The detailed information of human cutaneous melanoma cell lines and acral melanoma cells, growth media, and their metastatic state are in Table S1. All cells were grown in a humidified incubator at 37 °C with 5% CO2 and authenticated by STR analysis. Cells were maintained in culture for no more than 20 passages, excluding passaging prior to receipt in our lab. All cell lines were routinely tested negative for mycoplasma contamination.
Clinical sample collection
Human melanoma samples were obtained with informed consent, and the use of clinical samples was approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute. All samples were diagnosed via histopathological examination, including haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for melanoma markers (S-100, MART-1 and HMB-45).
Isolation of highly invasive HMY-1 cell sublines
Transwell chambers with 8 μm pores in 6-well plates (Corning, USA) were used to obtain highly invasive HMY-1 cell sublines. First, 7 × 105 HMY-1 cells in 1 mL serum-free DMEM were seeded into the upper chamber, which was precoated with Matrigel (BD Biosciences, USA), and 2 mL of DMEM supplemented with 25% foetal bovine serum was placed into the lower well. After incubation for 48 h, the invasive cells on the bottom of the chambers were harvested. Then, the harvested invasive cells were cultured and filtered for another 6 rounds using the Transwell chambers. After 7 continuous invasion assays, a highly invasive HMY-1 subline derived from the HMY-1 cell line was obtained. The invasive ability of these two cell lines was then confirmed by Transwell assays and lung metastasis models.
Purification of highly invasive HMY-1 clones
Five hundred highly invasive HMY-1 cells were evenly seeded onto a 10-cm dish. When a single cell grew to a visible cell cluster, a sterilized clone ring was used to get the clones. Different clones were separately cultured in a 24-well plate and passed on to 6-well plate when the plate was full.
RNA extraction and qRT–PCR
Total RNA was extracted from FFPE samples and cell lines using an RNeasy FFPE Kit (Qiagen, Germany) and TRIzol reagent (Life Technologies, USA), respectively. Then, cDNA was synthesized using a PrimeScript RT reagent kit (Takara, Japan) according to the manufacturer’s instructions. Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed using SYBR Green Real-time PCR Master Mix (TOYOBO, Japan). The sequence for each primer is listed in Table S2.
circRNA sequencing and microarray assay
For circRNA sequencing, total RNA was isolated from HMY-1 cells and highly invasive HMY-1 cells using TRIzol reagent (Life Technologies, USA) and purified with an RNease Mini kit (Qiagen, Germany). Then, circRNAs were sequenced using a human ceRNA Array V3.0 (4x180K, designed by Shanghai Biotechnology Corporation, and made by Agilent Technologies), which contains 105,509 circRNA probes.
Actinomycin D and RNase R treatment
Actinomycin D and RNase R treatment experiments were performed to assess the stability of circZNF609 and ZNF609 mRNA. In brief, cells were treated with 2 mg/ml actinomycin D in a 6-well plate. After incubation for several hours, the cells were collected. For RNase R treatment, total RNA (2 μg) was incubated for 15 min at 37 °C with 3 U/μg RNase R (Epicentre, WI, USA). After treatment with actinomycin D or RNase R, the circZNF609, ZNF609 and RAC1 mRNA expression was measured via qRT–PCR.
Small interfering RNA (siRNA) and transfection
siRNA duplexes for knockdown of circZNF609, ZNF609 and both were synthesized by GenePharma (Suzhou, China). Cells were transfected with the siRNAs using Lipofectamine RNAiMAX (Invitrogen, USA) according to the manufacturer’s instructions. The siRNA sequences are listed in Table S2.
circRNA plasmid construction and stable transfection
The circZNF609 overexpression plasmid and vector plasmid were obtained from Geneseed (Guangzhou, China). 293 T cells were transfected with the plasmids to package the lentivirus using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Human melanoma cells were infected with the packaged lentivirus and selected with 0.5–2 μg/ml puromycin for 4 days. Surviving melanoma cells exhibiting GFP expression were then selected for subsequent culture.
Nucleoplasmic separation assay
The nuclear and cytoplasmic fractions were extracted using a Minute™ Cytoplasmic and Nuclear Extraction Kit (Invent Biotechnologies, USA) according to the manufacturer’s instructions. In brief, cells were washed with cold PBS for 2 min. Then, cell lysis buffer was added to the cells on ice. After incubation for 5 min, the cell lysate was transferred to a prechilled 1.5 ml microcentrifuge tube. The tube was vigorously vortexed for 15 s. After centrifugation of the tube for 5 min at top speed at 4 °C, the supernatant was collected as the cytosolic fraction. Then, an appropriate amount of nuclear extraction buffer was added to the pellet, and the tube was vigorously vortexed for 15 s and incubated on ice for 1 min. The 15 s of vortexing and 1 min incubation were repeated 4 times. After that, the nuclear fraction was transferred to a prechilled filter cartridge with a collection tube and centrifuged at top speed in a microcentrifuge for 30 s.
Fluorescence in situ hybridization (FISH)
A Cy3-labelled specific probe for circZNF609 was designed and synthesized by RiboBio. The FISH experiment was performed using a FISH kit (RiboBio, China) according to the manufacturer’s instructions.
