MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene

Metastatic melanoma is the most aggressive form of skin cancer and is refractory to therapy. MicroRNAs have been recently discovered as novel molecules that provide therapeutic benefits against melanoma. This work aims to examine the effects of miR-26a and let-7a on the growth and invasiveness of malignant melanoma in vitro and in vivo. In addition, we elucidate the mechanism of action by identifying the target gene of miR-26a. Both miR-26a and let-7a inhibited proliferation and invasiveness and halted the cell cycle at the G1/G0 phase in SKMEL-28 and WM1552C malignant melanoma cell lines. Moreover, miR-26a potently induced apoptosis and downregulated the expressions of microphthalmia-associated transcription factor (MITF) and MAP4K3 in both cell lines. The luciferase reporter assay demonstrated that miR-26a suppresses MITF expression by binding the 3′-UTR, suggesting that MITF is a bona fide target of miR-26a. SiRNA knockdown of the MITF gene confirmed that miR-26a reduced cell viability and induced apoptosis by regulating MITF. Using a murine model, we also found miR-26a significantly retarded the growth of melanoma tumors in vivo. In conclusion, miR-26a and let-7a suppressed the growth and invasiveness of melanoma cells, suggesting that miR-26a and let-7a may represent novel therapies for malignant melanoma.


INTRODUCTION
The incidence of metastatic melanoma, the deadliest skin cancer, has increased in the past two decades. It is estimated that 76 380 new melanoma cases will be diagnosed in 2016. 1 The prognosis of melanoma is poor, with the lower than 20% of 5-year survival rates for patients who harbor metastatic melanoma. 2,3 Since about 50% patients with melanoma have been found to harbor the activating BRAF V600E mutation, 4 the current targeted therapy focuses on RAS-RAF-MEK-ERK signaling pathway. 5 Even though the initial response to BRAF inhibitors (vemurafenib and dabrafenib) and MEK inhibitors (trametinib and cobimetinib) is impressive, resistance usually develops in the patients within 6-8 months. 6 Thus, looking for alternative effective therapies for the metastasized melanoma is an imperative task.
MicroRNAs (miRNAs) are engaged in post-transcriptional regulation of gene expression by directly targeting the 3′-UTR of mRNA. 7 Among the microRNAs that are implicated in tumor growth and progression, miR-26a is involved in a variety of signaling pathways and can act as a tumor suppressor in carcinogenesis and cancer progression. 8 Previous studies found that miR-26a inhibits the proliferation of gallbladder cancer cells and breast cancer cells by downregulating high-mobility group AT-hook 2 (HMGA2) and MCL-1 expression, respectively. 9,10 In addition, enhancer of zeste homolog 2 was also directly regulated by miR-26a, leading to the inhibition of migration and invasion in osteosarcoma, 11 and tumorigenesis repression in nasopharyngeal cancer. 12 It has been identified that the expression of miR-26a is specifically downregulated in human melanoma cells. 13 It was reported that miR-26a induced apoptosis of melanoma cells by directly targeting the silencer of death domains (SODDs), which has a vital role in preventing apoptosis in the sensitive cell lines of melanoma. 13 Let-7a acts as tumor suppressor microRNA by negatively regulating the expressions of RAS, 14 HMGA, 14 C-MYC, 15 and NIRF 16 oncogenes in lung cancer, renal cell carcinoma, and colorectal cancer, respectively. Let-7a is also involved in many important signaling pathways in cancer progression. 17,18 Furthermore, let-7a induces apoptosis in a number of cancers including gastric carcinoma, 19 nasopharyngeal carcinoma, 20 hepatocellular cancer, 21 breast cancer, 22 cervical cancer, 23 brain cancer, 24 and prostate cancer. 25 For melanoma, let-7a was found to reduce invasive potential of melanoma cells by regulating the expression of integrin-β3. 26 In addition, let-7a increased melanoma cell sensitivity to doxorubicin. 27 Although the tumor suppressor functions of let-7a in other types of cancers is well documented, little research has been done on investigating the inhibitory effects of let-7a on the growth properties of malignant melanoma cells. The biological role of let-7a remains largely unknown in human malignant melanoma.
This study characterizes the tumor-suppressing effects of let-7a and miR-26a in malignant melanoma and identifies, for the first time, microphthalmia-associated transcription factor (MITF) as a novel gene target of miR-26a in malignant melanoma. In addition, this study is the first work that examines the inhibitory effect of miR-26a on melanoma growth in mice. This work establishes that miR-26a replacement is a potential therapeutic strategy for malignant melanoma.

