1 INTRODUCTION

Melanoma is the deadliest type of skin cancer and accounts for the majority of deaths from skin cancer.1-3 Recently, the incidence of melanoma has increased every year worldwide.2, 4, 5 Although the therapeutic methods available to melanoma patients have greatly improved with the improvement of medical standards, the mortality of melanoma remains high, particularly for patients with metastasis.6-8 Therefore, it is important to identify more biomarkers related to the mechanisms underlying melanoma progression through which more promising therapeutic targets for melanoma can be developed.

Radiotherapy is one of the major treatment modalities used in the management of cancer patients.9 Briefly, radiotherapy induces DNA damage, interrupts the cell cycle, and causes tumor cell death through apoptosis and necrosis.10 Radiotherapy provides survival benefits for advanced-stage patients with nasopharyngeal carcinomas.11 Radiotherapy improves survival for patients with locally advanced or inoperable esophageal squamous cell carcinomas.12 Moreover, radiotherapy plays an important role in the palliation of metastatic disease and as a treatment for malignant melanoma.13 However, radiotherapy may also exert serious toxic side effects on normal tissues, such as severe inflammatory reactions of skin and fibrosis of tissue.14 Low-dose radiotherapy is a recent advancement in the field of radiotherapy that is administered with a set of adjuvant therapies that sensitize cancer cells to radiation to achieve therapeutic effects similar to those observed with traditional high-dose radiotherapy but with lower toxicity induced in normal neighboring organs and tissues. Radiosensitivity is a hot topic in the field of oncology, and enhancing the efficacy of radiotherapy is important.15 Therefore, a key goal for realizing more effective radiosensitizing therapy is finding a suitable target for radiosensitizers.

Long noncoding RNAs (lncRNAs) belong to the noncoding RNA family, are longer than 200 nucleotides, and exert crucial effects on cancers through the regulation of the malignant phenotype of cancer cells.16 Previous research has established that lncRNAs are dysregulated in a variety of cancers and actively participate in the tumorigenesis of several cancers, including melanoma. For instance, the lncRNA SNHG8 acts as an oncogene in the development of non-small cell lung cancer by binding to miR-542-3p.17 The lncRNA HOXD-AS1 facilitates the migration of ovarian cancer cells through the miR-186-5p/PIK3R3 axis.18 LINC00520 increases the metastasis of malignant melanomas through the upregulation of EIF5A2.19 LINC01224 has been reported to be an oncogene in hepatocellular carcinomas.20 Nevertheless, the biological functions of LINC01224 in melanoma have not yet been studied.

In this study, we first determined the LINC01224 level in melanoma, and then, we investigated the role of LINC01224 in melanoma cells using functional assays. We subsequently explored the regulatory network of LINC01224. Overall, our findings may deepen our understanding of melanoma pathogenesis and lead to the development of novel methods for melanoma treatment.

2 MATERIALS AND METHODS 2.1 Tissues

Thirty-eight pairs of melanoma tissues and adjacent normal tissues, as well as 38 benign nevus tissues, were obtained from 38 patients with melanoma at Chengde Central Hospital (Hebei, China). Informed consent was given by all patients. None of the patients had received anticancer treatment before surgery. Tissue samples were stored at −80°C until use. Approval of this study was obtained from the Medical Ethics Committee of Chengde Central Hospital (Hebei, China). The approval number from the ethics committee is 2018-022.

2.2 Cell cultures

Melanoma cells (A-375, M21, SK-MEL-2, and A2058 cells) and normal human epidermal melanocytes (HEMa-LP cells) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultivated in RPMI 1640 medium with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 100 U/ml penicillin/streptomycin (Gibco, Grand Island, NY, USA) in a humidified incubator at 37°C with 5% CO2.

