Introduction

Melanoma is the most dangerous form of skin cancer [20, 29]. It originates from melanocytes, the pigment-producing cells in the skin. Melanoma is usually curable during the early stages. But once metastases have occurred, the prognosis is poor. Until recently, the systematic treatments for patient with advanced metastatic melanoma were mainly ineffective. Melanoma is known to be resistant to different types of chemotherapies with very few responders or consistent responses. Studies showed that its response rate to traditional chemotherapeutic treatment was below 10%. The 5-year survival rate of melanoma patients with distant metastases is less than 5% [22]. During the last 10 years, there have been some exciting improvements in melanoma treatments, especially in targeted and immune therapies. FDA has approved eight therapeutic agents, mainly the small molecular inhibitors of BRAF or MEK and antibodies against CTLA-4 and PD-1. The results of clinical trials for most of these agents have been positive, as compared to standard therapies [19].

Resveratrol (Res) is a natural polyphenol found in grapes, berries, chocolate and red wines, etc. It has been shown to have broad-spectrum beneficial effects for human health, including anti-oxidative, anti-infective and anti-inflammatory effects [11]. In cancer cells, Res exhibited various types of anti-cancer properties, such as suppressing tumor cell proliferation and inducing tumor cell apoptosis [25]. Further studies showed that Res could increase the chemical cytotoxicity to tumor cells and inhibit the growth of tumor cells by interrupting the cell cycle process and apoptosis [30]. Although the anti-cancer effect of Res has been reported both in vitro and in xenograft tumor models, its effect has not been demonstrated in cancer patients.

MicroRNAs (miRNAs) are small non-coding RNAs that play roles in posttranscriptional gene regulation. It has been shown to have different expression patterns between normal and cancer tissues [16, 18]. MiR-221, encoded on the X chromosome, is one of the identified microRNA markers overexpressed in glioblastoma, melanoma, pancreatic cancer, papillary thyroid carcinoma, gastric cancer, colorectal carcinoma, hepatocellular carcinoma and prostate carcinoma [5, 17, 27]. In several studies, miR-221 was showed to promote the progression of melanoma by decreasing the c-Kit receptor and p27Kip1 [1, 7].

One of the key signaling pathways regulated by miRNA is the NF-κB pathway. NF-κB is an important transcription factor that regulates various cellular signal pathways involved in cell death, proliferation and differentiations processes [26]. In many cancer studies, NF-κB acts to promote cancer cells, and create a microenvironment suitable for cancer cells to proliferate without being rejected by the immune system. Melanoma was showed to be activated by NF-κB via the lymphotoxin-beta receptor activation and kinase induction [24].

With the accumulated evidences of tumor suppressive functions of Res, deeper understanding of molecular mechanisms of this compound in cell development and cancer cell survival is needed. More importantly, the study of interaction of Res with key transcription factors and their downstream networks would facilitate its potential clinical applications.

Materials and methods

Cell culture and RNA extraction

Different human melanoma cells (SbCl2, HaCaT, A375, MV3, WM35, SK-MEL-3 and SK-MEL-5) were cultured as described in previous publication [31]. The A375, WM35, SK-MEL-3 and SK-MEL-5 cell lines were found to harbor the BRAFV600E mutation [10, 32], whereas the SbCl2, MV3 were BRAF wild type. HaCaT was reported to harbor mutations in p53 [15]. The cells were grown in an incubator at 5% CO2 and 37 °C. Total RNA was extracted from these cell lines using Ambion miRVana miRNA Isolation Kit (Ambion, USA).

Quantitative real time PCR

Total RNA was reverse transcribed with SuperScript III (Invitrogen, Carlsbad, CA, USA) and the produced cDNA was amplified (Applied Biosystems, Waltham, MA, USA) using universal PCR master mix. The quantification of mRNA was carried out by Taqman gene expression assay.

Western blotting

Western blot analysis was conducted as described previously [12] using the Bio-Rad kit. A total of 10 μg protein was loaded per lane. The primary antibody was incubated for overnight and secondary antibody was incubated for 1 h before the development with the ECL2 reagent (Pierce, WI, USA). Antibodies used were as follows: GAPDH (ab8245, Abcam, Cambridge, MA, USA) and NF-κB (ab16502, Abcam, USA).

Luciferase report assay

The luciferase report assay was performed using the dual-luciferase assay system (Promega, Madison, WI, USA). Both A375 and MV3 cells were seeded in 24-well plates (50,000 cells per well) 1 day before the transfection. The luciferase activity was measured 48 h after transfection.

