Contributed equally
Cutaneous melanoma is an aggressive malignant cancer associated with poor prognosis. Identification of reliable biomarkers for predicting prognosis of melanoma contributes to improved clinical outcome and disease management. Long non-coding RNAs (lncRNAs) serve a crucial regulatory role of oncogenesis and tumor suppression in melanoma. Using data from The Cancer Genome Atlas database, novel lncRNA 11β-hydroxysteroid dehydrogenase type 1-antisense RNA 1 (
Melanoma, a deadly malignant cancer originating from melanocytes, accounts for 5% of all skin cancers but 75% of skin cancer-associated mortality (
Long non-coding RNAs (lncRNAs) are a group of ncRNAs >200 nucleotides in length that have no protein-coding capacity due to the lack of open reading frames (
To date, melanoma has been regarded as a highly immunogenic tumor because of the key role of the immune system in its development and progression. For example, it demonstrates immunogenicity attributed to the recognition of antigens expressed by melanocytes (
The present study used publicly available microarray data to identify a novel tumor-suppressing lncRNA, 11β-hydroxysteroid dehydrogenase type 1-antisense RNA 1 (
RNA-Seq data and clinical information were obtained from University of California Santa Cruz Xena databases (xenabrowser.net/datapages/). Cases without clinical features were excluded. In clinical information, the Clark Level, which has five levels from Level 1 to Level 5, is a staging system that describes the depth of melanoma as it grows in the skin, and the Breslow Depth is a more standardized method to measure how far melanoma has invaded the body, which requires an optical micrometer fitted to the ocular position of a standard microscope (
Cutaneous melanoma cell lines and normal human epidermal melanocytes (HEMs) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (HyClone; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin in a sterile and humidified incubator at 37°C with 5% CO2. The constructed
Total RNA was extracted from the A375, AK-MEL-1 and HEM cell lines using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) after which the RNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed (10 µl reaction system) according to the instructions of PrimeScript™ RT reagent kit and SYBR Premix Ex Taq II (both Takara Biotechnology Co., Ltd.) was used to assess gene expression. The thermocycling conditions for RT-qPCR were as follows: Preheating for 10 min at 95°C; followed by 40 cycles at 95°C for 15 sec and 60°C for 60 sec. The following primers were used:
Cell proliferation was assessed by CCK-8 assay (BBI Life Sciences Corporation) according to the manufacturer's protocol. At 0, 24, 48, 72 and 96 h after transfection, A375 and SK-MEL-1 cells (2×104/ml) were seeded in 96-well plates overnight and 10 µl CCK-8 reagent was added to each well before cells were incubated for 1 h. The absorbance value at 450 nm was detected using a microplate reader (EL800; BioTek Instruments, Inc.).
At 6 h after transfection, A375 and SK-MEL-1 melanoma cell lines were seeded into a 6 cm plastic culture dish (1,000 cells/plate). Following 14 days incubation at 37°C with 5% CO2, the colonies were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 0.1% crystal violet at room temperature for 5 min. Then, the number of colonies (>50 cells) was counted manually under an Olympus IX71 inverted microscope (magnification, ×40; Olympus Corporation).
A375 and SK-MEL-1 cell migration was assessed using scratch wound healing assay. In brief, when the monolayer reached ~70% confluence, a 1-ml sterile pipetting tip was used to vertically scratch a 6-well plate with a cell monolayer adhered to the wall. Then, the plates were washed three times with PBS to remove the detached serum-starved cells. Images were captured under an Olympus IX71 inverted microscope (magnification, ×100; Olympus Corporation) at 0 and 48 h, and the wound closure ratio was calculated.
Transwell assay was performed using Transwell chambers (Costar; Corning, Inc.; pore size, 8 µm) coated with Matrigel (Sigma-Aldrich; Merck KGaA) at 37°C for 4 h. For the assay, 100 µl A375 or SK-MEL-1 cell suspensions (5×105/ml) in serum-free medium was seeded into the upper chamber and 600 µl DMEM containing 10% fetal bovine serum (HyClone; Thermo Fisher Scientific, Inc.) was added to the bottom chamber at 37°C. After 24 h cell culture at 37°C with 5% CO2, the migrated cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet solution at room temperature for 30 min. Cells in 10 random fields of view were counted and images were captured under an Olympus IX71 inverted microscope (magnification, ×100; Olympus Corporation).