2.3 Transfection

Short hairpin RNAs targeting LINC01224 (sh-LINC01224#1 and sh-LINC01224#2) and scrambled oligonucleotides used as negative controls (sh-NC) were chemically synthesized by GenePharma (Shanghai, China). To overexpress LINC01224 or NR1D2, the full-length cDNA sequence of LINC01224 or NR1D2 was amplified and inserted into a pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) that were then named pcDNA3.1/LINC01224 and pcDNA3.1/NR1D2, respectively. miR-193a-5p mimics and NC mimics were also purchased from GenePharma. M21 and A2058 cells were seeded into 24-well plates at a density of 2 × 104 cells/well, after which 50 nM synthetic oligonucleotide or 2 μg of vector was transfected into the cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The cells were collected 48 h after transfection for use in further experiments.

2.4 Reverse transcription–quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted from melanoma tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). SYBR Premix Ex Taq II (TaKaRa, Japan) was used to perform RT-qPCR on an ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA). The thermocycling conditions of the qPCR were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s and 60°C for 45 s. GAPDH and U6 served as internal controls. The relative expression levels were analyzed using the 2-ΔΔCt method.

2.5 Western blot analysis

Total protein from melanoma cells was extracted using radioimmunoprecipitation assay (RIPA) buffer (KeyGen Biotech, Nanjing, China), and the concentration of extracted protein was measured with a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, 20 μg of protein was separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the isolated proteins were transferred to polyvinylidene fluoride membranes. After being blocked with 1% bovine serum albumin, the membranes were maintained at 4°C overnight with primary antibodies against NR1D2, cleaved caspase-3, Bax, Bcl-2, and GAPDH. The membranes were then washed with Tris-buffered saline (TBS)-1% TBS with Tween 20 and further incubated with secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected using enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and analyzed using ImageJ v1.8.0 software (National Institutes of Health, Bethesda, MD, USA).

2.6 Subcellular fraction assay

Cytoplasmic and nuclear RNA was obtained using a nuclei isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. Briefly, M21 and A2058 cells were collected, and cell pellets were added with the proper amount of cytoplasm extraction buffer. The cells were vortexed vigorously for 15 s, and the mixtures were placed on ice for 10 min. Then, the mixtures were subjected to high-speed centrifugation for 5 min at 4°C. The cytoplasmic fraction was added to a prechilled 1.5-ml tube, and the remaining pellet was used as the nuclear fraction. RNA was extracted by TRIzol and detected by RT-qPCR.

2.7 Fluorescence in situ hybridization (FISH)

FISH assays were performed using a fluorescent in situ hybridization kit (RiboBio, Guangzhou, China). Briefly, cells were rinsed in PBS and fixed in 4% formaldehyde for 15 min at room temperature. Then, the cells were permeabilized in PBS containing 0.5% Triton X-100 at 4°C for 5 min and washed with PBS. Next, the sample was prehybridized at 55°C for 30 min. For hybridization, LINC01224 probes were added to a hybridization solution, and the cells were incubated overnight in this solution at 37°C in the dark. The cells were then counterstained with DAPI, and images were taken with a confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany).

2.8 MTT assays

M21 and A2058 cells were seeded in 96-well plates at 2 × 103 cells/well. At 0, 24, 48, and 72 h, 20 μl of MTT solution (5 mg/ml; Sigma Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h at 37°C. Next, 150 μl of dimethylsulfoxide was added to the medium to dissolve the formazan. The absorbance at 490 nm was detected with a microplate reader (Bio-Rad, Hercules, CA, USA).

2.9 Colony formation assay

M21 and A2058 cells were seeded into six-well plates at 500 cells/well. The medium was replaced every 3 days. The cells were cultured in complete medium for 2 weeks. After cell colonies were formed, the medium was removed. The cells were fixed with 4% paraformaldehyde at 4°C for 1 h and stained with 0.1% crystal violet staining solution for 20 min at room temperature. Finally, the number of colonies (colonies comprising >50 cells) was counted under a light microscope.