Soft agar assay

5 × 103 cells were suspended 0.3% agar (DNA grade agarose) mixed with DMEM and plated on the top of 0.5% agar (mix equal volumes of 1% agar and 2× DMEM). The plates were then incubated at 37 °C for 3 weeks. For staining, the plate was incubated by 0.1% crystal violet solution for 1 h and the colonies were visualized by microscope and photographed at 5× magnification.

Cell proliferation assay

CCK-8 assay was performed to measure the cell proliferation. The transfected cells were plated on 96-well plates at density of 5000/well before cells were serially diluted. A total of 10 μl CCK-8 solution was added to each well after treatment. The OD values were detected at 450 nm.

Mice xenograft assay

The immuno-deficient (SCID) mice were utilized in the study. A375 (2 × 106 cells per mouse) cells were subcutaneously injected into each side of posterior flank of the mice. Then (30 mg/kg) Res was added (n = 6) or not (n = 6) by peritoneal injection. The tumor volume (in mm3) was calculated by the formula: width2 × length/2. All animal procedures were approved by the Animal Care and Use Committee (IACUC) and Ethics Committee of Daqing Oilfield General Hospital.

Statistical analysis

Statistically significant differences between different treatments were determined by ANOVA analysis. For a single comparison, a p value < 0.05 was considered to be statistically significant.

Results

Resveratrol inhibits the growth of melanoma cell lines through its regulation of miR-221

Two melanoma cell lines (A375 and MV3) were treated with 50 μM Res for 24 h. By performing qRT-PCR, we quantitatively estimated the expression of miR-221 relative to control treatment. The results showed that Res could significantly inhibit the expression of miR-221 in both cell lines of A537 and MV3 (Fig. 1a, b). By validating this finding, miR-221 was overexpressed in both cell lines with or without treatment of Res. This overexpression could effectively reverse the inhibition of miR-221 by Res in these cells (Fig. 2a, b). Both the viability test and soft agar experiments further showed that overexpression of miR221 could rescue the cells from the inhibition of Res (Fig. 2c–f).

Fig. 1
figure 1

Resveratrol inhibits miR-221 expression. a, b Melanoma cell lines A375 and MV3 were treated with 50 μM resveratrol (Res) or DMSO, after 24 h the expression of miR-221 was analyzed. Data were presented as mean + SD from three independent experiments with triple replicates per experiment. **p < 0.01, indicates significant difference compared to DMSO group

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Fig. 2
figure 2

Overexpression of miR-221 reverses the suppression of melanoma by resveratrol. a, b A375 and MV3 cells were transfected with miR-221 mimics (miR-221) or control mimics (miR-NC), after which were treated with 50 μM Res or DMSO. After 24 h, the expression levels were analyzed by qRT-PCR. The data were normalized to the ratio of control group. **p < 0.01, indicates significant difference compared to DMSO + miR-NC group, ## p < 0.01, indicates significant difference compared to Res + miR-NC group. c, d Cells were treated as above, and the CCK-8 assay was employed to analyze the cell viability. The data were normalized to the ratio of control group. e, f Cells were treated as above, and the colony formation assay was used to analyze the cell growth ability of each group. **p < 0.01

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Resveratrol inhibits the expression of miR-221 by inhibiting the expression of NF-κB (RELA)

To investigate the cellular mechanism of inhibiting effects of Res to miR-221, we measured the level of expression level of NF-κB after treated with Res. The results showed that Res could significantly down-regulate NF-κB expression in both cell lines (Fig. 3a, b). The Western blot results confirmed this result at protein level (Fig. 3c). By transfected NF-κB activity reporter into two cell lines, we also observed that Res could inhibit the activation of NF-κB activation (Fig. 3d).

Fig. 3
figure 3

NF-κB (RELA) is down-regulated by resveratrol. a, b Melanoma cell lines A375 and MV3 were treated with 50 μM Res or DMSO, after 24 h the expression of RELA were analyzed by qRT-PCR. c Cell were treated as above, the protein expression levels of RELA were analyzed by Western blotting. d A375 and MV3 cells were transfected with NF-κB activity reporter, and were treated with 50 μM Res or DMSO. After 24 h, the luciferase activities were analyzed and the data were normalized to the DMSO control group. **p < 0.01

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We then examined the regulation of NF-κB to miR-221 by overexpressing or inhibiting the expression of NF-κB in both cell lines. We observed that miR-221 levels were induced or inhibited accordingly (Fig. 4a, b). Using the database from JASPAR, we identified the binding motif of NF-κB in the promoter region of miR-221 (Fig. 4c). Furthermore, by comparing the luciferase activities in both wild type and mutated miR-221 promoters (Fig. 4d), we confirmed that NF-κB could directly bind to the promoter region of miR-221.

