Integrated miRNA-mRNA analysis revealing the potential roles of miRNAs in chordomas

Abstract

Introduction: Emerging evidence suggests that microRNAs (miRNAs) are crucially involved in tumorigenesis and that paired expression profiles of miRNAs and mRNAs can be used to identify functional miRNA-target relationships with high precision. However, no studies have applied integrated analysis to miRNA and mRNA profiles in chordomas. The purpose of this study was to provide insights into the pathogenesis of chordomas by using this integrated analysis method.

Methods: Differentially expressed miRNAs and mRNAs of chordomas (n = 3) and notochord tissues (n = 3) were analyzed by using microarrays with hierarchical clustering analysis. Subsequently, the target genes of the differentially expressed miRNAs were predicted and overlapped with the differentially expressed mRNAs. Then, GO and pathway analyses were performed for the intersecting genes.

Results: The microarray analysis indicated that 33 miRNAs and 2,791 mRNAs were significantly dysregulated between the two groups. Among the 2,791 mRNAs, 911 overlapped with putative miRNA target genes. A pathway analysis showed that the MAPK pathway was consistently enriched in the chordoma tissue and that miR-149-3p, miR-663a, miR-1908, miR-2861 and miR-3185 likely play important roles in the regulation of MAPK pathways. Furthermore, the Notch signaling pathway and the loss of the calcification or ossification capacity of the notochord may also be involved in chordoma pathogenesis.

Conclusion: This study provides an integrated dataset of the miRNA and mRNA profiles in chordomas, and the results demonstrate that not only the MAPK pathway and its related miRNAs but also the Notch pathway may be involved in chordoma development. The occurrence of chordoma may be associated with dysfunctional calcification or ossification of the notochord.

Figure 1. Identification of chordoma and notochord tissues.

H&E stained section of a chordoma (A) showing moderately atypical physaliphorous (intracellular, bubble-like vacuoles) cells set within a myxoid matrix. The tumor cells demonstrated positive immunostaining for cytokeratin (B), S100 protein (C), and brachyury (D). H&E stained section of a notochord tissue (E) showed a clear boundary between the notochord and the surrounding tissue. The notochord cells demonstrated positive immunostaining for brachyury (F).

Figure 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.

Figure 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 category and the horizontal axis is the -lg p value of the GO category.

Figure 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.

Figure 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.

Figure 6. Quantitative analysis of miRNA expression in chordomas.

Differentially expressed 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).