2.10 Flow cytometry

Briefly, M21 and A2058 cells (6 × 104) were prepared in suspension after detachment. Two milliliters of cell suspension was incubated at 37°C in a six-well plate. After 48 h, the cells were harvested, digested with 0.25% trypsin, and then stained with 2 μl of Annexin-V-fluorescein isothiocyanate (FITC) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 μl of propidium iodide (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 4°C for 20 min in the dark. The number of apoptotic cells was determined by flow cytometry (BD Biosciences, San Jose, CA, USA).

2.11 RNA pull-down assay

Bio-NC, Bio-LINC01224, and Bio-miR-193a-5p-Wt/Mut constructs were synthesized by GenePharma. RIPA buffer containing RNase inhibitors was used to lyse cells. Next, 200 pmol probes were added to supernatants. Dynabeads M-270 Streptavidin (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added and incubated with the supernatants overnight at 4°C to isolate the RNA. Beads were isolated from the supernatant after centrifugation (2500 × g, 5 min, 4°C) and washed with wash buffer. This procedure was followed by another centrifugation (2500 × g, 5 min, 4°C), and then, the pellets were collected. The RNA complexes were eluted using Tris-EDTA buffer (Invitrogen, Carlsbad, CA, USA) and purified using ethanol. Finally, the level of RNA enrichment was analyzed by RT-qPCR.

2.12 Luciferase reporter assay

The sequences of LINC01224 or NR1D2 containing the binding site of miR-193a-5p were amplified and cloned into the psiCHECK-2 vector (Promega, Madison, WI, USA) and named LINC01224 or NR1D2-wild type (Wt). The putative common fragments were replaced and named LINC01224 or NR1D2-mutant (Mut). M21 and A2058 cells were seeded into 24-well plates at 5 × 10.4 The LINC01224-Wt/Mut or NR1D2-Wt/Mut vector (0.4 μg) was cotransfected into the M21 and A2058 cells with NC mimics (50 nmol/L) or miR-193a-5p mimics (50 nmol/L). Lipofectamine 3000 was utilized for transfection. After 48 h, the luciferase activity was detected using a luciferase reporter assay kit (Promega, Madison, WI, USA).

2.13 In vitro irradiation

M21 and A2058 cells were grown in six-well plates at 600 cells/well and irradiated at a dose of 0, 2, 4, 6, or 8 Gy. Two weeks later, the incubated cells were assessed for colony formation. The colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The ratio of the number of colonies to the number of total cells was the survival fraction.

2.14 Statistical analysis

Data analysis was performed with SPSS software (Version 20.0; IBM, Chicago, IL, USA). The data are expressed as the means ± SD. Student's t test and one-way analysis of variance were adopted for data comparison. Kaplan–Meier analysis was used to assess the correlation among genes. p < 0.05 was considered statistically significant.

3 RESULTS 3.1 LINC01224 knockdown promotes cell radiosensitivity in melanoma

To determine the role of LINC01224 in melanoma, we determined the LINC01224 level and found that LINC01224 expression was significantly upregulated in melanoma tissues compared to normal tissues (Figure 1A). Additionally, LINC01224 expression in melanoma tissues was higher than that in benign nevus tissues (Figure 1B). To determine the clinical significance of LINC01224 in melanoma patients, all patients were divided into a high LINC01224 expression group and a low LINC01224 expression group according to the mean expression value of LINC01224 as determined by RT-qPCR. As shown in Table 1, no significant difference was found by sex, age, histological subtype, body area, Breslow thickness, or ulceration in patients with differential expression of LINC01224. However, a high LINC01224 level was associated with the clinical stage of melanoma, distal metastasis, and lymphatic metastasis (p < 0.05 for all). Furthermore, LINC01224 was found to be notably upregulated in melanoma cells (A-375, M21, SK-MEL-2, A2058) compared to HEMa-LP cells (Figure 1C). Subsequently, we explored the biological role of LINC01224 in melanoma. First, M21 and A2058 cells were transfected with sh-LINC01224#1 or sh-LINC01224#2, and RT-qPCR showed that the LINC01224 level in the sh-LINC01224#1 group was significantly lower than that in the sh-NC group (Figure 1D). Through an MTT assay, we discovered that LINC01224 suppression reduced the viability of melanoma cells (Figure 1E). In addition, from the colony formation assay results, we observed that silencing LINC01224 hampered the proliferation of melanoma cells (Figure 1F,G). Next, we performed flow cytometry to evaluate the effect of LINC01224 depletion on cell apoptosis, and the results suggested that LINC01224 depletion boosted melanoma cell apoptosis (Figure 1H). In addition, the levels of proapoptotic proteins were increased upon LINC01224 silencing, while the levels of antiapoptotic proteins were decreased upon LINC01224 silencing (Figure 1I). Moreover, to further understand whether LINC01224 influences the radiosensitivity of melanoma cells, LINC01224-silenced M21 and A2058 cells received various doses of radiation. The results demonstrated that the survival fraction of cells was lower in the LINC01224-silenced cells than in the control cells after radiation exposure, implying an improvement in radiosensitivity after LINC01224 knockdown (Figure 1J). In summary, LINC01224 knockdown inhibits cell proliferation and promotes cell radiosensitivity in melanoma.