Fig. 4
figure 4

NF-κB (RELA) directly binds to the promoter region of miR-221 and induces the expression of miR-221. a Cells were transfected with RELA expression vector lacking of 3′ UTR region or control vector, after 24 h the expression of miR-221 was analyzed by qRT-PCR. b Cells were transfected with RELA interference vector (shRELA) or control vector (shNC), after 24 h the expression of miR-221 was analyzed by qRT-PCR. c JASPAR program was used to predict the bind site of RELA in the promoter of miR-221 region. d Dual-luciferase reporter assay was performed on A375 cells to detect the relative luciferase activities of WT and mut promoter reporters. **p < 0.01

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miR-221 interferes the induction of TFG by resveratrol

We then tried to identify the downstream factors of miR-221. The bioinformatics tool, TargetScan, was used to scan the potential targets which predicted two binding sites of miR-221 in the 3′ UTR region of TRK-fused gene (TFG) (Fig. 5a, b). TFG is a well-established melanoma suppressor gene which interested us. Based on these information, we transfected wild type or mutated promoter reporters into one of cancer cell lines before using dual-luciferase assay to show miR-221 could bind and inhibit the activation of TFG (Fig. 5c, d). Western blot results showed the same effects on the protein levels in both cell lines (Fig. 5e, f).

Fig. 5
figure 5

miR-221 targets TFG and reverses the induction of TFG by resveratrol. a, b TargetScan program was used to predict the bind site of miR-221 in the 3′ UTR region of TFG and putative seed-matching sites or mutant sites (red) between miR-221 and 3′-UTR of TFG were shown. c, d Dual-luciferase reporter assay was performed on A375 cells to detect the relative luciferase activities of WT and mut promoter reporters with the transfection of miR-221 or not. e, f A375 and MV3 cells were transfected with miR-221 or not and were treated with 50 μM Res or DMSO. After 48 h, the protein expression levels of TFG were analyzed by Western blotting. **p < 0.01

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Resveratrol suppresses melanoma growth in vitro and in vivo

We also conducted the function assay to further estimate the impact of Res on melanoma. The melanoma cell lines (BRAF wild type: SbCl2; BRAF-V600E mutation: WM35, SK-MEL-3 and SK-MEL-5) and normal skin HaCaT cells were treated with Res, and Res could significantly inhibit the proliferation of these cell lines (Online Resource 1). Since these cell lines either contain BRAF wild type and BRAF-V600E mutation, we observed a similar inhibitory effect of Res in both cells (Online Resource 1).

One of tumor cell lines was injected into nude mice before the animal was treated with Res. Compared with control (12 tumors each group), Res could significantly reduce the size of tumor (Fig. 6a–c), indicating clear anti-cancer effects. We then measured the expression level miR-221, RELA and TFG in the tumor tissue by qRT-PCR. Compared with control, we observed a decrease of miR-221 and RELA and an increase of TFG (Fig. 6d).

Fig. 6
figure 6

Resveratrol suppresses melanoma in vivo. A375 cells were injected into each side of posterior flank of the nude mice, then (30 mg/kg) Res was added (n = 6) or not (n = 6) by peritoneal injection once daily for consecutive 7 days. a The tumor sizes were measured every 2 days since day 10, and the volumes were calculated using the following formula: volume = 0.5 × length × width2. b, c After 20 days, the mice were killed and the tumors (12 tumors each group) were separated, and were photographed and weighed. d The miR-221, RELA and TFG expression levels of the tumors were analyzed by qRT-PCR. **p < 0.01

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We then tried to confirm the regulatory pathway between miR-221 and TFG from publicly available database. The TCGA melanoma expression data was used for the study, but we did not observe a significant inverse association between miR-221 and TFG expression (Online Resource 2).

Discussion

Melanoma is responsible for a majority of skin cancer-related deaths even with the development of various new treatment approaches. Currently, the incidence of melanoma is doubling almost every 10 years and it is becoming a major public health threats through the world. One major challenge of melanoma is its strong metastatic features, which make current treatment approaches ineffective. It is important to fully understand the molecular mechanism of melanoma progression.

Res, a polyphenol found in various natural products, was reported to regulate signal pathways that control cell division. Increasing evidence showed that Res has beneficial effects against different cancer types including melanoma both in vitro and in vivo. Multiple studies investigated the anti-cancer mechanism of Res. The major finding lay on several key cellular pathways, including inhibiting PI3K-AKT pathway, regulating the glucose uptake, and inducing autophagy-dependent cell death [13].

MiRNAs were showed to be involved in almost all the major cellular signal pathways including cancer genesis [9]. Both oncogenic and tumor suppressor roles of miRNAs were reported. In melanoma, several studies demonstrated the import regulatory functions of miRNAs. In one report from Dadras’ group, small RNAs from invasive melanoma and normal tissues were sequenced. A clear differential miRNA’s expression pattern was observed among different melanoma types, which strongly support that miRNAs were associated with invasive and aggressive of melanoma. Among functional studies, through a functional screen of human miRNAs, miR-339-3p was identified to control melanoma cell invasion. This is achieved by its regulation of MCL-1, a well-studied oncogene. In vivo study was also supporting this conclusion [34].

