MicroRNA-200 family members differentially regulate morphological plasticity and mode of melanoma cell invasion

Abstract

Background: A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression. The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis. Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others.

Principal findings: We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration. Individual tumor cells can invade in either elongated, "mesenchymal-type" or rounded, "amoeboid-like" modes and these two modes of invasion are inter-convertible [1]. In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion. MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion. Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions. In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology.

Significance: Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. microRNA-200 regulates morphological plasticity of melanoma cells.

(A) Schematic representation of the miR-200 family. (B) Relative levels of expression of miR-200a and -200c, normalized to the invariant miR-30b*, in melanoma cell lines compared to normal human epidermal melanocytes. (C) Transfection of miR-200a in Wm266.4 results in an elongated morphology of cells invading into collagen, while miR-200c transfection results in a rounded morphology (scale bars in um). Quantification of the percentage of cells with rounded morphology for melanoma cell lines invading into collagen. (D) miR-200 family members do not inhibit melanoma cell invasion, but can promote invasion. Each experiment was performed at least three times and representative examples are shown. Error bars represent ± SEM; unpaired t-test *p

Figure 2. microRNA-200c regulates MARCKS.

(A) The effect of miR-200c transfection on mRNA levels of predicted target genes in Wm266.4 melanoma cells was assessed using GAPDH as a control. Lower panel: immunoblot of MARCKS expression. (B) Brightfield imaging and morphological quantification of Wm266.4 cells transfected with siRNAs targeting miR-200c-responsive genes. (C) MARCKS knock-down by four individual siRNAs leads to rounded cell shape in Wm266.4 cells. (D) Levels of MARCKS mRNA in melanoma cell lines and normal melanocytes, measured by qPCR and normalized to levels of GAPDH. Error bars represent ± SEM; unpaired t-test *p

Figure 3. MARCKS expression leads to protrusion formation in melanoma cells.

Images of GFP-MARCKS or GFP expressing Wm266.4 cells plated on a thick layer of collagen-I. Error bars represent ± SEM; unpaired t-test **p

Figure 4. miR-200a associated elongation is associated with decreased actomyosin contractility.

(A) The effect of miR-200a transfection on mRNA levels was measured for predicted target genes in Wm266.4 melanoma cells using GAPDH as a control. (B) Transfection of Wm266.4 cells with miR-200a leads to decreased pMLC levels.