The Potential Role of Selected miRNA in Uveal Melanoma Primary Tumors as Early Biomarkers of Disease Progression

Abstract

Uveal melanoma (UM) is the most common primary tumor of the eye diagnosed in adults, associated with a high risk of metastasis and thereby, poor prognosis. Among known risk factors for the development of metastatic disease is the loss of BAP1 expression and chromosome 3 monosomy in the primary tumor. However, the expression levels of specific micro RNAs (miRNA) in tumor tissue may also serve as a valuable marker for determining the risk of metastatic disease in patients with primary uveal melanoma. In our study, we analyzed the miRNA expression data of cases selected from The Cancer Genome Atlas study on uveal melanoma, and determined a panel of 15 miRNAs differentially expressed between patients with primary and metastatic disease. Next, 6 miRNAs were validated on a group of 46 tumor samples from primary and metastatic patients. We have shown, that expression of hsa-miR-592, hsa-miR-346, and hsa-miR-1247 was significantly increased, while hsa-miR-506 and hsa-miR-513c were decreased in the tumors of patients with metastatic disease. Hsa-miR-196b expression did not differ between the two subgroups, however, we showed significant correlation with BAP1 expression. Moreover, hsa-miR-592 also showed correlation with monosomy 3 tumors. Gene ontology analysis revealed involvement of those miRNAs with cellular processes mediating the metastatic process. Our results showed that miRNAs play an important role in the deregulation of several oncogenic pathways in UM and can, thereby, promote metastatic spread to distant organs. Moreover, differentially expressed miRNAs may be used as an interesting biomarker for the assessment of metastatic risk in uveal melanoma patients.

Keywords: BAP1; chromosome 3 monosomy; metastasis; micro RNA; uveal melanoma.

Figure 1

Micro RNA differentially expressed between primary and metastatic uveal melanoma TCGA samples. Altogether, the expression of 37 miRNAs differed between analyzed samples, whereas 15 were considered to be statistically significant (marked with frames), with false discovery rate (FDR) < 0.05. The results are displayed as log2 transformation of normalized CPM values.

Figure 2

Analysis of miRNA expressed differentially between primary and metastatic uveal melanoma samples. The results are displayed as relative miRNA fold change calculated as log₂-ΔΔCt. Each dot represents an individual patient. The graph represents mean ± SD. The statistical differences between the two groups were analyzed with Mann–Whitney test where: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

The expression of BAP1 protein and status of chromosome 3 analyzed in formalin fixed and paraffin embedded (FFPE) primary tumor samples. (A) The BAP1 status was analyzed with immunohistochemistry (IHC) staining. Upper panel represents tumor with BAP1 expression, bottom panel represents one with loss of BAP1 (left: hematoxylin and eosin staining; right: IHC for BAP1). Scale bar represents 20 µm, magnification: 20×. (B) Chromosome 3 status was analyzed with fluorescent in situ hybridization (FISH). White arrows point out the nuclei with chromosome 3 disomy, red arrows with monosomy. Scale bar represents 50 nm, magnification: 100×. (C) Kaplan-Meier survival curves for progression free survival in group with and without BAP1 expression (left) and monosomy 3 (right), respectively.

Figure 4

The correlation between selected miRNAs with BAP1 expression and chromosome 3 monosomy in uveal melanoma primary tumors. The results are displayed as relative miRNA fold change calculated as log₂-ΔΔCt. The statistical differences between two groups were analyzed with the Mann–Whitney test where: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (left). Gene ontology (GO) enrichment analysis for hsa-miR-196b and has-miR-592. Results are displayed as −log₁₀(FDR) of given GO terms. The size of dots corresponds to the number of miRNA target genes detected in the given GO term (right). The analyses were performed for hsa-miR-196b (A) and hsa-miR-592 (B).