GSEA was performed using a gene expression matrix of melanoma extracted from RNA-Seq TCGA datasets to identify the distinct hallmarks of
Immune infiltration analysis was performed by single-sample GSEA method using the R package Gene Set Variation Analysis (GSVA) (
All data were presented as the mean ± standard deviation and all tests were performed in triplicate. The comparison of the mean values between two factors was performed using an independent sample t-test when variances were homogeneous. When variances were not homogeneous, rank sum statistical analysis was performed. The comparison of the mean values of multiple factors was performed using one-way ANOVA followed by a Bonferroni post hoc test. All bioinformatics analysis was performed in R (v.3.6.2). Kaplan-Meier method using the R package Survminer and univariate and multivariate Cox regression analyses were used to assess the prognostic value of
Significantly lower expression levels of
Association analysis of clinicopathological characteristics in low- and high-
Decreased
Kaplan-Meier survival analysis indicated that increased
The univariate Cox regression analysis demonstrated that elevated
Nomograms for 1-, 3-, and 5-year OS, PFI and DSS were constructed based on the independent variables obtained from the multivariable analysis (
CCK-8 assay showed that
Wound healing and Transwell migration assays were performed to assess the effect of
GSEA analysis revealed that 12 pathways were significantly enriched: ‘Interferon gamma response’, ‘IL-6/JAK/STAT-3 signaling’, ‘allograft rejection’, ‘inflammatory response’, ‘KRAS signaling up’, ‘complement’, ‘epithelial mesenchymal transition’, ‘TNF-α signaling via NF-κB, IL-2/STAT-5 signaling’, ‘coagulation’, ‘IFN-α response’, and ‘apoptosis’ (
Increasing evidence has suggested that lncRNAs serve as tumor suppressors or oncogenes and may be targets for developing selective anti-cancer therapeutic strategies owing to their cell type- and disease-specific expression profiles and their key role in tumor proliferation, migration and invasion (
The present study demonstrated that
To assess the accuracy of
To the best of our knowledge, the present study is the first to report that
Immune infiltration analysis revealed that
Although the findings of the present study provide understanding about the functions of
To the best of our knowledge, the present study was the first to demonstrate that
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
KL and LZ conceived and designed the present study. JZ and XL performed the literature review and analyzed the data. KL, LZ and XL drafted the initial manuscript and confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Flow chart demonstrating how long non-coding RNA
Differential expression of
Association of
Prognostic nomogram and calibration plot to predict the 1-, 3- and 5-year OS, PFI and DSS probabilities in patients with cutaneous melanoma. Nomograms for predicting the probability of 1-, 3-, and 5-year (A) OS, (B) PFI and (C) DSS. Calibration curves of the prognostic nomograms for predicting OS, PFI and DSS at 1, 3, and 5 years demonstrated no distinct departure from the ideal lines. OS, overall survival; PFI, progression free interval; DSS, disease specific survival.
Enrichment plots from Gene Set Enrichment Analysis. (A) Interferon gamma response, (B) allograft rejection, (C) complement, (D) inflammatory response, (E) epithelial mesenchymal transition, (F) TNF-α signaling via NF-κB, (G) IL-2/STAT-5 signaling, (H) KRAS signaling up, (I) coagulation, (J) IFN-α response, (K) IL-6/JAK/STAT-3 signaling, and (L) apoptosis were significantly enriched. The top 12 are presented and ordered by ascending adjusted P-value. Adj., adjusted. NES, normalized enrichment score; FDR, false discovery rate.
