Ivation of the MAPK signaling pathway plays a pivotal role in various human neoplasms [18] and that the MAPK signalingpathway has been association with chordomas [19], our findings prompted us to perform analyses to identify miRNAs with the potential to target the MAPK pathway. In the set of significantly dysregulated miRNAs, 5 downregulated miRNAs (miR-149-3p, miR-663a, miR-1908, miR-2861, and miR-3185) were predicted to target genes encoding 7 upregulated MAPK signaling pathway-related mRNAs (FGF2, JUND, DUSP4, MAP3K3, TGFB1, PRKACA and RAPGEF2) (Figure 5). The 5 differentially expressed miRNAs were selected on the basis of their involvement in the MAPK pathway and were subjected to qRT-PCR validation. Additionally, because HDAC-IN-3 chordoma is a primary bone tumor, miR762 and miR-1228 were also included for validation because they are involved in calcification [20] or PLV-2 web osteoblast differentiation [21,22]. All the 7 miRNAs were present in 13 chordoma samples (including 3 used for microarray analysis) and 3 notochord samples (used for microarray analysis). Differential expression wasIntegrated miRNA-mRNA Analysis of ChordomasFigure 5. miRNA-gene network of the MAPK signaling pathway in chordomas. Blue box nodes represent downregulated mRNAs, pink box nodes represent upregulated mRNAs, and blue cycle nodes represent downregulated miRNAs. doi:10.1371/journal.pone.0066676.gconfirmed for all the miRNAs analyzed, as shown in Figure 6. These 7 miRNAs may therefore play a role in the malignant progression of chordomas.Discussion 4.1 Integrated miRNA-mRNA Analysis of ChordomasCancer is a complex genetic disease that involves structural and regulatory abnormalities in both coding and non-coding genes, and abnormal expression of miRNA appears to be representative of aberrant gene expression in cancer cells [23]. Several miRNAs have been found to be involved in the initiation and progression of several types of human cancers [23]. Duan et al. [10] first established a direct connection between a cell signaling pathway implicated in the molecular pathogenesis of chordoma and the miRNA machinery; they profiled 21 miRNAs that were differentially expressed in chordoma tissues and chordoma cell lines when compared with normal muscle tissues and found that miR-1 and miR-206 were particularly downregulated in chordomas. Overexpression of miR-1 was found to suppress Met expression and inhibit the growth of chordoma cells. Therefore, miRNA-1 was suggested to have a functional effect on the pathogenesis of chordoma. Recently, it has been suggested that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision [24]. To our knowledge, the network of miRNA-mRNA interactions in chordomas has not been described. In this study, we have introduced integrated analysis of miRNA and mRNA expression profiles in classical primary chordoma tissues. Our miRNA microarray results revealed a set of miRNAs that are differentially expressed in chordoma tissue when compared with fetal notochord tissue. Our mRNA microarray results showed that ENO1, PKM2, and Gp96 were upregulated in chordoma tissue relative to notochord tissue. This result is consistent with our previousFigure 6. Quantitative analysis of miRNA expression in chordomas. Differentially expressed 1676428 miRNAs (miR-149-3p, miR-663a, miR-1908, miR-3185, miR-2861, miR-762, and miR-1228-5p) in chordomas (n = 13) relative to fetal notochords (n = 3). doi:10.1371/jour.Ivation of the MAPK signaling pathway plays a pivotal role in various human neoplasms [18] and that the MAPK signalingpathway has been association with chordomas [19], our findings prompted us to perform analyses to identify miRNAs with the potential to target the MAPK pathway. In the set of significantly dysregulated miRNAs, 5 downregulated miRNAs (miR-149-3p, miR-663a, miR-1908, miR-2861, and miR-3185) were predicted to target genes encoding 7 upregulated MAPK signaling pathway-related mRNAs (FGF2, JUND, DUSP4, MAP3K3, TGFB1, PRKACA and RAPGEF2) (Figure 5). The 5 differentially expressed miRNAs were selected on the basis of their involvement in the MAPK pathway and were subjected to qRT-PCR validation. Additionally, because chordoma is a primary bone tumor, miR762 and miR-1228 were also included for validation because they are involved in calcification [20] or osteoblast differentiation [21,22]. All the 7 miRNAs were present in 13 chordoma samples (including 3 used for microarray analysis) and 3 notochord samples (used for microarray analysis). Differential expression wasIntegrated miRNA-mRNA Analysis of ChordomasFigure 5. miRNA-gene network of the MAPK signaling pathway in chordomas. Blue box nodes represent downregulated mRNAs, pink box nodes represent upregulated mRNAs, and blue cycle nodes represent downregulated miRNAs. doi:10.1371/journal.pone.0066676.gconfirmed for all the miRNAs analyzed, as shown in Figure 6. These 7 miRNAs may therefore play a role in the malignant progression of chordomas.Discussion 4.1 Integrated miRNA-mRNA Analysis of ChordomasCancer is a complex genetic disease that involves structural and regulatory abnormalities in both coding and non-coding genes, and abnormal expression of miRNA appears to be representative of aberrant gene expression in cancer cells [23]. Several miRNAs have been found to be involved in the initiation and progression of several types of human cancers [23]. Duan et al. [10] first established a direct connection between a cell signaling pathway implicated in the molecular pathogenesis of chordoma and the miRNA machinery; they profiled 21 miRNAs that were differentially expressed in chordoma tissues and chordoma cell lines when compared with normal muscle tissues and found that miR-1 and miR-206 were particularly downregulated in chordomas. Overexpression of miR-1 was found to suppress Met expression and inhibit the growth of chordoma cells. Therefore, miRNA-1 was suggested to have a functional effect on the pathogenesis of chordoma. Recently, it has been suggested that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision [24]. To our knowledge, the network of miRNA-mRNA interactions in chordomas has not been described. In this study, we have introduced integrated analysis of miRNA and mRNA expression profiles in classical primary chordoma tissues. Our miRNA microarray results revealed a set of miRNAs that are differentially expressed in chordoma tissue when compared with fetal notochord tissue. Our mRNA microarray results showed that ENO1, PKM2, and Gp96 were upregulated in chordoma tissue relative to notochord tissue. This result is consistent with our previousFigure 6. Quantitative analysis of miRNA expression in chordomas. Differentially expressed 1676428 miRNAs (miR-149-3p, miR-663a, miR-1908, miR-3185, miR-2861, miR-762, and miR-1228-5p) in chordomas (n = 13) relative to fetal notochords (n = 3). doi:10.1371/jour.