RESULTS
Transfection of miR-26a and let-7a mimics significantly decreased cell viability in human malignant melanoma cell lines The expression of miR-26a and let-7a was detected by qRT-PCR in SKMEL-28 and WM1552C melanoma cell lines. The expression levels of let-7a in both cell lines were very low, especially in WM1552C cell line (data not shown). The data did not support our prediction that the expressions of miR-26a and let-7a were lower in SKMEL-28 cells, which was derived from a melanoma of more advanced stage than that of WM1552C cells. Transfection of miR-26a elevated the level of miR-26a by 511.4-and 1462.2-fold in SKMEL-28 and WM1552C melanoma cells, respectively. Similarly, transfection of let-7a mimics elevated the level of let-7a by 1535.5and 4733.3-fold in SKMEL-28 and WM1552C melanoma cells, respectively. Transfection of miR-26a or let-7a significantly reduced the viability of SKMEL-28 and WM1552C melanoma cell lines ( Figure 1). SKMEL-28 is more resistant to the cytotoxicity of miR-26a and let-7a compared with WM1552C.
MiR-26a and let-7a inhibited cell migration and invasion We further examined the effects of miR-26a on cell migration ability that was detected by the wound healing assay. SKMEL-28 melanoma cells that were transfected with 50 or 100 nM miR-26a showed a decreased ability to close the wound compared with untreated cells or cells transfected with negative control mimics, suggesting that miR-26a significantly inhibited cell migration (Figures 2a and b). The microRNA let-7a at 100 nM also displayed a strong inhibitory effect on cell migration (Supplementary Figure 1).
In addition, the effect of miR-26a and let-7a on cell invasion ability, which is a hallmark of malignancy, was examined using a Transwell assay. MiR-26a and let-7a significantly (P o 0.05) reduced the cell invasiveness in both SKMEL-28 and WM1552C melanoma cell lines ( Figure 2). MiR-26a and let-7a (100 nM) suppressed cell invasion in SKMEL-28 cells by 57% and 61%, respectively ( Figure 2c). In the WM1552C cell line, miR-26a and let-7a inhibited the cell invasion by 44% and 51%, respectively ( Figure 2d). Therefore, both miR-26a and let-7a significantly suppressed the migration and invasiveness of melanoma cells.
MiR-26a and let-7a caused the cell cycle to halt at the G 1 /G 0 phase We hypothesize that miR-26a and let-7a inhibit cell proliferation by inducing cell cycle arrest. In both SKMEL-28 ( Figure 3a) and WM1552C cell lines (Figure 3b), transfection of miR-26a or let-7a mimics showed a significant increase in the percentage of cells in the G 1 /G 0 phase with concomitant reduction in the percentage of cells in the S phase compared with control and negative control group. The results suggested that miR-26a and let-7a may inhibit melanoma cell proliferation by inducing cell cycle arrest at the G 1 phase.
MiR-26a induced apoptosis Cell cycle arrest for an extended time can cause apoptosis, which may be an important contributor to the cytotoxicity of miR-26a. We found that transfection of miR-26a at concentrations of 50 or 100 nM led to a significant increase in the percentage of Annexin V-positive cells in both SKMEL-28 ( Figure 4a) and WM1552C (Figure 4b), suggesting that miR-26a induced apoptosis in both melanoma cell lines.
MITF is suppressed by miR-26a in melanoma cell lines Three microRNA target search algorithms (Targetscan, miRDB, and microrna.org) were used to identify hypothetical gene targets of miR-26a in human malignant melanoma. Among all predicted target genes, mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) and MITF were chosen as the most likely targets for miR-26a. Results showed that miR-26a mimics remarkably reduced the expressions of MITF and MAP4K3 in both SKMEL-28 ( Figure 5a) and WM1552C melanoma cell lines ( Figure 5b). Figure 1. The effect of miR-26a and let-7a on the cell viability of SKMEL-28 and WM1552C melanoma cells. Cell viability was determined using the MTT assay after 48 h transfection with transfection reagent alone (control), AllStars negative control siRNA (100 nM), miR-26a mimics, and let-7a mimics at a final concentration of 50 or 100 nM. Each experiment was repeated six times and the results are presented as mean ± S.D. Asterisks indicate a significant difference (P o0.05) compared with control groups.

