Long non-coding RNA CYTOR promotes the progression of melanoma via the miR-485-5p/GPI axis

Patient information

The study was approved by the Medical Ethics Committee of The Affiliated Hospital of Chengde Medical University (CYFYLL2023241) and conducted in accordance with the Declaration of Helsinki. All patients were informed with the study and the signed consent forms were obtained.

Expression profile analysis of CYTOR

GEPIA 2 (http://gepia2.cancer-pku.cn/) was applied to analyze the difference of CYTOR mRNA expression between melanoma tumor and non-tumor control tissues based upon the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. |Log2FC| > 1 and p < 0.05 was considered as statistical significance. The “stage plot” module was selected, then we entered “CYTOR” and selected melanoma cancer to assess the correlation between CYTOR expression level and tumor pathological stages. The Kaplan–Meier (KM) plotter was used to analyze the prognostic value of CYTOR in melanoma.

Tissues and cells

Melanoma tissues and corresponding adjacent noncancerous tissues were obtained from the Affiliated Hospital of Chengde Medical University. None of the participants had received any preoperative anti-tumor treatments, including radiotherapy, chemotherapy, or surgery. This research received approval from the Ethics Committee of the Affiliated Hospital of Chengde Medical University (Approval No: CYFYLL2023241). All participants received informed consent. Clinical trial number: not applicable.

Four human melanoma cell lines (SK-MEL-28, MV3, A875, and A375), along with a normal human melanocyte cell line (PIG1), were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

Quantitative reverse transcriptase-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized separately using the FastKing RT Kit (containing gDNase) (TIANGEN, Beijing, China) and miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China). All qPCR reactions were conducted in accordance with the manufacturer’s guidelines for the SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China). The expression levels of CYTOR, glucose-6-phosphate isomerase (GPI), and miR-485-5p were normalized to either GAPDH or U6, as appropriate. The primers employed were as follows:

CYTOR: F: 5′-CCGAAAATCACGACTCAGCC-3′, R: 5′-CAGGCCCCAGGGAATCTTTC-3′;

miR-485-5p: 5′-CTGGCCGTGATGAATTCAAAA-3′;

U6: F: 5′-GGCTGGTAAGGATGAAGG-3′, R: 5′- TGGAAGGAGGTCATACGG-3′;

GPI: F: 5′- CCAGACCCAGCACCCCATA-3′, R: 5′-TCCGTCGATTTTCCCCTCA-3′.

RNA-fluorescence in situ hybridization

SK-MEL-28, MV3, A875, and A375 cells were fixed in 4% formaldehyde. Following this, the cells were rendered permeable through exposure to Triton X-100 for 5 min. They were then incubated with pre-hybridization buffer and exposed overnight to a 37 °C pre-hybridization buffer containing 20 μM CYTOR FISH probe (Servicebio, Wuhan, China). DAPI staining was applied and the cells were analyzed using a fluorescence microscope.

Transfection

The siRNA sequences (si-CYTOR-1: F: 5′-CCGUCUGCAUCCCUCGAAUTT-3′, R: 5′-AUUCGAGGGAUGCAGACGGTT-3′; si-CYTOR-2: F: 5′-GAAACCUCUUGACUCUUCUTT-3′, R: 5′-AGAAGAGUCAAGAGGUUUCTT-3′; si-CYTOR-3: F: 5′-GGCUUGAACAUUUGGUCUUTT-3′, R: 5′-AAGACCAAAUGUUCAAGCCTT-3′) targeting CYTOR were synthesized by GenePharma (Shanghai, China), with a scramble sequence serving as the negative control (NC). Full-length cDNA of CYTOR was synthesized and cloned into the pcDNA3.1 vector, also obtained from GenePharma, with the empty vector serving as the negative control. All miR-485-5p mimics (F: 5′-AGAGGCUGGCCGUGAUGAAUUC-3′, R: 5′-AUUCAUCACGGCCAGCCUCUUU-3′), miR-485-5p inhibitors (5′-GAAUUCAUCACGGCCAGCCUCU-3′), and their respective controls were also sourced from GenePharma. All cells were transfected using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) following the manufacturer’s instructions.

Cell viability assay

Cell proliferation was assessed employing the CCK-8 assay (Dojindo, Tokyo, Japan) according to the manufacturer’s protocol. Cells that had undergone transfection for 24 h were plated into five 96-well plates at a density of 2 × 10³ cells per well and incubated at 37 °C. At 24-h intervals, one plate was removed, and 10 μL of CCK-8 reagent was added to each well. The plates were incubated for 2 h at the same conditions, and the cell viability was determined by measuring the optical density (OD) at 450 nm. A proliferation curve was then generated based on the OD values. These experiments were performed in triplicate.

Scratch wound assay

Following 24 h post-transfection, the cells were seeded into 6-well plates at a concentration of 4 × 10⁵ cells per well and incubated for another 24 h. A wound was made in the middle of each well with a 200 µL pipette tip, followed by washing and an additional 24-h incubation in serum-free medium. Images of the wound areas were captured, and the degree of wound closure was assessed through ImageJ software.

