Cated that 7 GOs were significantly regulated by the downregulated genes, whereas 184 GOs were significantly regulated by the upregulated genes. The mainFigure 2. Hierarchical clustering of differentially expressed miRNAs and mRNAs in chordoma tissues (Ch1, Ch2, Ch3) and notochord tissues (N1, N2, N3). (A) The 33 miRNAs listed above were differentially expressed (P,0.05) between the chordoma tissues and notochord tissues. (B) In total, 2,791 mRNAs differed between the two sample groups. The color scale shown on the top illustrates the relative expression level of the indicated miRNA across all samples: red Chebulagic acid denotes high expression levels, whereas green denotes low expression levels. doi:10.1371/journal.pone.0066676.gIntegrated miRNA-mRNA Analysis of ChordomasGO categories targeted by the upregulated genes included gene expression, axon guidance, and apoptotic processes (Figure 3). In contrast, significant GOs corresponding to the downregulated genes included positive regulation of the action potential, multicellular organismal development, and cerebral cortex regionalization (Figure 3).3.4 Pathway AnalysisPathway analyses showed that 44 different pathways corresponded to the significantly upregulated intersecting genes. Overall, a genetic cluster summarizing the functions of focal AKT inhibitor 2 web adhesion, pathways in cancer, and ECM-receptor interactions wasfound to have the highest relationship with the chordoma group (Figure 4, Table S6). By considering the genetic pathways listed in KEGG as being involved in cancer development, we identified several significantly related pathways, including MAPK signaling, neurotrophin signaling, TGF-beta signaling, Wnt signaling, insulin signaling, p53 signaling, ErbB signaling, Notch signaling, chemokine signaling, Jak-STAT signaling, T cell receptor signaling, calcium signaling, RIG-I-like receptor signaling, mTOR signaling, and GnRH signaling (Figure 4, Table S6). In addition to these classical pathways, several clusters of genes associated with the following major cancer entities were overrepresented, which suggests a common oncogenic basis: small cellFigure 3. miRNA targeted significant GOs. The upper chart shows the GOs targeted by downregulated miRNA, and the lower chart shows the GOs targeted by overexpressed miRNA. The vertical axis is the GO 23148522 category and the horizontal axis is the -lg p value of the GO category. doi:10.1371/journal.pone.0066676.gIntegrated miRNA-mRNA Analysis of ChordomasFigure 4. Pathway analysis based on miRNA-targeted genes. Significant pathways targeted by downregulated miRNA are shown. The vertical axis is the pathway category, and the horizontal axis is the enrichment of pathways. doi:10.1371/journal.pone.0066676.glung cancer, prostate cancer, glioma, renal cell carcinoma, pancreatic cancer, thyroid cancer, and non-small cell lung cancer (Figure 4, Table S6). Notably, the Notch signaling pathway was dysregulated in chordoma; aberrant Notch signaling is associated with tumorigenesis in many types of tumors [16,17]. Six genes (NOTCH2, NCOR2, CREBBP, JAG1, KAT2A and NCSTN) related to the Notch signaling pathway were upregulated in chordoma tissues.3.5 Validation of miRNA Array DataTo validate the microarray data, 1676428 7 miRNAs were selected and subjected to qRT-PCR validation. Our pathway analysis showed that the most highly overrepresented genetic pathway involved in chordoma development was the MAPK signaling pathway, which had the lowest P value (P = 4.79E-8). Given that constitutive act.Cated that 7 GOs were significantly regulated by the downregulated genes, whereas 184 GOs were significantly regulated by the upregulated genes. The mainFigure 2. Hierarchical clustering of differentially expressed miRNAs and mRNAs in chordoma tissues (Ch1, Ch2, Ch3) and notochord tissues (N1, N2, N3). (A) The 33 miRNAs listed above were differentially expressed (P,0.05) between the chordoma tissues and notochord tissues. (B) In total, 2,791 mRNAs differed between the two sample groups. The color scale shown on the top illustrates the relative expression level of the indicated miRNA across all samples: red denotes high expression levels, whereas green denotes low expression levels. doi:10.1371/journal.pone.0066676.gIntegrated miRNA-mRNA Analysis of ChordomasGO categories targeted by the upregulated genes included gene expression, axon guidance, and apoptotic processes (Figure 3). In contrast, significant GOs corresponding to the downregulated genes included positive regulation of the action potential, multicellular organismal development, and cerebral cortex regionalization (Figure 3).3.4 Pathway AnalysisPathway analyses showed that 44 different pathways corresponded to the significantly upregulated intersecting genes. Overall, a genetic cluster summarizing the functions of focal adhesion, pathways in cancer, and ECM-receptor interactions wasfound to have the highest relationship with the chordoma group (Figure 4, Table S6). By considering the genetic pathways listed in KEGG as being involved in cancer development, we identified several significantly related pathways, including MAPK signaling, neurotrophin signaling, TGF-beta signaling, Wnt signaling, insulin signaling, p53 signaling, ErbB signaling, Notch signaling, chemokine signaling, Jak-STAT signaling, T cell receptor signaling, calcium signaling, RIG-I-like receptor signaling, mTOR signaling, and GnRH signaling (Figure 4, Table S6). In addition to these classical pathways, several clusters of genes associated with the following major cancer entities were overrepresented, which suggests a common oncogenic basis: small cellFigure 3. miRNA targeted significant GOs. The upper chart shows the GOs targeted by downregulated miRNA, and the lower chart shows the GOs targeted by overexpressed miRNA. The vertical axis is the GO 23148522 category and the horizontal axis is the -lg p value of the GO category. doi:10.1371/journal.pone.0066676.gIntegrated miRNA-mRNA Analysis of ChordomasFigure 4. Pathway analysis based on miRNA-targeted genes. Significant pathways targeted by downregulated miRNA are shown. The vertical axis is the pathway category, and the horizontal axis is the enrichment of pathways. doi:10.1371/journal.pone.0066676.glung cancer, prostate cancer, glioma, renal cell carcinoma, pancreatic cancer, thyroid cancer, and non-small cell lung cancer (Figure 4, Table S6). Notably, the Notch signaling pathway was dysregulated in chordoma; aberrant Notch signaling is associated with tumorigenesis in many types of tumors [16,17]. Six genes (NOTCH2, NCOR2, CREBBP, JAG1, KAT2A and NCSTN) related to the Notch signaling pathway were upregulated in chordoma tissues.3.5 Validation of miRNA Array DataTo validate the microarray data, 1676428 7 miRNAs were selected and subjected to qRT-PCR validation. Our pathway analysis showed that the most highly overrepresented genetic pathway involved in chordoma development was the MAPK signaling pathway, which had the lowest P value (P = 4.79E-8). Given that constitutive act.