LINC01224 knockdown inhibits melanoma cell proliferation and promotes cell radiosensitivity. (A) LINC01224 expression in melanoma tissues and adjacent normal tissues was measured by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). (B) LINC01224 expression in melanoma tissues and benign nevus tissues was measured by RT-qPCR. (C) The expression of LINC01224 in melanoma cells (A-375, M21, SK-MEL-2, and A2058 cells) and HEMa-LP cells was determined by RT-qPCR. (D) Sh-LINC01224#1 or sh-LINC01224#2 was transfected into M21 and A2058 cells, and RT-qPCR analysis was performed to evaluate the transfection efficiency. (E) MTT was adopted to examine the viability of M21 and A2058 cells. (F and G) A colony formation assay was used to assess the proliferation of M21 and A2058 cells. (H) Apoptosis of M21 and A2058 cells was detected by flow cytometry. (I) The expression levels of proteins associated with cell apoptosis were assessed by western blot analysis. (J) Clonogenic survival of M21 and A2058 cells after exposure to radiation. OD, Optical density. *p < 0.05; **p < 0.01; ***p < 0.001

TABLE 1. Correlation between LINC01224 expression and the clinicopathological characteristics of melanoma patients Characteristics LINC01224 expression p value High n = 19 Low n = 19 Gender Male 7 8 1.000 Female 12 11 Age <60 12 10 0.743 ≥60 7 9 Histologic subtype Superficial 7 6 1.000 Nodular 8 10 Acral 4 3 Body area Upper extremities 4 4 0.557 Lower extremities 7 5 Rear trunk 4 8 Anterior trunk 4 2 Breslow thickness <4 mm 11 13 0.737 ≥4 mm 8 6 Ulceration Absent 13 12 1.000 Present 6 7 TNM stage I-II 2 12 0.002 III-IV 17 7 Distal metastasis No 6 14 0.022 Yes 13 5 Lymphatic metastasis No 4 12 0.02 Yes 15 7 Note: p < 0.05 is considered significant (Fisher's exact test). Abbreviation: TNM, tumor node metastasis. 3.2 LINC01224 interacts with miR-193a-5p

We then aimed to probe the molecular regulatory mechanism of LINC01224 in melanoma. First, the location of LINC01224 in melanoma cells was detected by FISH and subcellular fraction assays. The results indicated that LINC01224 was preferentially located in the cytoplasm (Figure 2A,B). Hence, we predict that LINC01224 acts as a ceRNA in melanoma. Subsequently, with the help of the starBase database, we found two miRNAs sharing binding sites with LINC01224 (condition: pan-cancer: eight cancer types). The pull-down assay results indicated that miR-193a-5p was significantly enriched in both M21 and A2058 cells transfected with Bio-LINC01224 (Figure 2C). Then, we adopted a luciferase reporter assay to validate the binding strength between LINC01224 and miR-193a-5p, and their predicted binding site is presented in Figure 2D. The results showed that miR-193a-5p overexpression weakened the luciferase activity of the LINC01224-Wt vector, but the LINC01224-Mut vector exhibited no obvious change in luciferase activity, confirming the binding ability of LINC01224 and miR-193a-5p (Figure 2D). Next, the RT-qPCR results revealed that miR-193a-5p was expressed at a low level in melanoma tissues and cells (Figure 2E and F). Furthermore, we observed that miR-193a-5p expression was negatively correlated with LINC01224 expression in melanoma tissues (Figure 2G). In conclusion, these results reveal that LINC01224 acts as a ceRNA to sponge miR-193a-5p in melanoma cells.