Transwell invasion assay

The invasive capacity of the transfected cells was evaluated through a transwell invasion assay with 8 μm pores (Corning, NY, USA). The upper chamber was pre-coated with Matrigel matrix gel (BD Biosciences, San Jose, USA). Following 24 h post-transfection, the cells were resuspended in a serum-free medium. Subsequently, 200 µL of the cell suspension containing 4 × 10⁴ cells was introduced into the upper chamber, and 600 µL of medium supplemented with fetal bovine serum (FBS) was introduced in the lower chamber. After a 24-h incubation, cells that crossed the membrane were fixed in 4% paraformaldehyde and stained using 0.1% crystal violet. The cells were subsequently rinsed with PBS, and three random regions were selected under a microscope (magnification, ×100) for imaging and quantification.

Xenograft tumor model experiments

Lentiviral vectors encoding sh-CYTOR were constructed using the LV3-H1-GFP+PURO vector (GenePharma, Shanghai, China) in order to achieve stable knockdown of CYTOR in A375 cells. Male BALB/c nude mice, aged 5 weeks, were obtained from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The mice were acclimated for 1 week prior to the experiment. Each cage contained 2–4 mice of the same gender. Mice were housed at 20–24 °C under a 12-h light-dark cycle (lights on at 07:00, lights off at 19:00), with food and water available ad libitum. Equal volumes of LV3-sh-CYTOR or LV3-sh-NC A375 cell suspensions were injected subcutaneously into the axillary region of the mice (0.2 mL, 1 × 10⁶ cells per 100 µL). Each group consisted of 5 mice. Tumor progression was assessed every 3 days. On the 18th day post-injection, the tumors were excised. Statistical analyses were conducted using GraphPad Prism 8.0 (San Diego, CA, USA), and results were expressed as mean ± standard deviation (SD). All animal experiments were reviewed and approved by the Animal Welfare Committee of the Affiliated Hospital of Chengde Medical University. The experiments were carried out following ethical standards and established animal care guidelines.

Western blot analysis

Cells or melanoma tissues were lysed in RIPA buffer (Solarbio, Beijing, China) containing a protease inhibitor cocktail (Solarbio, Beijing, China). The extracted proteins were separated using gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China), and blocked in 5% skimmed milk. The membranes were exposed to primary antibodies targeting GPI (A Rabbit Anti-Human monoclonal IgG antibody, Abcam, UK, ab167394) and α-Tubulin (A Rabbit Anti-Human polyclonal IgG antibody, Proteintech, China, 11224-1-AP) at 4 °C overnight. Afterward, the membranes were exposed to a secondary antibody (A Goat Anti-Rabbit IgG antibody, Affinity Biosciences, Cincinnati, OH, USA, S0001). The detection of protein bands was performed via electrochemiluminescence (ECL, Biosharp, Hefei, China).

Dual-luciferase reporter gene assay

When cells reached 60% confluence, they were co-transfected with 1 μg of GP-mirGLO-CYTOR/GPI 3′UTR and 50 pmol of miR-485-5p mimics (F: 5′-AGAGGCUGGCCGUGAUGAAUUC-3′, R: 5′-AUUCAUCACGGCCAGCCUCUUU-3′) using GP-transfect-Mate. 48 h post-transfection, the cells were lysed to assess luciferase activity with the Dual-Luciferase Reporter Assay System. The luciferase reporter constructs were obtained from GenePharma (Shanghai, China). All experiments were conducted in triplicate.

Statistical analysis

Statistical analyses were conducted with SPSS 17.0 (Chicago, IL, USA) and GraphPad Prism 8.0 (San Diego, CA, USA), and results were expressed as mean ± standard deviation (SD). Differences between two groups were assessed using the Student’s t-test, while one-way analysis of variance (ANOVA) was applied for comparisons involving three or more groups, followed by the least significant difference (LSD) multiple comparison test. Spearman’s correlation was applied to assess relationships between variables (*p < 0.05, **p < 0.01, and ***p < 0.001).

High expression of CYTOR in melanoma

Utilizing the Gene Expression Profiling Interactive Analysis (GEPIA2) with |Log₂FC| > 1 and p < 0.05 as target selection standard, we observed a significant upregulation of CYTOR in melanoma tissues compared to normal tissues (Average Log₂FC 5.75 vs 3.82, p < 0.05) (Fig. 1A). CYTOR expression levels were quantified in 18 melanoma clinical specimens and melanoma cell lines (SK-MEL-28, MV3, A875, and A375) via qRT-PCR. The results showed that the CYTOR expression was highest in A375 cells (nearly six times that seen in the PIG1 melanocyte cells) and was also significantly elevated in SK-MEL-28, MV3 and A875 cells (two times, two times and three times respectively compared with that of PIG1 melanocyte cells) (Fig. 1C). The findings revealed elevated CYTOR expression in both melanoma cell lines and tissues in comparison to the control groups (Figs. 1B and 1C). Furthermore, CYTOR expression correlated with the prognosis of melanoma patients. Those exhibiting elevated CYTOR levels demonstrated reduced overall survival (OS) in comparison to patients with lower expression levels (Fig. 1D). These findings indicate that CYTOR could serve as a potential prognostic marker in melanoma.

