Learning from Embryogenesis-A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development

Abstract

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial-mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells.

Keywords: embryogenesis; melanoblasts; melanoma; miRNAs.

Figure 1

miRNA sequencing in NHEMs, MBrCs, and melanoma cell lines reveals a group of miRNAs, which are strongly regulated only in melanoma. (A) Principal component analysis of miRNA-seq data plot of the first three principal components (PCs). Samples with related gene expression profiles are closer in the three-dimensional embedding. Each sphere represents an RNA-seq sample, and replicates are shown in the same color: NHEM (n = 4), MBrC (n = 2), melanoma primary tumor (PT) cell lines Mel Juso, Mel Wei and Mel Ei, melanoma metastasis (MET) cell lines Mel Im, Mel Ju and Hmb2. (B) Differential gene expression analysis of miRNA-seq data in the indicated sample pairs is shown as a heatmap of log2fold values. The arrow indicates miRNAs, which are equally expressed in MBrCs and NHEMs but strongly upregulated in melanoma cell lines. (C) Venn diagram of miRNAs that are not significantly differentially expressed in MBrCs vs. NHEMs (p-value > 0.05) but significantly differentially expressed (p-value < 0.05) in MBrCs vs. melanoma primary tumor (PT) or metastasis-derived (MET) melanoma cell lines.

Figure 2

Significantly differentially expressed miRNAs in melanoma cells. Heatmap of log2fold values of all miRNAs that were not regulated in MBrCs vs. NHEMs (p > 0.05), but differentially expressed (p < 0.05) in MBrCs vs. melanoma primary tumor (PT), and MBrCs vs. melanoma metastasis-derived (MET) cell lines. miRNAs, which were upregulated in melanoma compared to MBrCs are shown in red, and miRNAs downregulated in melanoma are shown in blue. Both are indicated by black arrows. Mentions in the literature of functional relevance of the respective miRNA in melanoma or other cancers are depicted by +.

Figure 3

Target gene analysis of strongly upregulated miRNAs in melanoma compared to MBrCs and NHEMs. (A) Venn diagram of predicted targets of miR-105-5p and miR-767-5p. (B) Venn diagram of predicted target genes of miR-3689f, miR-6840-5p, miR-4652-5p, miR-4706, and miR-4435. (C) STRING network analysis of selected target genes of the 24 shared target genes of miR-3689f, miR-6840-5p, miR-4652-5p, miR-4706, and miR-4435 (red), and additional genes involved in the network (grey).

Figure 4

Significant downregulation of miRNA target genes in melanoma cells. Enrichment plot for (A) miR-105-5p, (B) miR-767-5p, (C) miR-96-5p, (D) miR-4425, (E) miR-182-5p, and (F) miR-3689F. Profile of the running enrichment score (green) and positions of gene set members of the respective miRNA target genes on the rank-ordered list of genes differentially regulated in melanoma cells compared to MBrCs and NHEMs identified by gene expression array analysis. Genes upregulated in melanoma are shown on the left side of the graph in red, genes downregulated in melanoma on the right side in blue.

Figure 5

QRT-PCR shows a strong downregulation of miRNAs of the miR-506–514 cluster and miR-1299. Analysis of miR-508-3p, miR-509-3p, miR-514a-3p, and miR-1299 expression in normal human epidermal melanocytes (NHEM), melanoblast-related cells (MBrC), and at least 6 different melanoma cell lines (MM) via qRT-PCR. Bars represent mean + SEM, statistical significance was calculated using one-way ANOVA and subsequent Tukey’s Multiple Comparison Test with ΔCP values before normalization to NHEM and is indicated as * p ≤ 0.05 and *** p ≤ 0.001.

Figure 6

Target gene analysis of strongly downregulated miRNAs in melanoma compared to MBrCs and NHEMs. (A) Venn diagram of shared predicted targets of miR-1299, miR-199b-3p and miR-664a-5p. (B) STRING network analysis of selected target genes of the 99 shared targets of miR-1299, miR-199b-3p, and miR-664a-5p (blue), which belong to one network and additional genes involved in the network (grey).

Figure 7

miRNAs are significantly regulated in only metastasis-derived melanoma cell lines. (A) miRNAs that are not regulated in MBrCs vs. NHEMs (p > 0.05) and only significantly differentially expressed (p < 0.05) in MBrCs vs. melanoma metastasis-derived (MET) cell lines with log2fold in MET vs. PT > 1 or < −1 and in MBrC vs. PT > −1 and simultaneously < 1. Mentions in the literature of functional relevance of the respective miRNA in melanoma or other cancers are indicated (+). (B) Venn diagram of shared predicted targets of upregulated miR-4426, miR-203a-3p, and miR-4516. (C) Venn diagram of shared predicted targets of downregulated miR-20b-3p, miR-363-3p, and miR-211-5p. (D) STRING network analysis of connected target genes of the 51 shared targets of miR-20b-3p, miR-363-3p, and miR-211-5p (blue) and additionally involved genes (grey).