Knockdown of circular RNA VANGL1 inhibits TGF‐β‐induced epithelial‐mesenchymal transition in melanoma cells by sponging miR‐150‐5p

Abstract Melanoma is one of the most aggressive and life‐threatening skin cancers, and in this research, we aimed to explore the functional role of circular RNA VANGL1 (circVANGL1) in melanoma progression. The expression levels of circVANGL1 were observed to be significantly increased in clinical melanoma tissues and cell lines. Moreover, circVANGL1 knockdown suppressed, while circVANGL1 overexpression promoted the proliferation, migration and invasion abilities of melanoma cells. Further investigations confirmed the direct binding relation between circVANGL1 and miR‐150‐5p in melanoma, and restoration of miR‐150‐5p blocked the effects of circVANGL1 overexpression in melanoma cells. We further found that circVANGL1 was up‐regulated by TGF‐β treatment, and the enhanced EMT of TGF‐β‐treated melanoma cells was blocked by circVANGL1 knockdown. In conclusion, these results indicated that circVANGL1 might serve as a promising therapeutic target for melanoma.


| INTRODUC TI ON
Melanoma is one of the most lethal cutaneous cancers with a highly aggressive and metastatic phenotype. 1 The incidence of melanoma has risen dramatically over past decades. For earlystage melanoma, surgical resection is still recognized as the mainstay of therapeutic method, 2 but this disease has the proclivity to metastasize. At present, the long-term survival rate for patients with metastatic melanoma remains unfavourable. 3 Therefore, it is of great clinical significance to clarify the molecular mechanisms and genetic alterations underlying melanoma growth and metastasis.
More than 90% of transcripts from human genome are not translated into proteins. 4 Circular RNAs (circRNAs), a large group of widespread and diverse endogenous non-coding RNAs, are characterized by a covalently closed loop structure without a 5' cap or a 3' Poly A tail. 5 circRNAs were first considered as products of splicing errors, 6 but till now, emerging evidence indicates that they play important roles in many human diseases, including cancers. Considering their conserved sequences, stable structures and tissue-specific expression, circRNAs were deemed to be promising biomarkers for cancer diagnosis. 7 circRNA VANGL1 (circVANGL1) is generated from two exons of the Van Gogh-like 1 (VANGL1) gene and serves as a tumour promoter in bladder cancer. 8 Up-regulated circVANGL1 also contributes to the progression of non-small cell lung cancer. 9 In this research, we aimed to explore the functional role of circVANGL1 in melanoma progression.

| Patients and tissue samples
Sixty-nine melanoma tissues and twenty benign nevi tissues were obtained from patients who underwent surgical resection at the Seventh Affiliated Hospital of Sun Yat-sen University. None of these patients underwent chemotherapy or radiotherapy prior to surgery. After collection, all tissue samples were immediately snap-frozen in liquid nitrogen and stored at −80℃. This study was approved by the Ethics Committee of The Seventh Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki.

| Cell culture and treatments
Human melanoma cell lines, including WM-35, WM-115 and A375, and normal human epidermal melanocyte HEMa-LP cells were purchased from the Cell Bank of Chinese Academy of Sciences and maintained in RPMI-1640 Medium (Thermo Fisher Scientific, Inc.) containing 10% foetal bovine serum (FBS; Invitrogen) and 1% penicillin/streptomycin at 37℃ in a humidified incubator with 5% CO 2 .

| RT-qPCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen) and then treated with RNase-free DNase I (Promega). The RNA was first reversely transcribed to cDNA using the PrimeScript™ RT reagent kit (TaKaRa). Thereafter, qPCR reactions were carried out using a SYBR Green PCR Kit (TaKaRa) on a 7500HT Real-Time PCR System (Applied Biosystems). The 2 −ΔΔCt method was used to calculate the relative gene expression, 10 with GAPDH or U6 as an internal control.

| MTT assay
Cells were plated into 96-well plates at a density of 5 × 10 3 cells/ well and cultured for 24-72 h. Thereafter, 20 µl MTT solution (5 mg/L; Sigma-Aldrich) was added to each well. After incubation for additional 4 h, 150 μl DMSO (Sigma-Aldrich) was added to dissolve the formazan crystals. The absorbance of each well was read at 570 nm on an ELx808 microplate reader (BioTek Instruments, Inc.).

| Colony formation assay
Cells (1,000 cells/well) were suspended and seeded onto six-well plates. After 14 days, the medium was removed, and the colonies were fixed with methanol and stained with 0.1% crystal violet at room temperature. The number of colonies containing >10 cells was counted manually.

| Western blot analysis
Total protein was extracted using RIPA lysis buffer (Beyotime). Equal amounts of protein samples were separated by SDS-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking in 5% nonfat milk for 1 h, the membranes were incubated with the specific primary antibodies overnight at 4℃, followed by incubation with the HRPconjugated secondary antibody for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescent detection kit (Beyotime). GAPDH was used as a protein-loading control.

| Dual-luciferase reporter assay
The circVANGL1 sequence containing the putative target sites for miR-150-5p was amplified by PCR and inserted into the psiCHECK-

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software Inc.) and SPSS 18.0 software (SPSS Inc.). Continuous data are expressed as mean ± standard deviation (SD) and compared using Student's t test or one-way ANOVA.
The differences between groups were undertaken using Student's t test or one-way ANOVA. Statistical significance was set at p < 0.05.

