Malignant germ cell tumors display common microRNA profiles resulting in global changes in expression of messenger RNA targets

Abstract

Despite their extensive clinical and pathologic heterogeneity, all malignant germ cell tumors (GCT) are thought to originate from primordial germ cells. However, no common biological abnormalities have been identified to date. We profiled 615 microRNAs (miRNA) in pediatric malignant GCTs, controls, and GCT cell lines (48 samples in total) and re-analyzed available miRNA expression data in adult gonadal malignant GCTs. We applied the bioinformatic algorithm Sylamer to identify miRNAs that are of biological importance by inducing global shifts in mRNA levels. The most significant differentially expressed miRNAs in malignant GCTs were all from the miR-371-373 and miR-302 clusters (adjusted P < 0.00005), which were overexpressed regardless of histologic subtype [yolk sac tumor (YST)/seminoma/embryonal carcinoma (EC)], site (gonadal/extragonadal), or patient age (pediatric/adult). Sylamer revealed that the hexamer GCACTT, complementary to the 2- to 7-nucleotide miRNA seed AAGUGC shared by six members of the miR-371-373 and miR-302 clusters, was the only sequence significantly enriched in the 3'-untranslated region of mRNAs downregulated in pediatric malignant GCTs (as a group), YSTs and ECs, and in adult YSTs (all versus nonmalignant tissue controls; P < 0.05). For the pediatric samples, downregulated genes containing the 3'-untranslated region GCACTT showed significant overrepresentation of Gene Ontology terms related to cancer-associated processes, whereas for downregulated genes lacking GCACTT, Gene Ontology terms generally represented metabolic processes only, with few genes per term (adjusted P < 0.05). We conclude that the miR-371-373 and miR-302 clusters are universally overexpressed in malignant GCTs and coordinately downregulate mRNAs involved in biologically significant pathways.

Figure 1. Unsupervised hierarchical clustering analysis of miRNA expression in 42 pediatric tissue samples and 6 GCT cell lines

Sample numbers refer to those in Supplementary Table S1.

Figure 2. Differential expression of the miR-371~373 and miR-302 clusters in malignant GCTs

Samples are color-coded as in Figure 1. A) Expression of the eight main members of the miR-371~373 and miR-302 clusters in all tissue samples in the pediatric and adult datasets. Circles represent microarray expression M-values from individual pediatric samples (normalized within and across arrays), while triangles represent normalized Δ CT values for individual adult samples. B) Hierarchical clustering analysis based on the miR-371~373 and miR-302 clusters in (i) pediatric and (ii) adult samples.

Figure 3. Relationships between expression of miR-302 and miR-371~373 clusters and protein-coding genes

The graphs show linear regression analysis for 21 pediatric samples with matching mRNA and miRNA expression data, plotting levels of protein-coding gene expression (y-axis) against the matched median expression values for members of the miR-371~373 and miR-302 clusters (x-axis). Panel A shows data for the transcription factors SOX17 and TEAD4, plotted against all eight main members of the miRNA clusters. Panel B shows data for four representative common SCR-containing genes, from 22 under-expressed in both pediatric and adult malignant GCTs, plotted against the six miRNAs that contain the common 2-7nt seed. Analogous plots for the remaining 18 genes are shown in Supplementary Figure S9. Samples are color-coded as in Figure 1.

Figure 4. Sylamer landscape plot analysis for the SCR words corresponding to the common seed of miR-372~373 and miR-302a~302d

Log₁₀-transformed and sign-adjusted enrichment p-values for each SCR word, relative to p-values of all other words, are plotted on the y-axis, against the ranked genelist on the x-axis (down-regulated genes to left; up-regulated genes to right). For each comparison, the left hand plot shows the data for the three hexamers complementary to 1-8nt in the common seed region, the central plot the two heptamers and the right hand plot the octamer. The single summed significance score and p-value for all six SCR words in each comparison is given.