Human melanoma cells were fixed with 4% paraformaldehyde for 15 min and then blocked with 5% BSA for 30 min. The melanoma cells were incubated with primary antibodies at 4 °C overnight and fluorescence-conjugated secondary antibodies at room temperature for 1 h. After washing with PBS, the cells were counterstained with DAPI for 10 min and imaged under a microscope (Olympus Corp).
Wound healing assays
Wound healing assays were performed using Culture–Insert (Ibidi, Germany) according to the manufacturer’s instructions. In brief, 1 × 105 human melanoma cells in 70 μl complete medium were seeded into each culture well and incubated for 24 h, and then, the culture insert was gently removed using sterile tweezers. After washing with PBS, the cells were cultured in culture medium with 1% FBS. Images were taken at several time points.
Human melanoma cells were cultured in serum-free culture medium 24 h before performing Transwell assays. Cell culture medium supplemented with 25% foetal bovine serum was added to the bottom chambers of a 24-well plate. For the invasion and migration assays, 1 × 105 melanoma cells in serum-free medium were seeded into the top chamber with or without Matrigel (BD Biosciences, USA), respectively. After incubation for several hours, the Transwell inserts were fixed in 4% paraformaldehyde for 10 min and then stained with 1% crystal violet for 10 min. Cells that did not migrate through the 8 μm pores of the Transwell chambers were removed using cotton swabs. Migratory or invasive melanoma cells located on the bottom of the chamber were counted using an inverted phase-contrast microscope.
3D spheroid-based Matrigel invasion assay
3D spheroid-based Matrigel invasion assays were performed as previously described [25,26,27]. In brief, 1 × 104 LM-MEL-45 cells in 200 μl complete culture medium were grown in ultralow attachment (ULA) 96-well round-bottom plates (Corning, USA) for 3 days. After tumor spheroids formed, 100 μl of growth medium was gently removed from the spheroid plates. Then, 100 μl Matrigel (BD Biosciences, USA) was gently dispensed into the U-bottom well. After incubation in an incubator at 37 °C for 1 h, 100 μl complete growth medium was gently added to each well. Three days later, images of invasive tumor spheroids were taken with an inverted phase-contrast microscope.
All animal experiments were approved by the Medical Ethics Committee of the Beijing Cancer Hospital & Institute. For in vivo cutaneous melanoma lung metastasis assays, 4-week-old female BALB/c nude mice were intravenously injected with 5 × 105 A2058 cells in 100 μl PBS via the tail vein. The mice were sacrificed when they became weak, hunched and emaciated. For in vivo acral melanoma lung metastasis assays, 5-week-old female NOD/SCID mice were intravenously injected with 2 × 106 HMY-1 cells in 200 μl PBS via the tail vein. The mice were sacrificed when they became weak, hunched and emaciated. For in vivo acral melanoma liver metastasis assays, 1 × 106 HMY-1 cells suspended in 50 μl PBS were injected into the inferior hemispleen of 4-week-old female NOG mice. The mice were euthanized after 4 weeks. The lungs and livers were excised, and then, the number of metastatic tumor nodules in the lungs and livers was carefully counted. Subsequently, all the samples were fixed with 4% paraformaldehyde and analysed via H&E staining. To detect tumors in live animals, mice were injected with 100 μl D-luciferin (15 mg/ml) and then anaesthetized with isoflurane. Ten minutes later, bioluminescence images were acquired and analysed using an IVIS Spectrum In Vivo Imager.
RNA pull-down assay
The biotin-labelled circZNF609 probe was synthesized by RiboBio, and the RNA pull-down assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, USA) according to the manufacturer’s instructions. First, the biotin-labelled circZNF609 probe or control probe was incubated with streptavidin magnetic beads at room temperature for 30 min. Then, the melanoma cell lysates were incubated with probe-bead complexes at 4 °C for 1 h to allow binding of RBPs to RNAs. Subsequently, the RNA-protein complexes were washed and eluted from beads via incubation for 30 min at 37 °C with agitation. The eluted proteins were finally analysed via silver staining and Western blotting. The circZNF609 probe and control probe sequences used are listed in Table S2.
Silver staining was performed using a Fast Silver Stain Kit (Beyotime, China) according to the manufacturer’s instructions.
RNA immunoprecipitation (RIP)
RIP assays were performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) as described in the protocol. Briefly, human melanoma cells were lysed in RIP lysis buffer on ice for 5 min. After washing, 50 μl magnetic beads were incubated with 5 μg antibodies with rotation for 30 min at room temperature. After incubation, the melanoma cell lysates were incubated with bead-antibody complexes at 4 °C overnight. Subsequently, the immune complexes were washed six times with washing buffer, and the remaining RNA was purified and extracted for later qRT–PCR analysis and RIP sequencing.
All experiments were carried out at least three times, and the results are presented as the mean ± standard deviation (S.D.). Statistical analysis was performed using Student’s two-tailed t test or one-way analysis of variance (ANOVA). The expression correlations in melanoma samples were assessed using Pearson correlation analysis. Survival curves were assessed using the Kaplan–Meier method and compared with log-rank tests. Statistical significance was defined as a P value of less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001); ns: not significant (P > 0.05).
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