Mesenchymal stem cells (MSCs) are believed to be the cell of origin for most sarcomas including osteosarcoma and malignant fibrous histiocytoma (MFH/UPS). transcription was performed using T7 RNA polymerase to obtain Cy3-labeld cRNA. 2.3. Microarray Hybridization and Data Analysis To investigate the difference in gene expression between sarcoma cells and MSCs, gene expression profiling was performed by Agilent array analysis (Agilent Technologies, B?blingen, Germany). Briefly, the Cy3-labeled cRNA were fragmented with hybridization answer and hybridized with Agilent Expression Array (Whole rat genome array) for 17 hrs at 65C and 10?rpm and subsequently washed at room temperature. The microarray slides were scanned on an Agilent DNA Microarray Scanner. Data analysis was carried out using Agilent Feature Extraction software, analyzing pathways that were differentially expressed in sarcoma cells compared to MSCs. 3. Results 3.1. Gene Expression Profiling Distinguishes Rat Sarcomas from MSCs In this study, oligonucleotide microarray made up of probe units for 26,930 rat genes were used to obtain genome-wide expression profiles of chemically induced rat osteosarcoma, MFH cells, and MSCs. To gain insight into the biological processes participating in sarcomagenesis, we performed pathway analysis and hierarchical clustering analysis. The hierarchical clustering analysis of expression profiling data clearly showed individual clusters among osteosarcoma, MFH, and MSCs (Physique 1). We subsequently focused on the genes strongly expressed in MSCs compared to both sarcomas for pathway analysis. Open in a separate window Physique 1 Taxifolin inhibitor The hierarchical clustering analysis of expression profiling data clearly shows individual clusters among osteosarcoma, MFH, and MSCs. 3.2. Differential Gene Expression Profiles Between Rat Osteosarcoma and MFH, Indicating the Altered Pathways Firstly, we analyzed stem cell markers including mesenchymal stem cell markers. Mesenchymal stem cell markers, CD44, CD73, CD90, and CD105 were all strongly expressed in MSCs compared to both sarcomas. Other stem cell markers such as STAT3 and Sox4 were also strongly expressed in MSCs, while angiopoietin-1 was highly expressed in osteosarcoma cells (Physique 2). Open in a separate window Physique 2 The ratio of scale transmission intensity of mesenchymal stem cell markers and other stem cell markers. The graph indicates the fold changes in rat sarcomas in comparison to rat MSCs. Genes that were differentially expressed were analyzed in the context of the pathways in which they function using the KEGG pathway map. Pathways with a higher incidence of strongly expressed genes in MSCs were cell adhesion molecules, cytokine-cytokine-receptor conversation, ECM-receptor conversation, chemokine signaling, Jak-STAT signaling, and Wnt signaling pathways. Those showing higher incidence in both sarcomas were DNA replication, cell cycle, mismatch repair, and Hedgehog signaling pathways (Table 1). Table 1 Pathways with higher incidence of strongly expressed genes. scale signal intensity of cell adhesion and extracellular matrix Taxifolin inhibitor conversation molecules in both sarcomas compared to MSCs are shown in Physique 3. Open in a separate window Physique 3 The ratio of scale transmission intensity of cell KIAA1704 adhesion and extracellular matrix conversation molecules in both sarcomas compared to MSCs are shown. Integrins and their ligands CAMS were strongly expressed in MSCs, especially differential expression of CAMs between sarcomas and MSCs was significant. Matrix metalloproteinase 2 and 9 also showed higher expression in MSCs. Integrins and their ligands CAMS were strongly expressed in MSCs. In particular, differential expression of CAMs between sarcomas and MSCs was significant (Table 2). Table 2 Genes expressed significantly differentiate between rat sarcomas and MSCs. Scale transmission intensities in rat sarcomas comparing to MSCs. Matrix metalloproteinase 2 and 9 also showed higher expression in Taxifolin inhibitor MSCs. Interleukins and their receptors also showed higher expression in MSCs compared to both sarcomas, except for IL6 and its receptor IL6Ra in osteosarcoma, suggesting the possible involvement of IL6 signaling in osteosarcoma development (Physique 4). Open in a separate window Physique 4 Interleukins and their receptors showed higher expression in Taxifolin inhibitor MSCs compared to both sarcomas, except for IL6 and its receptor IL6Ra in osteosarcoma. The ratio of most chemokines and their receptors also showed predominant expression in MSCs compared to both sarcomas, except for CXCR 3 and 7 (Physique 5). CXCR7 is one of the receptors for CXCL12 other than CXCR4, therefore the CXCL12-CXCR7 axis might be involved in sarcoma development through tumor cell and stromal cell conversation. Open in a separate window Physique 5 The ratio of most chemokines and their receptors also showed predominant expression in MSCs compared to both sarcomas, except.