Nal.pone.0066676.gIntegrated miRNA-mRNA Analysis of Chordomasfindings [25]. However, these genes were

Nal.pone.0066676.gIntegrated miRNA-mRNA Analysis of Title Loaded From File Chordomasfindings [25]. However, these genes were not identified as target of the dysregulated miRNAs. These results support our previous findings that ENO1, PKM2, and Gp96 may play roles in chordomas, but also imply that these genes are not directly regulated by any of the 33 differentially expressed miRNAs.4.2 Selection of Fetal Notochord as the ControlDifferential miRNA expression profiling analysis of human tumor and normal tissues allows the identification of miRNAs that may provide key information regarding carcinogenesis [10]. Chordomas are the most frequent primary tumors of the spine and account for 1? of all primary bone tumors [1,2]. Chordomas were originally thought to develop from cartilage [1]; however, chordomas localize at the same sites as notochordal remnants [3], and immunohistochemical [26] and ultrastructural [27] similarities between chordomas and notochordal tissue suggest that chordomas arise from remnants of the embryonic notochord [28]. Recently, brachyury, which is crucial in notochord development, was also observed in tumor cells of primary chordomas [14], which provide compelling evidence to support the hypothesis that chordomas originate from the remnant notochord. In comparative studies of chordomas, it is difficult to find corresponding normal tissues to use as a control. During embryogenesis, most notochordal cells die and are replaced by bone in the vertebral bodies and by the nucleus pulposus in the intervertebral discs [29]. In humans, notochordal cells are believed to disappear by the age of 10 years [30]. Additionally, the nucleus pulposus expresses neither cytokeratins nor brachyury [14], whereas both are coexpressed in the embryonic notochord [14]. Therefore, whether the nucleus pulposus is formed directly by notochordal cells is unclear [29]. In a previous study [10], normal muscle tissues were used as a control. In our study, we choose fetal notochord as a control for chordomas. The previous study identified 4 differentially expressed miRNAs that overlapped with the 33 differentially expressed miRNAs in the present study, including two miRNAs (miR-1228 and miR-1268) with the same downregulation trend that we observed and two miRNAs (miR23a and miR-26a) with dysregulation trends opposite to those that we observed. We believe that the differences between our results and those of the previous study mainly arise from differences in the control and small sample size. Our microarray results identified a set of miRNAs that were differentially expressed in chordoma tissue relative to fetal notochord tissue. The differentially expressed miRNAs were clearly different between the chordoma and notochord tissue and therefore may, together with their target genes, be involved in the pathogenesis of chordomas. On the other hand, only 3 (33/1,105) of all analyzed miRNAs showed a difference in the expression level, which suggests a close relationship between these 2 tissues. However, it should be emphasized that the differences that we found may be influenced by the different developmental Title Loaded From File stages for the chordoma and fetal notochord tissues. Additionally, because of the limited number of samples (only 6 tissue samples) in this study, the results may have been influenced by individual differences as well.The GO analysis showed that the apoptotic process was the critical GO term. Evading apoptosis is an essential alteration for the malignant growth of cells [31.Nal.pone.0066676.gIntegrated miRNA-mRNA Analysis of Chordomasfindings [25]. However, these genes were not identified as target of the dysregulated miRNAs. These results support our previous findings that ENO1, PKM2, and Gp96 may play roles in chordomas, but also imply that these genes are not directly regulated by any of the 33 differentially expressed miRNAs.4.2 Selection of Fetal Notochord as the ControlDifferential miRNA expression profiling analysis of human tumor and normal tissues allows the identification of miRNAs that may provide key information regarding carcinogenesis [10]. Chordomas are the most frequent primary tumors of the spine and account for 1? of all primary bone tumors [1,2]. Chordomas were originally thought to develop from cartilage [1]; however, chordomas localize at the same sites as notochordal remnants [3], and immunohistochemical [26] and ultrastructural [27] similarities between chordomas and notochordal tissue suggest that chordomas arise from remnants of the embryonic notochord [28]. Recently, brachyury, which is crucial in notochord development, was also observed in tumor cells of primary chordomas [14], which provide compelling evidence to support the hypothesis that chordomas originate from the remnant notochord. In comparative studies of chordomas, it is difficult to find corresponding normal tissues to use as a control. During embryogenesis, most notochordal cells die and are replaced by bone in the vertebral bodies and by the nucleus pulposus in the intervertebral discs [29]. In humans, notochordal cells are believed to disappear by the age of 10 years [30]. Additionally, the nucleus pulposus expresses neither cytokeratins nor brachyury [14], whereas both are coexpressed in the embryonic notochord [14]. Therefore, whether the nucleus pulposus is formed directly by notochordal cells is unclear [29]. In a previous study [10], normal muscle tissues were used as a control. In our study, we choose fetal notochord as a control for chordomas. The previous study identified 4 differentially expressed miRNAs that overlapped with the 33 differentially expressed miRNAs in the present study, including two miRNAs (miR-1228 and miR-1268) with the same downregulation trend that we observed and two miRNAs (miR23a and miR-26a) with dysregulation trends opposite to those that we observed. We believe that the differences between our results and those of the previous study mainly arise from differences in the control and small sample size. Our microarray results identified a set of miRNAs that were differentially expressed in chordoma tissue relative to fetal notochord tissue. The differentially expressed miRNAs were clearly different between the chordoma and notochord tissue and therefore may, together with their target genes, be involved in the pathogenesis of chordomas. On the other hand, only 3 (33/1,105) of all analyzed miRNAs showed a difference in the expression level, which suggests a close relationship between these 2 tissues. However, it should be emphasized that the differences that we found may be influenced by the different developmental stages for the chordoma and fetal notochord tissues. Additionally, because of the limited number of samples (only 6 tissue samples) in this study, the results may have been influenced by individual differences as well.The GO analysis showed that the apoptotic process was the critical GO term. Evading apoptosis is an essential alteration for the malignant growth of cells [31.