MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma

Abstract

Background: Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma.

Methods: Human and canine osteosarcoma patient's samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance.

Results: Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544.

Conclusions: We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease.

Figure 1

14q32 miRNA expression levels and metastasis in human osteosarcoma. (A) Correlation network of 14q32 miRNAs expression in human osteosarcoma samples. Correlation network was constructed with Pearson correlation R >0.9 as the edges. The resulting correlation sub-network contained thirty-six miRNAs and 33 of them mapped to the 14q32 locus. All pairwise correlations used to generate this image are given in Additional file 8: Table S7. (B) Microarray based transcript levels of highly correlated 14q32 miRNAs in osteosarcoma tumors shown relative to the level observed in FT-14 (human osteosarcoma primary tumor). miRNA profiling data from human osteosarcoma tumors where metastasis was observed show stronger downregulation of 14q32 miRNAs (highlighted in red) than primary osteosarcoma tumors where metastasis was not observed. (C) Microarray based transcript levels for miR-382 and miR-154. miR-382 and miR-154 show a high level of correlation (R² = 0.95). (D) qRT-PCR validation for miR-382 and miR-154 expression levels obtained in primary osteosarcoma tumors (FT-14, -7) where metastasis was not observed and three primary osteosarcoma tumors with low levels of (FT-18, -13, -12) where metastasis was observed. miR-382 and miR-154 show a high level of correlation (R² = 0.99). Measurements were carried out in triplicate and were normalized to the expression levels in two independent normal bone tissues that were also carried out in triplicate.

Figure 2

Prognostic significance of genes correlated with 14q32 miRNAs in canine osteosarcoma. (A) Unsupervised clustering of human mRNAs (n = 385) that shows high-level direct correlations to miR-382 for two normal bone tissues and 11 osteosarcoma tumors, for which we have both mRNA profiles and miRNA profiles. A positive correlation to miR-382 is shown in yellow (n = 288), and a negative correlation to miR-382 is shown in blue (n = 97), at the right of the heatmap. Directional Functional enrichment analyses (Ingenuity Pathways Analyses) shows that correlating transcripts are enriched in transcripts involved in metastasis, and direction of change is consistent with increased activity of metastatic function in tumors with decreased levels of 14q32 miRNA member, miR-382. Identities of positive and negatively correlating mRNA are provided in Additional file 6: Table S5 and Additional file 7: Table S6. (B) Unsupervised hierarchical clustering heatmap of canine mRNA found in 26 canine osteosarcoma-derived samples that correspond to human mRNAs correlating to miR-382. The yellow and blue bars represent correlations to miR-382 (positive = yellow or negative = blue) observed in the human data indicating that the direction of change shows similar trends between human and canine data.(C) Kaplan-Meier survival curves generated using the groups shown in 2B. Survival times are significantly different between the two groups of tumors (p-value 0.02).

Figure 3

14q32 miRNA expression level, metastasis and outcome in human osteosarcoma. (A) miR-382 expression levels determined by qRT-PCR in osteosarcoma primary tumor samples with clinical follow-up information. The miRNA expression levels were normalized relative to normal bone tissues. Osteosarcoma samples were ranked based on expression levels of miR-382 from highest to lowest. Primary osteosarcoma patient samples that later developed metastases are highlighted in red and samples from patients who died due to disease are shown in black. Samples were grouped based on whether they were above or below the median expression level. (B) Kaplan-Meier analysis of metastasis in osteosarcoma patients based on miR-382 expression. Group 1 and Group 2 are significantly different (p-value 0.01). Patients with lowest levels of miR-382 expression showed increased likelihood of metastasis. (C) Kaplan-Meier analysis of survival in osteosarcoma patients based on miR-382 expression. Group 1 and Group 2 are different (p-value 0.08). Patients with lowest levels of miR-382 expression showed decreased likelihood of survival.

Figure 4

14q32 orthologous region transcript levels and outcome in canine osteosarcoma. (A) miR-134 and B) miR-544 expression levels determined by qRT-PCR in canine osteosarcoma primary tumor samples with clinical follow-up information. The miRNA expression levels were normalized relative to reactive canine osteoblasts. Osteosarcoma samples were ranked based on expression levels of miR-134 from highest to lowest. Samples were grouped based on whether they were above or below the median expression levels. (C and D) Kaplan-Meier analysis of survival in osteosarcoma patients based on miR-134 and miR-544 expression. Survival of dogs with osteosarcoma in Group 1 and Group 2 are significantly different (miR-134 p-value 0.004, miR-544 p-value 0.01). Dogs with lowest levels of miR-134 or miR-544 expression showed decreased likelihood of survival.