O gain insights into the functional targets of the 33 differentially expressed miRNAs, GO and KEGG pathway annotation were applied to the putative target gene pool.4.5 The Role of Calcification/Osteoblast Differentiation in ChordomasChordoma is a primary bone tumor and originates from remnants of the embryonic notochord, which normally becomes ossified in regions of forming vertebrae and contributes to theIntegrated miRNA-mRNA Analysis of Chordomascenter of the IQ1 biological activity intervertebral discs [29,43,44]. In the present study, we observed that three miRNAs (miR-762, miR-1228, and miR2861) that have been previously reported to be associated with calcification or osteoblast differentiation were significantly dysregulated. MiR-1228 has been found to be upregulated in many malignancies [45,46] and is involved in the inhibition of cellular apoptosis by repressing MOAP1 expression [47]; however, it was downregulated in our study and in previous miRNA study on chordomas [10]. One possible explanation of this discrepancy is that the same miRNA could have different targets and the same mRNA could be targeted by different miRNAs in different cell types [23]. Thus, the same miRNA can participate in distinct pathways and have different effects on cell survival, growth and proliferation, that are dependent on cell type and the gene expression pattern. MiR-1228 is also involved in osteoblast differentiation. Evidence suggests that miR-1228 is involved in 1,25-dihydroxyvitamin D (1,25D)-mediated regulatory effects in bones. BI-78D3 Inactivation of miR-1228 alone was sufficient to abrogate 1,25Dmediated downregulation of BMP2K protein expression [48]. The role of miR-1228 in chordomas has not been elucidated thus far. MiR-2861 is another miRNA that has been shown to play an important physiological role in osteoblast differentiation [21,22]. Overexpression of miR-2861 enhances BMP-induced osteoblastogenesis, and silencing of miR-2861 inhibits bone formation [21]. Altered expression of miR-762 is associated with calcification [20]. A previous study showed that overexpression of miR-762 decreased the protein levels of NCX1, PMCA1, and NCKX4, which are involved in calcium transport [20]. Inhibition of miR762 restores the protein level of calcium pumps and reduces the degree of Pi- and Ca-induced calcification [20]. Therefore, the three downregulated miRNAs in the chordoma group in our study may have an effect on the calcification or osteoblast differentiation of notochord tissues. Loss of the capability of calcification or osteoblast differentiation may result in the formation of undifferentiated notochordal remnants and play a role in the occurrence of chordoma. In conclusion, to our knowledge, this investigation is the first to integratively analyze miRNAs that are differentially expressed between chordomas and notochord tissue. Based on preliminary microarray data, we defined a set of miRNA candidates that are dysregulated in chordomas; however, these findings also imply a close relationship between chordomas and notochord tissue in another way. Our results also demonstrate that not only the MAPK signaling pathway and its inversely related miRNAs (miR149-3p, miR-663a, miR-1908, miR-2861, and miR-3185) but also the Notch signaling pathway may play a role in chordoma development. In addition, the occurrence of chordomas may beassociated with the dysfunction of notochord ossification. However, it should be emphasized that the miRNAs and their target genes integratively analyz.O gain insights into the functional targets of the 33 differentially expressed miRNAs, GO and KEGG pathway annotation were applied to the putative target gene pool.4.5 The Role of Calcification/Osteoblast Differentiation in ChordomasChordoma is a primary bone tumor and originates from remnants of the embryonic notochord, which normally becomes ossified in regions of forming vertebrae and contributes to theIntegrated miRNA-mRNA Analysis of Chordomascenter of the intervertebral discs [29,43,44]. In the present study, we observed that three miRNAs (miR-762, miR-1228, and miR2861) that have been previously reported to be associated with calcification or osteoblast differentiation were significantly dysregulated. MiR-1228 has been found to be upregulated in many malignancies [45,46] and is involved in the inhibition of cellular apoptosis by repressing MOAP1 expression [47]; however, it was downregulated in our study and in previous miRNA study on chordomas [10]. One possible explanation of this discrepancy is that the same miRNA could have different targets and the same mRNA could be targeted by different miRNAs in different cell types [23]. Thus, the same miRNA can participate in distinct pathways and have different effects on cell survival, growth and proliferation, that are dependent on cell type and the gene expression pattern. MiR-1228 is also involved in osteoblast differentiation. Evidence suggests that miR-1228 is involved in 1,25-dihydroxyvitamin D (1,25D)-mediated regulatory effects in bones. Inactivation of miR-1228 alone was sufficient to abrogate 1,25Dmediated downregulation of BMP2K protein expression [48]. The role of miR-1228 in chordomas has not been elucidated thus far. MiR-2861 is another miRNA that has been shown to play an important physiological role in osteoblast differentiation [21,22]. Overexpression of miR-2861 enhances BMP-induced osteoblastogenesis, and silencing of miR-2861 inhibits bone formation [21]. Altered expression of miR-762 is associated with calcification [20]. A previous study showed that overexpression of miR-762 decreased the protein levels of NCX1, PMCA1, and NCKX4, which are involved in calcium transport [20]. Inhibition of miR762 restores the protein level of calcium pumps and reduces the degree of Pi- and Ca-induced calcification [20]. Therefore, the three downregulated miRNAs in the chordoma group in our study may have an effect on the calcification or osteoblast differentiation of notochord tissues. Loss of the capability of calcification or osteoblast differentiation may result in the formation of undifferentiated notochordal remnants and play a role in the occurrence of chordoma. In conclusion, to our knowledge, this investigation is the first to integratively analyze miRNAs that are differentially expressed between chordomas and notochord tissue. Based on preliminary microarray data, we defined a set of miRNA candidates that are dysregulated in chordomas; however, these findings also imply a close relationship between chordomas and notochord tissue in another way. Our results also demonstrate that not only the MAPK signaling pathway and its inversely related miRNAs (miR149-3p, miR-663a, miR-1908, miR-2861, and miR-3185) but also the Notch signaling pathway may play a role in chordoma development. In addition, the occurrence of chordomas may beassociated with the dysfunction of notochord ossification. However, it should be emphasized that the miRNAs and their target genes integratively analyz.