Even lots of evidences are pointing to the significant roles of miRNA in melanoma progression, fewer studies showed the regulatory between miRNAs and nature products. We initiated this study, intending to investigate the relationship between Res and miR-221, an identified oncogenic miRNA. Several previous studies found that miR-221 is involved in tumor angiogenesis. Mattia et al. showed that overexpressed miR-221 and miR-222 could activate some major cellular pathways to promote the cell survival and block melanogenesis in melanoma [21].

We used two melanoma cell lines, A75 and MV3, to study the underline mechanism of RES against melanoma. From both in vitro and in vivo experiments, RES showed significant tumor suppression abilities. In both cell lines, Res decreased the colony formation in soft agar and reduced the growth of tumor in nude mice, which is consistent with its anti-cancer functions in previous studies. Based on this, we were focusing on studying the miR-221 as the potential target of Res during this process. Supporting our hypothesis, we observed a clear inhibiting effect of Res to miR-221. According to our knowledge, this is the first time to identify the downstream regulation of miR-221 by Res, which help for the better standing the tumor suppression mechanism of Res and other nature products and their derivatives.

Based on the above discoveries, we performed a series of studies to further address the Res-miR-221 pathway. An important question is key regulator between these two. A good candidate is the NF-κB, which regulates a broad spectrum of genes involved in cell death, cell proliferation and differentiation processes. Previous studies showed the regulatory relationship between Res and NF-κB [28]. It was proved that NF-κB could contribute to the resistance to target therapies through promoting the cell cycle [2, 6]. Lots of cancer genesis factors can be activated by this factor. Studies were also showed that miRNA could be involved in NF-κB pathway to affect the progression of malignant melanoma [35]. For instance, miR-7-5p was recently reported to inhibit melanoma cell proliferation and metastasis by suppressing RelA/NF-κB [8]. Our experiment indicated that NF-κB is a downstream factor of Res, through which to inhibit the miR-221. Given the critical functions of NF-κB in cell development-related functions, this finding could also lead Res to many other important cell fate processes. Our binding motif study and luciferase report further confirmed the regulation between NF-κB and miR-221.

Our next experiment completed our understanding of miR-221 in cancer development. We used bioinformatics tools to do the high throughput screening and identified TFG, a well-studied tumor suppressor [4], as an interesting downstream factor. TFG was showed to be the target miR-221, which can also form the negative feedback loop to the Res effects. TFG was also proved to play a role in the NF-κB pathway in other studies, which makes the regulation network in our model more convincing [23].

Our in vivo studies fully supported the pathways we discovered in the cell lines, suggesting that this network could exist in the real physiological condition. We have chosen the concentration of Res in vivo based on previous studies and used the intraperitoneal injection to achieve the best anti-cancer effects [3]. That would provide some useful information for identifying new potential diagnosis and therapeutic candidates for melanoma. Moreover, in several cancer models, the resistance to Res and other natural compounds were observed. The combination treatments were tested in several reports. There were also efforts in enhancing the anti-cancer effects of Res by collaborating some key signal inhibitors [14]. The better understanding of downstream networks of Res in cancer cells will provide the valuable information for designing the better treatment strategy when using natural compound as the agent.

Given the rich studies of both Res and miRNAs in cancer field, by connecting them together in our model, we provided some interesting thoughts for the anti-cancer signaling pathway. Beyond that, Res and its derivatives were showed to have many beneficial effects involved in enhancing chronic symptoms such as diabetes, cardiovascular diseases and aging [33]. But the actual mechanism underlying these processes is still lacking. The study of regulation between Res and miRNA can significantly expand our knowledge on these compounds and help to explain its complex downstream effects.

Due to the scope of this study and limited resources, we did not validate our model on human tissues. Also, the result from TCGA expression data did not reflect the regulatory effects between miR-221 and TFG we observed in our study. This could be due to our confounding factors which reduced the significance. It will be extremely important to prove some of our results on patient-level studies, which could bring some real reward to melanoma patients.

Conclusions

In the present study, Res and its tumor suppression effects on melanoma cell lines and mouse model were showed and the mechanisms were further studied. MiR-221, a miRNA which could promote the oncogenesis, was inhibited by Res. This regulation is mediated by NF-κB pathway. TFG was showed to be bound by miR-221 to reduce its tumor suppression function. Overall, our work is suggesting a complicated downstream network of Res in melanoma cell lines.