CYTOR downregulation inhibits melanoma cell proliferation, migration, and invasion

To investigate the functional role of CYTOR in melanoma cells, knockdown experiments were conducted through transfecting A375 and A875 cells with either si-NC or si-CYTOR. Both si-CYTOR-2 and si-CYTOR-3 effectively reduced CYTOR expression (Fig. 2A). The CCK-8 assay demonstrated that CYTOR silencing significantly inhibited cell proliferation in contrast to si-NC transfection (Fig. 2B). Furthermore, the impact of CYTOR on cellular migration and invasion was evaluated through scratch wound and transwell assays. CYTOR silencing significantly diminished the migratory and invasive capacities in the cell lines (Figs. 2C and 2D). These findings suggest that CYTOR silencing may reduce the malignant phenotype of melanoma cells.

Upregulation of CYTOR stimulates melanoma cell proliferation, migration, and invasion

We next performed overexpression assays, demonstrating that CYTOR overexpression significantly enhanced proliferation (Figs. 3A and 3B), migration (Figs. 3C and 3D), and invasion (Figs. 3C and 3D) of SK-MEL-28 and MV3 cells.

MiR-485-5p is a target of CYTOR

Starbase (ENCORI) (https://rnasysu.com/encori/) was used to verify interaction of lncRNA CYTO and miR-485-5P (p value of 2.283e−5 and FDR of 9.134e−5) and to predict the binding site of CYTO to miR-485-5P. To further elucidate the role of CYTOR in melanoma tumorigenesis, we utilized a competitive endogenous RNA (ceRNA) model. Fluorescence in situ hybridization (FISH) assays demonstrated that CYTOR primarily localizes within the cytoplasmic compartment (Fig. 4A). Bioinformatic analyses predicted possible interaction sites between CYTOR and miR-485-5p, which were subsequently validated using a luciferase reporter assay (Figs. 4B and 4C). In melanoma cells, miR-485-5p expression was observed to be reduced compared to normal cells (Fig. 4D). Furthermore, the expression of miR-485-5p in MV3 and A875 cells was markedly increased or decreased following CYTOR knockdown or overexpression, respectively (Fig. 4E). In clinical melanoma specimens, miR-485-5p levels were diminished and exhibited an inverse relationship with CYTOR expression (Figs. 4F and 4G).

GPI is a target of miR-485-5p

TargetScan (https://www.targetscan.org/vert_80/) was used to verify the interaction of miR-485-5P and GPI and to predict the binding site of GP to miR-485-5P. miR-485-5p targeting GPI was corroborated by luciferase reporter assays (Figs. 5A and 5B). A marked upregulation of GPI expression was detected in both melanoma cells and tissues (Figs. 5C–5E). MiR-485-5p mimic transfection led to a reduction in GPI mRNA levels and protein expression in MV3 and A875 cells, whereas miR-485-5p inhibitor transfection produced the converse effect (Figs. 5F and 5G). Furthermore, GPI levels exhibited an inverse correlation with miR-485-5p expression, and elevated GPI expression was linked to reduced OS in melanoma patients (Figs. 5H and 5I).

CYTOR enhances GPI expression by sequestering miR-485-5p

To investigate the impact of the CYTOR/miR-485-5p/GPI signaling pathway in melanoma development, we simultaneously overexpressed CYTOR and miR-485-5p mimics in MV3 and A875 cell lines. In CCK-8 essays, CYTOR overexpression significantly enhanced cell viability. However, co-transfection with pcDNA CYTOR and miR-485-5p mimics resulted in decreased cell proliferation in MV3 and A875 cell lines (Fig. 6A). Similarly, CYTOR overexpression facilitated migration and invasion, but this impact was markedly reduced when cells were co-transfected with pcDNA CYTOR and miR-485-5p mimics (Figs. 6B and 6C). Furthermore, Western blot analysis demonstrated that miR-485-5p mimics effectively reversed the alterations in GPI protein levels induced by CYTOR overexpression in melanoma cells (Fig. 6D). In conclusion, our findings suggest that the CYTOR/miR-485-5p/GPI pathway contributes to the regulation of melanoma aggressiveness.

CYTOR suppression impedes the growth of melanoma cell xenograft tumors in vivo

Finally, we aimed to investigate the role of CYTOR in regulating tumorigenesis in vivo by establishing a xenograft model. Nude mice were implanted with A375 cells transduced with either control or sh-CYTOR lentiviral vectors. The results revealed that CYTOR silencing significantly inhibited tumor growth (Figs. 7A–7C). Tumors transduced with the sh-CYTOR lentivirus exhibited significantly higher levels of miR-485-5p compared to control tumors (Fig. 7D). Furthermore, Western blotting verified that GPI levels were markedly decreased in tumors infected with the sh-CYTOR lentivirus compared to control tumors (Fig. 7E). These findings demonstrate that CYTOR downregulation is sufficient to suppress melanoma tumor growth in vivo.