Unraveling the role of microRNA/isomiR network in multiple primary melanoma pathogenesis

Abstract

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial-mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|-2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.

Fig. 1. Single and multiple melanoma classification based on the miRNA profile.

a Unsupervised principal component analysis (PCA) of 24 samples based on the expression profile of all the miRNAs detected at NGS analysis. Familial (yellow) and non-familial (cyan) multiple primary melanomas display a similar microRNA profile, which is different from single cutaneous melanoma (red) and benign nevi (gray). b Cluster analysis and heatmap representation of multiple and single melanoma based on the expression of 22 differentially expressed miRNAs (moderated t-test, adjusted p < 0.05). Red and green color represent the increased or reduced expression across samples. c Cluster analysis and heatmap representation of the first and second melanomas from the same patient based on the expression of 37 differentially expressed miRNAs (paired t-test, p < 0.05). Red and green color represent the increased or reduced expression across samples. d Volcano plot showing the differentially expressed miRNAs at the selected p-value and fold-change combinations.

Fig. 2. Differential microRNA expression in benign nevi (BN), cutaneous melanoma (CM), and in multiple primary melanoma (MPM).

Box and whiskers graph representation of nine microRNAs differentially expressed in single and multiple primary melanomas (P < 0.05). MPM shows higher expression levels of miR-205-5p, miR-200b-3p, and miR-149-5p compared to CM, and higher expression of miR-92b-3p compared to BN. MPM downregulates miR-21-5p compared to CM and miR-146a-5p compared to CM and BN. BN upregulates miR-25-3p and miR-211-5p compared to CM and MPM. Each miRNA was tested in triplicate by quantitative RT-PCR. Relative miRNA expression was normalized on invariant miR-16-5p. The bar shows minimum and maximum values; superimposed points represent all individual values.

Fig. 3. Differential microRNA expression in first vs. second melanoma from the same patient.

Before–after plot showing the paired expression of 4 selected microRNAs in 17 multiple primary melanoma (MPM) patients. miR-92b-3p, miR-205-5p, miR-200b-5p, and miR-149-5p are significantly downregulated in the first melanoma compared to the second melanoma. Each miRNA was tested in triplicate by quantitative RT-PCR. Relative miRNA expression was normalized on invariant miR-16-5p. Paired t-test p-value is reported.

Fig. 4. MetaCore pathway analysis showing the involvement of differently expressed miRNAs in epithelial–mesenchymal transition (EMT).

EMT pathway representation with regulating miRNAs. Log ratio of miRNA expression level in CM/BN (1), MPM/BN (2), and MPM/CM (3) is visualized on the maps as a thermometer-like figure. Upward thermometers have a red color and indicate upregulated signals, and downward (blue) ones indicate downregulated expression level of specific microRNAs. “M” indicates microRNA binding (regulation of gene expression by binding of microRNA to target mRNA), whereas “TR” indicates Transcription regulation (physical binding of a transcription factor to target gene’s promoter). MPM showed higher expression levels of microRNAs involved in the inhibition of epithelial–mesenchymal transition (EMT), including miR-205-5p and miR-200b-3p. (BN, benign nevi; CM, cutaneous melanoma; MPM multiple primary melanoma).

Fig. 5. Comparison of miR-125a-5p and miR-200b-3p isomiR expression in benign nevi (BN), cutaneous melanoma (CM), and multiple primary melanoma (MPM).

a Floating bar chart of miR-125a-5p isomiR expression. Canonical miR-125a-5p (hsa-miR-125a-5p|0|0) shows a lower expression level and opposite expression trend in BN, CM, and MPM if compared to its shorter isomiR (hsa-miR-125a-5p|0|−2). Bars represent min–max and median values. b Floating bar chart of miR-200b-3p isomiR expression showing the same expression trend for canonical miR-200b-3p (hsa-miR-200b-3p|0|0) and its longer isomiR (hsa-miR-200b-3p|0|+1) in MPM groups. Canonical miR-200b-3p was not detected in CM samples. Bars represent min–max and median values. c Box and whiskers graph representation of canonical miR-125a-5p expression assessed with miRCURY LNA assay and overall expression of all miR-125a-5p isoforms, detected using miSCRIPT assay. Results show that the combined expression of miR-125a-5p isoforms of levels is higher in MPM compared to CM. The bar shows minimum and maximum values; superimposed points represent all individual values. d Before–after plot of canonical miR-125a-5p expression (miRCURY LNA assay) and all miR-125a-5p isoforms (miSCRIPT assay) show an opposite trend in the first and second melanoma from the same patient. e Box and whiskers graph representation of canonical miR-200b-3p expression assessed with miRCURY LNA assay and overall expression of all miR-200b-3p isoforms, detected using miSCRIPT assay. Results show that in both there is a higher expression in MPM compared to CM. The bar shows minimum and maximum values; superimposed points represent all individual values. f Before–after plot of canonical miR-200b-3p expression (miRCURY LNA assay) and all miR-200b-3p isoforms (miSCRIPT assay) show both a higher expression in the second melanoma compared to the first and from the same patient.

Fig. 6. Canonical hsa-miR-125a-5p|0|0) and hsa-miR-125a-5p|0|−2 isomiR expression across 32 TCGA tumor types.

miR-125a isomiR is most expressed in many cancer types. Box and whiskers graphs of canonical and shorter isoform of miR-125a-5p show variable expression levels, represented here as log2 RPKM data, across 32 different cancer type. miR-125a-5p isoform is most abundant in many cancer types and shows a specifically high canonical/isoform ratio in the melanoma (SKCM) group. The bar shows 1–99 percentile values. TCGA abbreviations: ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphom; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.