An Integrative Analysis of The Micro-RNAs Contributing in Stemness, Metastasis and B-Raf Pathways in Malignant Melanoma and Melanoma Stem Cell

Abstract

Objective: Epithelial-mesenchymal transition (EMT) and the stemness potency in association with BRAF mutation are in dispensable to the progression of melanoma. Recently, microRNAs (miRNAs) have been introduced as the regulator of a multitude of oncogenic functions in most of tumors. Therefore identifying and interpreting the expression patterns of these miRNAs is essential. The present study sought to find common miRNAs regulating all three important pathways in melanoma development.

Materials and methods: In this experimental study, 18 miRNAs that importantly contribute to EMT and have a role in regulating self-renewal and the BRAF pathway were selected based on current literature and cross-analysis with available databases. Subsequently, their expression patterns were evaluated in 20 melanoma patients, normal tissues, serum from patients and control subjects, and melanospheres. Pattern discovery and integrative regulatory network analysis were used to find the most important miRNAs in melanoma progression.

Results: Among 18 selected miRNAs, miR-205, -141, -203, -15b, and -9 were differentially expressed in tumor samples than normal tissues. Among them, miR-205, -15b, and -9 significantly expressed in serum samples and healthy donors. Attribute Weighting and decision trees (DT) analysis presented evidence that the combination of miR-205, -203, -9, and -15b can regulate self-renewal and EMT process, by affecting CDH1, CCND1, and VEGF expression.

Conclusion: We suggested here that miR-205, -15b, -203, -9 pattern as the key miRNAs linked to melanoma status, the pluripotency, proliferation, and motility of malignant cells. However, further investigations are required to find the mechanisms underlying the combinatory effects of the above mentioned miRNAs.

Keywords: Epithelial-Mesenchymal Transition; Melanoma; MicroRNA; Network Analysis.

Fig.1

Schematic illustration of miRNA selection procedure. MiRNAs were selected using literature and database mining. Ultimately, 18 miRNAs were selected following cross-analysis.

Fig.2

The expression pattern of selected miRNAs in melanoma and normal adjacent tissue. A. The significant down regulation of miR-205, -141, -203, -15b was observed in melanoma tissues (n=20, Log 10, *; P<0.05, **; P<0.01) and B. Scatter-plots of the expression levels of the selected miRNAs show a significant higher expression of miR-9 (n=20, Log 10, *; P<0.05) in melanoma samples compared with normal adjacent tissues.

Fig.3

The expression pattern of selected miRNAs in serum obtained from melanoma patients and healthy donors. Among five miRNAs, miR-205, -15b, and -9 showed significant differences (Log 10, *; P<0.05) between serum obtained from melanoma patients (n=11) and control subjects (n=5).

Fig.4

Pattern discovery distinguishing tumor from normal samplesusing machine learning and multivariate analytical models. A. Decision tree (DT) model of Random Forest Gain Ratio predicts normal/tumor status based on miRNA expression levels. Random Forest is able to find the threshold in expression of each miRNA. As shown in the results, miR-205 was the key regulator of healthy and malignant status, B. Clustering of miRNAs, based on their expression levels, indicates that the expression patterns of miR-205/ miR-200a, miR-200c/ miR-155, and miR-222/ miR-10b in cancer samples were over 95% similar, and C. PCA analysis of expression of miRNAs in relation to tumor/normal status exhibited high diversity in tumor samples.

Fig.5

Validation of the most important common regulators and targets of differentially expressed miRNAs. A. Analysis of common targets revealed that MET, CDH1, VEGFA, TNF, ZEB, CDH1, and CCND1 represent the key targets of differentially expressed miRNAs, B, C. The protein expression levels of SOX2 and CDH1 obtained from Tissue Micro Array data and presented as H-Score. SOX2 expression was elevated in melanoma tissues (n=12) despite of normal skin (n=7), and D. mRNA expression levels of CCND1, SOX2, CDH1, VIM, BRAF, TNF, VEGF. CCND1 was significantly lower expressed in tumor samples, whereas SOX2, BRAF, TNF, and VEGF were overexpressed in malignant tissues compared with normal samples (n≤15, *; P<0.05, **; P<0.01, ***; P<0.001).

Fig.6

The expression analysis of stemness transcripts, selected miRNAs, and their target genes in melanospheres by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A. The morphology of melanospheres derived from NA8 (left), D10 (middle), and A375 (right) revealed that, NA8- melanospheres were compact with defined borders. However, D10 and A375 formed loose, grapelike melanospheres, B. Relative expression levels of SOX2, KLF4, OCT4 and NANOG transcripts in NA8-, D10- and A375-melanospheres compared to adherent cells. There were significant upregulations in KLF4 and OCT4 expression in NA8-melanospheres. D10 showed upregulation in SOX2, KLF4, OCT4 and NANOG levels and significant upregulation was observed in SOX2 and NANOG in melanosphere derived from A375 cells (n=3, *; P<0.05, **; P<0.01), C. The expression of miR-205, -203, -141, -15b, -22, -9, and -155 in melanospheres originating from all three cell lines. The expression of miR-205 was upregulated in all melanospheres compared with parental cells. MiR-203 was significantly upregulated in D10 and A375 melanospheres, unlike in spheres derived from NA8 cells. MiR-15b expression was significantly increased in D10 and A375 melanospheres compared to parental cells. MiR-141 was upregulated in D10 and downregulated in A375. Although miR-9 showed significantly higher expression in D10 melanospheres in comparison with parental cells (n=3, log 2, *; P<0.05, **; P<0.01), and D. Relative expression of CDH1, VIM, TNFA, VEGF, and CCND1 transcript in three cell lines. TNFA was significantly downregulated in D10- and A375- and upregulated in NA8-melanospheres compared to parental cells. VIM shows higher and CDH1 lower expression in NA8- and A375-melanospheres. CCND1 was significantly downregulated only in NA8- melanospheres. Expression of VEGF was markedly lower in D10 and A375 spheres (n=3, *; P<0.05, **; P<0.01).