miR-26a inhibits melanoma by targeting MITF H Qian et al
MiR-26a directly targets the 3′-UTR of MITF To further investigate whether the MITF gene is the direct target of miR-26a in melanoma, a luciferase reporter assay was performed by using a Secrete-Pair Dual Luminescence Assay Kit (Genecopoeia Biotechnology Co., Rockville, MD, USA). The luminescence was measured after co-transfection of miRNA mimics and plasmid vectors into the two malignant melanoma cell lines. After cotransfection for 48 h, the negative control microRNA did not decrease the luciferase activity. At the same time, the transfection of miR-26a mimics did not reduce the luciferase activity produced by negative control vector without the MITF 3′-UTR sequence. However, the cells transfected with miR-26a mimics and the MITF 3′-UTR vector showed a significantly lower luciferase activity compared with the control and the negative controls ( Figure 5d). The Secrete-Pair Dual Luminescence Assay (Genecopoeia Biotechnology Co.) indicated that miR-26a directly binds the 3′-UTR of the MITF gene, resulting in lower expression of Gaussia luciferase. Therefore, it was validated that MITF is a bona fide target of miR-26a.
Knockdown of MITF reduced cell viability, and induced cell apoptosis in melanoma cell lines To test whether miR-26a targeting MITF was responsible for the reduced cell viability observed from miR-26a mimics, we knocked down the MITF gene expression in both SKMEL-28 and WM1552C melanoma cell lines using siRNAs. Cell lines were treated with siRNA (40 nM) against MITF for 72 h and subjected to western blot, MTT and apoptosis assays. Both siMITF-a and siMIFT-b successfully reduced the expression of MITF ( Figure 5e). In addition, both siRNAs against MIFT significantly decreased the cell viability of SKMEL-28 and WM1552C melanoma cell lines (Figure 5f). Transfection of siMITF-a or siMITF-b (40 nM) markedly increased the Annexin V-positive cell populations in both SKMEL-28 and WM1552C melanoma cells, suggesting that the knockdown of MIFT induced apoptosis in both cell lines ( Figure 5g). Thus, results from both the cell viability assay and the apoptosis assay upon siMITF treatment were generally consistent with results for miR-26a treatments.
MiR-26a retarded the growth of melanoma in mice While the results of the cell-based assays showed that miR-26a inhibited the growth of melanoma cells, it is important to determine whether miR-26a inhibited the tumor growth in vivo. B16-F10 mouse melanoma cells transfected with miR-26a (50 or 100 nM) or negative control were subcutaneously implanted into the hind flank of mice for tumor formation. Tumor size was then measured every 3 days for 16 days to observe the effect of miR-26a on tumor growth. MiR-26a at the concentrations of 50 and 100 nM significantly reduced the average size of tumors on 10th, 13th and 16th day in comparison with the control and negative control groups (Figures 6a and b), suggesting that miR-26a retarded the growth of melanoma in vivo.

