MiR-206 inhibits reorganization of the cytoskeleton in melanoma cells by targeting DDX5

Purpose: To investigate the role and mechanism of microRNA-206 (miR-206) in cytoskeleton reorganization in melanoma cells. Methods: MiR-206 and RNA helicase p68 (DDX5) expression levels were measured in A375, A875, and HEM-M cells by quantitative real time polymerase chain reaction (qRT-PCR). A DDX5 overexpression cell line was constructed, and DDX5 overexpression, A375, and A875 cells were transfected with miR-206 mimic or DDX5 small interfering RNA (siRNA). Transwell assay was used to assess cell migration and invasion of A375 and A875 cells, while Luciferase reporter assay was used to determine the putative target of miR-206. DDX5, miR-206, vinculin, coronin3, and ezrin expression levels were evaluated by qRT-PCR. Protein expressions of DDX5, vinculin, coronin3, and ezrin were evaluated by western blot analysis. Results: DDX5 expression was higher and miR-206 expression lower in A375 and A875 cells when compared to HEM-M cells (p < 0.05). Knockdown of DDX5 and overexpression of miR-206 repressed invasion and migration, and inhibited expression of vinculin, coronin3, and ezrin in A375 and A875 cells (p < 0.05). However, overexpression of DDX5 reversed the effect of miR-206 on cytoskeletal protein expression. Luciferase reporter assay data confirmed that DDX5 is a direct target of miR-206 (p < 0.05). Conclusion: MiR-206 suppresses reorganization of the cytoskeleton in melanoma cells by targeting DDX5, and is thus, a promising target for the treatment of melanoma.


INTRODUCTION
Malignant melanoma results from proliferation of abnormal melanocytes, has become a common malignant tumor in the past decades, and its incidence and mortality rate are rising [1]. Exposure to the sun and genetic factors are risk factors for melanoma progression [1]. Surgery and traditional first-line treatments do not prolong survival, and there is no preventative vaccine [2]. In addition, the underlying mechanism of melanoma development and metastasis has not been clarified yet. Therefore, it is necessary to elucidate the biological mechanism of melanoma to develop a promising therapeutic remedy.
MicroRNAs (miRNAs), common exosomal constituents, have been implicated in diverse biological processes through their effects on various target genes [3]. It was shown that miRNA affects the cytoskeletal reorganization of cancer cells and regulates the actin cytoskeleton [4]. miR-206 was found to suppress cell migration and affect the actin cytoskeleton and cell morphology by targeting Coronin 1C in triplenegative breast cancer [5]. Moreover, miR-206 inhibited cell invasion and migration of MDA-MB-231 breast cancer cells partially through regulation of actin cytoskeleton reorganization, especially filopodia formation [6]. However, the effect of miR-206 on actin cytoskeleton reorganization, cell morphology, and migration in melanoma cells has not been reported.
The RNA helicase DDX5, also known as RNA helicase p68, belongs to a family of highly conserved proteins involved in tumorigenesis, metastasis, and proliferation of many human malignancies [7]. Abnormal expression of DDX5 has been observed in various cancers [8,9]. DDX5 contributed to cytoskeletal reorganization in basal breast cancer cells via modulation of the DDX5-miR-182-actin cytoskeleton pathway [10]. However, the role of DDX5 in cytoskeletal reorganization in melanoma cells remains unclear. In this study, the effects of overexpression and knockdown of miR-206 and DDX5 and their interactions were analyzed in A375 and A875 melanoma cells.

EXPERIMENTAL
Cell culture A375, A875, and HEM-M cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) with 5 % CO2 at 37 °C.

DDX5 expression construct
cDNA encoding DDX5 was amplified by polymerase chain reaction (PCR) and subcloned into an overexpression vector (Invitrogen, USA). The primers used to amplify and clone DDX5 are shown in Table 1. Subsequently, Lipofectamine 2000 (Thermo Fisher, USA) was used to transfect the DDX5 overexpression plasmid into A375 and A875 cells according to the manufacturer's instructions.
Subsequently, psicheck2-DDX-WT or psicheck2-DDX-MUT were co-transfected with a miR-206 mimic or the NC mimic using Lipofectamine 2000 (Invitrogen). A dual-reporter luciferase assay system (Switchgear Genomics) was used to measure expression 24 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.

Transwell assay
After transfection of A375 and A875 cells with DDX5 siRNA, miR-206 mimic, or the NC mimic for 24 h, the cells were harvested and washed once with PBS (Invitrogen, USA). Next, 8 µm pore inserts (Costar, High Wycombe, UK), plain inserts (for migration), or matrigel-coated inserts (for invasion) were placed into the wells of 24well culture plates to separate the upper and lower chambers. The upper chamber was seeded with 1 × 10 5 cells and the lower chamber was filled with serum-free medium. Extracellular matrix gel was used in the cell invasion assay. Finally, after 48 h of incubation, cells that migrated and invaded were stained with hematoxylin and eosin and quantified by light microscopy (Olympus Corporation, Tokyo, Japan).

Quantitative real-time reverse transcriptasepolymerase chain reaction (qRT-PCR)
Total RNA was exacted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and reverse-transcribed into cDNA using the RNeasy kit (Qiagen, Valencia, CA) and the Omniscript reverse transcription kit (Qiagen). Real-time quantitative PCR was performed with the SYBR Green PCR kit (Takara, Dalian, China) and the LightCycler 480 System (Roche Applied Science) with a thermocycle profile of 95°C for 10 min and then 40 cycles of 95°C for 10 s and 60°C for 20 s. Gene expression was normalized to U6 snRNA expression using the 2 -ΔΔCT method [11]. Primers used for qRT-PCR are shown in Table 2.

