title: Differential circRNA expression profiles during the BMP2-induced osteogenic differentiation of MC3T3-E1 cells.
abstract: OBJECTIVE
  Recent studies have indicated that circular RNAs (circRNAs) might play important roles in various diseases. However, little is known about the functions of circRNAs in the skeletal system, and the role of circRNAs in the mechanism by which bone morphogenetic protein 2 (BMP2) promotes bone differentiation remains unknown. Here, we performed RNA-seq to analyze differential expression of circRNA during different osteoblast differentiation stages and investigated the relevant mechanisms.
  
  
  METHODS
  Alkaline phosphatase (ALP) staining and activity were performed to assess osteogenic differentiation in MC3T3-E1 cells. The expression of osteogenic markers in MC3T3-E1 cells and the differential expression levels of circRNAs were measured and validated by qRT-PCR. Osteogenic marker proteins were measured by western blot. RNA-seq was performed to detect differential expression of circRNAs during the osteogenic differentiation of MC3T3-E1 cells induced by BMP2. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and PANTHER pathway analyses were performed to predict the functions of differentially expressed circRNAs and potentially co-expressed target genes. The microRNA (miRNA) targets of the circRNAs and circRNA-miRNA interactions were predicted by miRanda. The circRNA-miRNA co-expression network was constructed based on the correlation analysis between the differentially expressed circRNAs and miRNAs. A graph of the circRNA-miRNA network was created using Cytoscape 3.01.
  
  
  RESULTS
  The Cell Counting Kit 8 (CCK-8) assay showed that BMP2 promoted the proliferation of osteoblasts in vitro. Both the intracellular ALP content and activity were increased in BMP2-treated MC3T3-E1 cells. In addition, the mRNA and protein levels of the osteoblastic markers ALP, Sp7 transcription factor (SP7) and runt-related transcription factor 2 (RUNX2) were substantially up-regulated. In the present study, 158 circRNAs were differentially expressed by a fold-change ≥2.0, P<0.05 and false discovery rate <0.05. Among these, 74 circRNAs were up-regulated, while 84 circRNAs were down-regulated. In addition, the expression levels of circRNA.5846, circRNA.19142 and circRNA.10042 were significantly increased in the BMP2 group. Furthermore, by analyzing the target mRNAs of miR-7067-5p using GO and PANTHER pathway analyses, circ19142 and circ5846 were found to be not only strongly associated with the biological process of the positive regulation of developmental processes but also related to the fibroblast growth factor, epidermal growth factor, platelet-derived growth factor and Wnt signaling pathways, which are involved in cell growth and differentiation.
  
  
  CONCLUSIONS
  The present study identified circ19142 and circ5846 as being associated with osteoblast differentiation and BMP2 may induce osteogenic differentiation through a circ19142/circ5846-targeted miRNA-mRNA axis.
authors.affiliation: Southern Medical University, Guangzhou 510515, China; Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Dong-Yang
authors.initials: DY
authors.lastname: Qian
authors.affiliation: Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Guang-Bin
authors.initials: GB
authors.lastname: Yan
authors.affiliation: Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Bai
authors.affiliation: Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Shu-Jiang
authors.initials: SJ
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedics, the First Affiliated Hospital, Guangzhou Medical University/Guangdong key Laboratory of Orthopaedic Technology and Implant Materials, Guangzhou 510120, China.
authors.forename: Yong-Chang
authors.initials: YC
authors.lastname: Yao
authors.affiliation: Southern Medical University, Guangzhou 510515, China; Department of Orthopedics, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou 510010, China. Electronic address: gzxiahong2@126.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Xia
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics*; D002452:Cell Count / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D063990:Gene Ontology; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000235:genetics; D010006:Osteoblasts / Q000378:metabolism; D010012:Osteogenesis / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D000079962:RNA, Circular; D015398:Signal Transduction / Q000235:genetics; D015854:Up-Regulation / Q000235:genetics
chemical_list: D015415:Biomarkers; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D036341:Intercellular Signaling Peptides and Proteins; D035683:MicroRNAs; D000079962:RNA, Circular; D012313:RNA
keywords: BMP2; Osteogenic differentiation; circRNAs