DISCUSSION
Prior studies in melanoma have identified several miRNAs that are involved in melanoma progression. 28,29 The inhibitory effect of miR-26a has been observed in gallbladder cancer, nasopharyngeal cancer, esophageal squamous cell cancer, and melanoma. 9,12,13,28 Our study reported for the first time that, in malignant melanoma cells, miR-26a directly targets on MITF, which is a master regulator of melanocytes and sustains the cell viability. 29 Therefore, miR-26a may be used to as a novel therapeutic small molecule against human malignant melanoma, possibly by regulating the expression of MITF. In addition, MITF is a potential therapeutic target for melanoma treatment. The inhibitory effect of let-7a has been observed in human papillomavirus-induced cervical carcinogenesis and Burkitt lymphoma cells. 30,31 Previous research also found that let-7a induced apoptosis in various cancers. 19,20,21,25 However, the role of let-7a on melanoma has been largely unknown. We report for the first time that a let-7a mimics significantly reduced the cell viability in several melanoma cell lines. We showed that miR-26a and let-7a decreased the cell viability by causing a cell cycle arrest at the G1/S phase. This mechanism is in agreement with ability for miR-26a to arrest the cell cycle in ACTH-secreting pituitary adenomas, lung carcinoma 32 and hepatocellular carcinoma. 33 It is also consistent with the findings that let-7a arrests the cell cycle of human hepatocellular carcinoma. 19,34 In addition, the miR-26a and let-7a mimics decreased the ability of malignant melanoma cells to migrate across the Transwell membrane, suggesting that miR-26a and let-7a may inhibit invasiveness and metastasis of melanoma. Similar results were found in nasopharyngeal cancer, gastric cancer, esophageal squamous cell cancer, and pancreatic cancer cells where miR-26a inhibited metastasis. 28,35,36 Similarly, let-7a has been shown to inhibit metastatic properties of nasopharyngeal carcinoma, gastric cancer, breast cancer, and lung cancer. 14,17,37,38 Several genes have been identified and validated to be the targets of miR-26a in multiple cancers including melanoma. 12,13,39 Reuland et al. 13 reported that the expression of miR-26a is significantly downregulated in melanomas compared with melanocytes and miR-26a mediates apoptosis in melanoma by targeting the SODD in a few sensitive cell lines of melanoma. 13 This study reports that MITF is a bona fide target of miR-26a. MITF has an important role in melanoma initiation and development due to its oncogenic function specifically for the melanocytes. 40 MITF drives the progression of melanomas harboring BRAF V600E mutation by promoting melanocytes to synthesize the melanin to retain melanoma and activating the overexpression of antiapoptotic genes for survival. 41 Therefore, MITF is a potential novel therapeutic target for malignant melanoma, and miR-26a may exert its antimelanoma effects by targeting MITF. It is also worth mentioning that miR-26a targets other genes including SODD and SMAD1. Therefore, like any other tumor suppressor microRNA, the inhibitory effect of miR-26a may be the combinatorial effect of targeting several oncogenic genes. In addition, this study showed that miR-26a reduced the expression of MAP4K3 in both melanoma cell lines. MAP4K3 is a Ser/Thr kinase that is believed to regulate the activation of mTOR pathway, 42 which is crucial in regulating cell proliferation, cell growth, and inducing autophagy. 43 A variety of cancers can be caused by hyperactivation of the mTOR pathway. [44][45][46] Owing to the importance of mTOR pathway and the major role of MAP4K3 in activating mTOR pathway, miR-26a may inhibit cell viability and invasiveness by downregulating MAP4K3.
In conclusion, miR-26a and let-7a showed several strong anticancer properties. Both microRNAs significantly decreased the proliferation and invasion of melanoma cells. Along with targeting other oncogenic target genes such as SODD, SMAD1, and MAP4K3, miR-26a inhibited the growth of melanoma cells by directly downregulating MITF. We have found that miR-26a and let-7a act as tumor suppressors in melanoma cells, and replacement therapy with miR-26a and let-7a represents a promising novel therapeutic strategy against human malignant melanoma. The hsa-let-7a-5p mimic has a mature miRNA sequence of 5′-UGAGGUAGUAGG UUGUAUAGUU-3′, and the miR-26a-5p mimic has a mature miRNA sequence of 5′-UUCAAGUAAUCCAGGAUAGGCU-3′. The hsa-let-7a-5p (MSY0000062), miR-26a-5p (MSY0000082) and AllStars negative control siRNA were purchased from Qiagen (Germantown, MD, USA) and transfected into the cells using DharmFECT transfection reagent 2 (Thermo Scientific, Pittsburgh, PA, USA) at a final concentration of 50 or 100 nM. FlexiTube siRNAs (Qiagen) specific to MITF were used for knockdown of the MITF gene as per the manufacturer's instructions. Two siRNAs specific to MITF gene were used to guard against the possibility of off-target effects. These were Hs_MITF_1 (cat. no. SI00005362) and Hs_MITF_5 (cat. no. SI02781212). These siRNAs are referred to hereafter as siMITF-a and siMITFb, respectively. Cells were transfected with siRNAs (40 nM) using HiPerFect (QIAGEN Inc., Valencia, CA, USA). AllStars negative control siRNA was also used as a control. Both MTT and Annexin V apoptosis assays were conducted 72 h after transfection.

MicroRNA isolation and quantitative RT-PCR
Total microRNA was isolated from WM1552C and SKMEL-28 human melanoma cell lines after transfection for 48 h using mirVana miRNA

Plasmid construction and dual luminescence assay
Secrete-Pair Dual Luminescence Assay (Genecopoeia Biotechnology Co.) was applied to investigate whether miR-26a directly regulates the expression of MITF in two malignant melanoma cell lines. The recombinant MITF vector, containing MITF 3′-UTR that contains miR-26a binding site was purchased from GeneCopoeia (Rockville, MD, USA). The recombinant control vector without the MITF 3′-UTR was used as a negative control vector. MiR-26a mimics or negative control mimics at a final concentration of 100 nM were co-transfected with the MITF vector or a negative control vector (1 μg/μl) using the TransIT-X2 Dynamic Delivery System (Madison, WI, USA) into SKMEL-28 and WM1552C melanoma cells. After 48 h transfection, cells were trypsinized and the luminescence assay was performed by using Secrete-Pair Dual Luminescence Assay Kit (Genecopoeia Biotechnology Co.) according to the manufacturer's instructions. Luminescence activity was measured by a microplate reader.
In vivo tumor growth model All animal protocols were approved by Animal Care and Use Committee of Emporia State University. Six-week-old male C57BL/6 mice were obtained from Harlan Laboratories Inc. (Indianapolis, IN, USA) and housed in controlled animal facility with 12 h light-dark cycle and allowed to have ad libitum access to rodent pellet and fresh tap water. A total of 32 mice were randomly divided into four groups (eight mice per group). Cells were transfected with miR-26a at concentrations of 50 or 100 nM, or with AllStars negative control siRNA for 48 h. A total of 2 × 10 5 transfected cells were subcutaneously implanted into the hind flank of mice to produce tumor. The size of the tumor was measured every 3 days for 16 days.

Statistical analysis
All values were represented as the mean ± S.D. from at least three independent experiments. Statistical difference between groups was measured using the Student's T-test. P-value in all experiments were considered significant at ⩽ 0.05.