| CircVANGL1 is up-regulated in melanoma
Through RT-qPCR analysis, we observed that circVANGL1 expression was notably increased in melanoma tissues, compared with benign nevi tissues ( Figure 1A). In addition, circVANGL1 was also up-regulated in melanoma cell lines (WM-35, WM-115 and A375), compared with normal HEMa-LP cells ( Figure 1B). Moreover, we observed that circVANGL1 was predominantly localized in the cytoplasm of WM-35, WM-115 and A375 cells ( Figure 1C).
We then investigated the correlation between circVANGL1 expression and clinicopathological characteristics in 69 melanoma patients. According to the median circVANGL1 expression level, these patients were allocated into two groups, including low expression group (n = 39) and high expression group (n = 30). As exhibited in Table 1, melanoma patients with high circVANGL1 expression exhibited a close association with larger tumour thickness (p = 0.035), advanced TNM stage (p = 0.006) and lymphatic metastasis (p = 0.031).

| CircVANGL1 promotes melanoma cell proliferation and invasion
Functional assays were further carried out to validate the role of circVANGL1 in melanoma progression. As shown in Figure 2A assay, circVANGL1 knockdown also notably inhibited the migratory and invasive abilities of A375 cells, whereas these abilities were enhanced by circVANGL1 overexpression in WM-115 cells ( Figure 2D).

| CircVANGL1 directly binds to miR-150-5p in melanoma
Since circVANGL1 was mainly located in the cytoplasm, we therefore speculated that circVANGL1 might serve as microRNA sponges in melanoma. By using the online software program Starbase (http:// starb ase.sysu.edu.cn/index.php), we noticed that circVANGL1 formed complementary base pairing with miR-150-5p ( Figure 3A).
We also found that circVANGL1 knockdown increased, while circ-

VANGL1 overexpression decreased miR-150-5p expression in A375
and WM-115 cells, respectively ( Figure 3B). Dual-luciferase reporter assay further indicated a significant reduction in the luciferase activities of circVANGL1-wt after co-transfection with miR-150-5p mimics in A375 andWM-115 cells, but the luciferase activities of circVANGL1-mut were not obviously affected ( Figure 3C)  Figure 3D). Furthermore, miR-150-5p expression was significantly decreased in melanoma tissues, compared with benign nevi tissues ( Figure 3E), and a negative correlation was observed between the expression of circVANGL1 and miR-150-5p in melanoma tissues ( Figure 3F).

| MiR-150-5p restoration blocks the oncogenic role of circVANGL1 in melanoma cells
We then performed rescue experiments to verify whether miR-150-5p was involved in the functions of circVANGL1 in melanoma.
As demonstrated in Figure 4A,B, the enhanced proliferation and clonogenic capacity of circVANGL1-overexpressing WM-115 cells were notably blocked by co-transfection with miR-150-5p mimics.
MiR-150-5p restoration also remarkably suppressed the migratory and invasive abilities of circVANGL1-overexpressing WM-115 cells ( Figure 4C). In addition, we confirmed that the effects of circ-VANGL1 knockdown in A375 cells were largely diminished by miR-150-5p inhibition.

| CircVANGL1 knockdown inhibits EMT of TGFβ-treated melanoma cells
As shown in Figure 5A We also observed that the increased circVANGL1 expression and decreased miR-150-5p expression in TGFβ-treated WM-35 and WM-115 cells were obviously reversed by SB431542, a TGFβ receptor antagonist ( Figure 5C,D). Moreover, as exhibited in Figure 5E,F, the TGFβ-induced EMT-related characteristics were also markedly diminished by circVANGL1 knockdown, and these effects were obviously blocked by miR-150-5p inhibition.

| DISCUSS ION
Melanoma is the most dangerous type of skin cancer, and its aetiology is a complex process involving multiple environmental, phenotypic and genetic factors. In recent years, many circRNAs have been identified to function as important drivers of tumorigenesis or tumour suppressors in melanoma. 12 The oncogenic functions of circVANGL1 were previously reported, and in the present study, we aimed to investigate the functional role of circVANGL1 in melanoma progression.
CircVANGL1 expression levels were observed to be significantly increased in clinical melanoma tissues and cell lines. We then conducted a series of functional experiments and demonstrated that circVANGL1 knockdown remarkably suppressed, while circVANGL1 overexpression enhanced the proliferation, migration and invasion abilities of melanoma cells. EMT is identified as a major determinant of melanoma metastasis, and therefore, modulation of EMT is a potential therapeutic strategy for reducing the aggressive progression of metastatic melanoma. 13,14 TGFβ is a secreted cytokine that stimulates tumour cells to undergo EMT. 15 This study further showed that TGFβ treatment enhanced EMT in melanoma cells, and the EMT-related characteristics were obviously diminished by circVANGL1 knockdown.
Next, we explored the mechanisms by which circVANGL1 serves as an oncogene in melanoma. It has been widely recognized that cir-cRNAs can function as microRNA sponges, indicating that circRNAs bind to miRNAs and consequently repress their functions. 16,17 Bioinformatic tools identified that miR-150-5p harbours a complementary sequence in circVANGL1 sequence. Previous studies have reported the inhibitory role of miR-150-5p in melanoma progression, 18,19 and through experimental validations, this study verified that circVANGL1 could directly interact with miR-150-5p and negatively regulated its expression in melanoma. By rescue experiments, we further confirmed that miR-150-5p restoration could block the oncogenic role of circVANGL1 in melanoma cells.
In conclusion, the findings of our study clearly demonstrated that circVANGL1 is up-regulated in melanoma and that it can enhance the malignant traits of melanoma cells partly by sponging miR-150-5p, indicating that targeting circVANGL1/miR-150-5p axis may be a promising therapeutic strategy for melanoma patients in the future.

CO N FLI C T O F I NTE R E S T
None.