Differential expression of microRNAs during melanoma progression: miR-200c, miR-205 and miR-211 are downregulated in melanoma and act as tumour suppressors

Abstract

Background: The incidence of malignant melanoma is increasing faster than that for any other cancer. Histological examination of skin excision biopsies remains the standard method for melanoma diagnosis and prognosis. Significant morphological overlap between benign and malignant lesions complicates diagnosis, and tumour thickness is not always an accurate predictor of prognosis.

Methods: To identify improved molecular markers to support histological examination, we used microarray analysis of formalin-fixed and paraffin-embedded samples from different stages of melanomagenesis to identify differentially expressed microRNAs (miRNAs). Differential expression was validated by qRT-PCR, and functional studies were carried out after transfection of miRNA precursors or inhibitors into melanoma cells to modulate miRNA expression.

Results: In all, 20 miRNAs showed highly significant differential expression between benign naevi and either primary or metastatic melanomas, the majority being downregulated in melanoma, whereas only 2 miRNAs, namely miR-203 and miR-205, were differentially expressed between primary and metastatic melanomas. In functional in vitro assays, overexpression of miR-200c and miR-205 inhibited anchorage-independent colony formation and overexpression of miR-211 inhibited both anchorage-independent colony formation and invasion.

Conclusion: We have identified a series of differentially expressed miRNAs that could be useful as diagnostic or prognostic markers for melanoma and have shown that three miRNAs (namely miR-200c, miR-205 and miR-211) act as tumour suppressors.

Figure 1

Top differentially expressed microRNAs between benign naevi, primary and metastatic melanoma. Venn diagram showing the relationship between the differentially expressed miRNAs from the comparisons shown in Table 1. For each X vs Y comparison, up means expression was higher in X, down means expression was lower in X. Naevus group, n=11; primary group, n=20; metastasis group, n=21.

Figure 2

microRNA expression changes during melanoma progression. (A) Downregulated miRNAs miR-200a, miR-200b, miR-200c, miR-141 and miR-211. (B) Downregulated miRNAs miR-203 and miR-205. It must be noted that the Y axis is on a log₁₀ scale. (C) Upregulated miRNAs miR-20b and miR-675. Mean expression (±s.e.m.) of each miRNA, determined by qRT–PCR, is shown relative to miR-92 and normalised to the Naevus group mean. Naevus group, n=12–17; primary, n=20; metastasis, n=14. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 3

Ectopic expression of miR-200c in melanoma cells causes ZEB2 downregulation and E-cadherin upregulation. Expression was determined by qRT–PCR after transfection of A375 melanoma cells with miR-200c precursor, or a combination of miR-200c and miR-205 precursors (dark shading), or a scrambled control miRNA (intermediate shading); non-transfected negative control (light shading). Mean relative expression levels (±s.e.m.) from three independent experiments are shown (A) Expression of miR-200c relative to miR-92 and normalised to the mean of the negative control. (B) Expression of ZEB2 mRNA after transfection with miR-200c precursor relative to β-actin and normalised to the mean of the negative control. (C) Expression of ZEB2 mRNA after transfection with miR-200c and miR-205 precursors relative to β-actin. (D) Expression of E-cadherin mRNA after transfection with miR-200c precursor relative to β-actin. ^*P<0.05; ^***P<0.001.

Figure 4

Ectopic expression of miR-200c in melanoma cells causes reduced anchorage-independent colony formation. A375 cells were transfected with miR-200c precursor, or a combination of miR-200c and miR-205 precursors, or a scrambled control miRNA and colony formation in methylcellulose was determined after 6 days. Non-transfected A375 cells were used as a negative control. (A) Histogram showing the mean size (±s.e.m.) of 30 colonies from each group, normalised to the mean of the negative control group. ^*P<0.05; ^***P<0.001. (B) Representative colony images on day 6.

Figure 5

Ectopic expression of miR-211 in melanoma cells causes reduced anchorage-independent colony formation and invasion. A375 cells were transfected with miR-211 precursor, or a scrambled control miRNA and colony formation in methylcellulose and invasion in a transwell migration assay was determined. (A) Expression of miR-211 in melanoma and melanocyte cell lines. Immortalised melanocyte lines Hermes 1 (HER1) and Hermes 4a (HER4); melanoma lines EDMEL3, G361, C32 and A375. Mean miR-211 expression from two independent experiments relative to miR-92 and normalised to HER1 is shown. (B) Overexpression of miR-211 in A375 melanoma cells after transfection of precursor miR-211. Mean relative expression levels (±s.e.m.) of miR-211 relative to miR-92 and normalised to the mean of the negative control from three independent experiments are shown. (C) Reduced methylcellulose colony formation in A375 cells after transfection of miR-211 precursor. Histogram shows the mean size (±s.e.m.) of 30 colonies from each group on day 6, normalised to the mean of the negative control group. (D) Representative colony images on day 6. (E) Reduced invasion in A375 cells after transfection of miR-211 precursor. Cells migrating through Matrigel in a transwell inverse invasion assay were quantified after 5 days at the 40 μM layer. The relative cell number was determined as the ratio of cell number in the 40 μM layer/cell number in the origin (0 μM layer). Results represent average ratios (±s.e.m.) of six microscope fields for each group, normalised to the negative control group. (F) Representative stacked confocal images from the origin (0 μM layer) to the tenth layer (90 μM). ^*P<0.05; ^***P<0.001.