Association analysis of
Association between
Characteristic | Low (n=234) | High (n=234) | P-value |
---|---|---|---|
Sex | 0.849 | ||
Female | 88.00 (37.60) (%) | 91.00 (38.90) (%) | |
Male | 146.00 (62.40) (%) | 143.00 (61.10) (%) | |
Median age, years (IQR) | 60.00 (48.25-72.00) | 58.00 (47.00-68.00) | 0.036 |
Breslow depth | 0.005 |
||
≤3 mm | 77.00 (43.80) (%) | 107.00 (59.10) (%) | |
>3 mm | 99.00 (56.20) (%) | 74.00 (40.90) (%) | |
Melanoma ulceration | 0.003 |
||
No | 62.00 (38.30) (%) | 83.00 (55.70) (%) | |
Yes | 100.00 (61.70) (%) | 66.00 (44.30) (%) | |
T stage | 0.015 |
||
T1 | 13.00 (7.30) (%) | 28.00 (15.30) (%) | |
T2 | 37.00 (20.80) (%) | 41.00 (22.40) (%) | |
T3 | 40.00 (22.50) (%) | 50.00 (27.30) (%) | |
T4 | 88.00 (49.40) (%) | 64.00 (35.00) (%) | |
N stage | 0.717 | ||
N0 | 120.00 (59.10) (%) | 114.00 (54.80) (%) | |
N1 | 36.00 (17.70) (%) | 38.00 (18.30) (%) | |
N2 | 24.00 (11.80) (%) | 25.00 (12.00) (%) | |
N3 | 23.00 (11.30) (%) | 31.00 (14.90) (%) | |
M stage | 0.305 | ||
M0 | 206.00 (93.20) (%) | 210.00 (95.90) (%) | |
M1 | 15.00 (6.80) (%) | 9.00 (4.10) (%) | |
Pathological stage | 0.003 |
||
I | 27.00 (13.50) (%) | 49.00 (23.40) (%) | |
II | 83.00 (41.50) (%) | 57.00 (27.30) (%) | |
III | 76.00 (38.00) (%) | 94.00 (45.00) (%) | |
IV | 14.00 (7.00) (%) | 9.00 (4.30) (%) | |
Melanoma Clark level | 0.002 |
||
I | 3.00 (1.90) (%) | 3.00 (1.90) (%) | |
II | 4.00 (2.50) (%) | 14.00 (8.70) (%) | |
III | 29.00 (18.40) (%) | 47.00 (29.20) (%) | |
IV | 88.00 (55.70) (%) | 80.00 (49.70) (%) | |
V | 34.00 (21.50) (%) | 17.00 (10.60) (%) | |
Radiation therapy | 0.213 | ||
No | 194.00 (85.10) (%) | 187.00 (80.30) (%) | |
Yes | 34.00 (14.90) (%) | 46.00 (19.70) (%) |
P<0.05.
Characteristic | Total number of cases | OR | P-value |
---|---|---|---|
Sex, female vs. male | 468 | 1.06 (0.73-1.53) | 0.775 |
Age, ≤60 vs. >60 years | 460 | 1.29 (0.90-1.87) | 0.170 |
T stage, T2, 3 and 4 vs. 1 | 361 | 0.44 (0.21-0.86) | 0.019 |
N stage, N1, 2 and 3 vs. 0 | 411 | 1.19 (0.81-1.76) | 0.378 |
M stage, M1 vs. 0 | 440 | 0.59 (0.24-1.35) | 0.221 |
Pathological stage, IV, III and II vs. I | 409 | 0.51 (0.30-0.85) | 0.011 |
Melanoma Clark level, IV and V vs. I, II and III | 319 | 0.45 (0.27-0.72) | 0.001 |
Breslow depth, >3 vs. ≤3 mm | 357 | 0.54 (0.35-0.82) | 0.004 |
Melanoma ulceration, yes vs. no | 311 | 0.49 (0.31-0.77) | 0.002 |
Radiation therapy, no vs. yes | 461 | 0.71 (0.44-1.16) | 0.172 |
P<0.05.
Association between overall survival and clinicopathological features using univariate and multivariate Cox regression.
Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|
Characteristic | Total | HR (95% CI) | P-value | HR (95% CI) | P-value |
Sex, male vs. female | 453 | 1.164 (0.872-1.554) | 0.301 | - | - |
Age, >60 vs. ≤60 years | 453 | 1.678 (1.266-2.225) | <0.001 |
1.166 (0.781-1.741) | 0.452 |
T stage, T3 and 4 vs. 1 and 2 | 358 | 2.040 (1.468-2.836) | <0.001 |
0.909 (0.517-1.600) | 0.742 |
N stage, N1, 2 and 3 vs. 0 | 399 | 1.711 (1.271-2.304) | <0.001 |
3.764 (1.140-12.425) | 0.030 |
M stage, M1 vs. 0 | 427 | 1.734 (0.915-3.287) | 0.092 | - | - |
Pathological stage, III and IV vs. I and II | 407 | 1.579 (1.177-2.118) | 0.002 |
0.598 (0.182-1.968) | 0.398 |
Melanoma Clark level, IV and V vs. I, II and III | 312 | 2.117 (1.472-3.045) | <0.001 |
1.291 (0.797-2.090) | 0.299 |
Melanoma ulceration, yes vs. no | 310 | 2.087 (1.494-2.916) | <0.001 |
1.315 (0.865-1.999) | 0.201 |
Breslow depth, ≤3 vs. >3 mm | 352 | 0.386 (0.281-0.528) | <0.001 |
0.537 (0.314-0.916) | 0.023 |
Radiation therapy, yes vs. no | 447 | 0.953 (0.674-1.348) | 0.785 | - | - |
453 | 0.534 (0.407-0.700) | <0.001 |
0.618 (0.423-0.903) | 0.013 |
P<0.05. HR, hazard ratio;
Association between with progression-free interval and clinicopathological features using univariate and multivariate Cox regression.
Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|
Characteristic | Total | HR (95% CI) | P-value | HR (95% CI) | P-value |
Sex, male vs. female | 454 | 1.027 (0.813-1.298) | 0.821 | - | - |
Age, >60 vs. ≤60 years | 454 | 1.600 (1.258-2.035) | <0.001 |
1.398 (0.988-1.978) | 0.059 |
T stage, T3 and 4 vs. 1 and 2 | 359 | 1.655 (1.259-2.175) | <0.001 |
0.903 (0.569-1.433) | 0.665 |
N stage, N1, 2 and 3 vs. 0 | 400 | 1.853 (1.451-2.365) | <0.001 |
2.611 (0.991-6.880) | 0.052 |
M stage, M1 vs. 0 | 428 | 1.942 (1.188-3.175) | 0.008 |
1.176 (0.481-2.877) | 0.722 |
Pathological stage, III and IV vs. I and II | 408 | 1.717 (1.349-2.187) | <0.001 |
0.868 (0.322-2.343) | 0.781 |
Melanoma Clark level, IV and V vs. I, II and III | 312 | 1.762 (1.302-2.386) | <0.001 |
0.956 (0.628-1.455) | 0.832 |
Melanoma ulceration, yes vs. no | 310 | 1.635 (1.233-2.169) | <0.001 |
1.182 (0.831-1.681) | 0.351 |
Breslow depth, ≤3 vs. >3 mm | 352 | 0.498 (0.378-0.655) | <0.001 |
0.566 (0.359-0.892) | 0.014 |
Radiation therapy, yes vs. no | 448 | 1.201 (0.906-1.593) | 0.203 | - | - |
454 | 0.691 (0.552-0.866) | 0.001 |
0.704 (0.507-0.978) | 0.036 |
P<0.05. HR, hazard ratio;
Association between disease-specific survival and clinicopathological features using univariate and multivariate Cox regression.
Univariate analysis | Multivariate analysis | ||||
---|---|---|---|---|---|
Characteristic | Total | HR (95% CI) | P-value | HR (95% CI) | P-value |
Sex, male vs. female | 447 | 1.151 (0.847-1.564) | 0.368 | ||
Age, >60 vs. ≤60 years | 447 | 1.728 (1.278-2.337) | <0.001 |
1.096 (0.715-1.681) | 0.674 |
T stage, T3 and 4 vs. 1 and 2 | 353 | 1.842 (1.308-2.594) | <0.001 |
0.913 (0.513-1.625) | 0.757 |
N stage, N1, 2 and 3 vs. 0 | 393 | 1.620 (1.179-2.227) | 0.003 |
5.961 (1.369-25.957) | 0.017 |
M stage, M1 vs. 0 | 421 | 2.013 (1.059-3.828) | 0.033 |
2.244 (0.755-6.665) | 0.146 |
Pathological stage, III and IV vs. I and II | 402 | 1.495 (1.093-2.045) | 0.012 |
0.345 (0.078-1.536) | 0.163 |
Melanoma Clark level, IV and V vs. I, II and III | 307 | 2.075 (1.419-3.035) | <0.001 |
1.382 (0.829-2.304) | 0.215 |
Melanoma ulceration, yes vs. no | 306 | 1.949 (1.369-2.775) | <0.001 |
1.349 (0.871-2.089) | 0.179 |
Breslow depth, ≤3 vs. >3 mm | 347 | 0.452 (0.323-0.633) | <0.001 |
0.571 (0.331-0.985) | 0.044 |
Radiation therapy, yes vs. no | 441 | 0.966 (0.667-1.400) | 0.856 | ||
447 | 0.520 (0.389-0.695) | <0.001 |
0.623 (0.418-0.928) | 0.020 |
P<0.05. HR, hazard ratio;