoHubba was used to identify that only Ptgs2 was involved in the biological process of bone mineralization, and Ptgs2 was defined as a key gene in the process of osteogenic differentiation. (2) The upstream miRNA of Ptgs2 was predicted by Targetscan, and it was found that mmu-miR-107-3p could regulate Ptgs2 in a targeted way. (3) Knockdown Ptgs2 in MC3T3-e1 cells significantly inhibited cell proliferation and osteogenic differentiation (P < 0.05). After transfection of mmu-miR-107-3p, cell proliferation and osteogenic differentiation were also inhibited. (4) Dual luciferase reporter gene results showed that mmu-miR-107-3p could directly inhibit the transcription of Ptgs2 (P < 0.05). (5) mmu-miR-107-3p can inhibit the proliferation and osteogenic differentiation of MC3T3-E1 cells by targeting Ptgs2. Ptgs2 may be a key gene in the osteogenic differentiation of MC3T3-E1 cells.

Key words: bioinformatics, differentially expressed genes, MC3T3-E1 preosteoblast, prostaglandin-endoperoxide synthase 2, mmu-miR-107-3p, osteogenic differentiation, proliferation