Statistical analysis
SPSS software (version 18.0, Chicago, IL) was used for statistical analysis. Data were presented as mean ± standard deviation. One-way ANOVA with the Tukey's post-hoc test was utilized to compare multiple groups. p < 0.05 was considered statistically significant.

DDX5 expression increased and miR-206 expression decreased in malignant melanoma cells
qPCR analysis showed that DDX5 expression was higher in A375 and A875 cells than in HEM-M cells (p < 0.01; Figure 1A), whereas miR-206 was lower in A375 and A875 cells than in HEM-M cells (p < 0.01; Figure 1B). These data indicate that DDX5 and miR-206 expression differs in malignant melanoma cells and normal human epidermal melanocytes.

MiR-206 inhibits cell invasion and migration of malignant melanoma cells
To investigate the role of DDX5 in melanoma cells, DDX5 siRNA was used to knock down DDX5 expression in A375 and A875 cells, and this knockdown was confirmed by qPCR analysis and western blot (p < 0.01; Figure 2A, B). In addition, a miR-206 mimic was used to overexpress miR-206 in A375 and A875 cells, and this overexpression was confirmed by qPCR analysis (p < 0.01; Figure 2C). Transwell assays showed that knockdown of DDX5 and overexpression of miR-206 repressed invasion and migration of A375 and A875 cells when compared to the NC siRNA or NC miR-206 groups (p < 0.01; Figure 2D). Thus, miR-206

MiR-206 repressed expression of cytoskeletal proteins
To investigate the effects of miR-206 and DDX5 on cytoskeleton remodeling, protein and mRNA levels of the cytoskeletal proteins vinculin, coronin3, and ezrin were determined after knockdown of DDX5 or overexpression of miR-206. Vinculin, coronin3, and ezrin levels were lower after knockdown of DDX5 in A375 and A875 cells (p < 0.01; Figure 3A, B). Vinculin, coronin3, and ezrin levels were also lower following overexpression of miR-206 in A375 and A875 cells (p < 0.01; Figure 4A, B). These findings suggest that inhibition of DDX5 or overexpression of miR-206 inhibits expression of cytoskeletal proteins.

Overexpression of DDX5 reverses the effect of miR-206 on cytoskeletal proteins
Relative expression levels of vinculin, coronin3 and, ezrin were downregulated upon overexpression of miR-206, but upregulated upon overexpression of DDX5 and miR-206 in A375 and A875 cells when compared the to control (p < 0.01; Figure 5A). Western blot analysis also showed that protein expression of vinculin, coronin3, and ezrin decreased upon overexpression of miR-206, but increased upon overexpression of DDX5 and miR-206 in A375 and A875 cells when compared to the control (p < 0.01; Figure 5B). Thus, overexpression of DDX5 reversed the effect of miR-206 overexpression on cytoskeletal protein levels.

DISCUSSION
Cell migration plays a key role in tumor invasion and metastasis, and the control of cell migration may provide a promising strategy for the eradication of cancer [12]. Increasing evidence suggests that actin reorganization is a major factor in cell motility and is critical for most types of cell migration [12]. Thus, targeting cytoskeleton reorganization may be an antimetastatic remedy. In this study, the role and mechanism of miR-206 on cytoskeleton remodeling in melanoma cells were investigated. The results indicate that miR-206 represses cytoskeleton reorganization via inhibition of DDX5 expression in melanoma cells.
It was reported that repression of miR-206 leads to abnormal cell proliferation and migration, which contributes to the development of nonsmall cell lung cancer [13]. In addition, evidence showed that reduced serum miR-206 levels associate with poor prognosis in patients with melanoma [14]. Recently, transfection of breast cancer cells with miR-206 mimics inhibited cell invasion and migration through regulation of actin cytoskeleton remodeling [6]. This study revealed that reduced miR-206 levels significantly suppressed cell invasion and migration of A375 and A875 cells. miR-206 also repressed expression of the cytoskeletal proteins, vinculin, coronin3, and ezrin.
A previous study demonstrated that DDX5 promotes cell migration and metastasis [15]. It was also shown that DDX5 may function as a target for suppression of cell migration and invasion of basal cell carcinoma via inhibition of the JAK2/STAT3 pathway [16]. More importantly, DDX5 was shown to be involved in actin cytoskeleton reorganization in breast cancer cells [17]. These findings indicated that DDX5 contributes to the conformational changes in migrating cells [18]. In this study, elevated DDX5 levels were observed in A375 and A875 cells. Overexpression of DDX5 significantly increased vinculin, coronin3, and ezrin expression, and knockdown of DDX5 reduced expression of these cytoskeletal proteins and inhibited cell invasion and migration. These results revealed that DDX5 may promote cytoskeletal reorganization in A375 and A875 cells and that inhibition of DDX5 may repress reorganization of the cytoskeleton and cell invasion and migration of melanoma cells.
To investigate the relationship between miR-206 and DDX5 in cytoskeleton reorganization, target genes of miR-206 were predicted and analyzed by luciferase reporter assay. A sequence complementary to a sequence in miR-206 was found in the 3'-UTR of WT DDX5, indicating that DDX5 is a target gene of miR-206, and this was confirmed by luciferase assay. This study is the first to investigate the effect of miR-206 on cytoskeleton remodeling in melanoma cells in vitro. The results of this study need to be confirmed in vivo.

CONCLUSION
MiR-206 suppresses reorganization of the cytoskeleton in melanoma cells. Therefore, it is potential target for an anticancer therapy for melanoma.