LINC01224 interacts with miR-193a-5p. (A and B) FISH and subcellular fraction assays were used to analyze the location of LINC01224 in melanoma cells. (C) The StarBase tool was applied to search for potential miRNAs that share binding sites with LINC01224, and a pull-down assay was used to assess the binding of LINC01224 to miRNAs. (D) A luciferase reporter assay was performed to evaluate the binding of LINC01224 to miR-193a-5p. (E and F) The miR-193a-5p expression level in melanoma tissues and cells was measured by reverse transcription–quantitative polymerase chain reaction. (G) Kaplan–Meier analysis was used to assess the correlation between LINC01224 expression and miR-193a-5p expression in melanoma tissues. *p < 0.05; **p < 0.01; ***p < 0.01

3.3 NR1D2 is a target of miR-193a-5p

To identify the downstream targets of miR-193a-5p, we examined the starBase database and found one mRNA that shared a binding site with miR-193a-5p (condition: CLIP data: strict stringency [> = 5], pan-cancer: 10 cancer types, prediction program: microT + RNA22) (Figure 3A). Through RT-qPCR, NR1D2 was found to be upregulated in melanoma tissues and cells (Figure 3B and C). Additionally, the luciferase reporter assay results demonstrated that the luciferase activity of the NR1D2-Wt plasmid was significantly reduced by miR-193a-5p overexpression, but miR-193a-5p overexpression exhibited no distinct influence on the NR1D2-Mut plasmid. These results imply that NR1D2 can be targeted by miR-193a-5p (Figure 3D). Subsequently, RT-qPCR and western blot analysis revealed that overexpression of miR-193a-5p reduced NR1D2 expression at the mRNA and protein levels in M21 and A2058 cells (Figure 3E). Similarly, silencing LINC01224 reduced NR1D2 expression at the mRNA and protein levels in M21 and A2058 cells (Figure 3F), which indicates that NR1D2 positively regulates LINC01224 expression in melanoma cells. Furthermore, both LINC01224 and NR1D2 were enriched in the Bio-miR-193a-5p-Wt group compared to the Bio-miR-193a-5p-Mut group (Figure 3G). We overexpressed LINC01224 in M21 and A2058 cells by transfection of pcDNA3.1/LINC01224, and RT-qPCR confirmed the transfection efficiency (Figure 3H). As shown in Figure 3I,J, LINC01224 overexpression significantly upregulated NR1D2 mRNA and protein expression, which was reversed after miR-193a-5p was overexpressed. These results suggest that LINC01224 positively regulates NR1D2 by controlling the availability of miR-193a-5p. Moreover, a positive relationship between LINC01224 expression and NR1D2 expression in melanoma tissues was found (Figure 3K).

Nuclear receptor subfamily 1 group D member 2 (NR1D2) is a target of miR-193a-5p in melanoma cells. (A) The StarBase tool was adopted to identify the targets of miR-193a-5p. The Venn diagram shows one mRNA has a binding site for miR-193a-5p. (B) The NR1D2 expression level in melanoma tissues was measured by reverse transcription–quantitative polymerase chain reaction (RT-qPCR). (C) The NR1D2 expression level in melanoma cells was measured by RT-qPCR. (D) A luciferase reporter assay was used to evaluate the binding ability of NR1D2 with miR-193a-5p. (E) The influence of miR-193a-5p mimics on the mRNA expression and protein level of NR1D2 was analyzed by RT-qPCR and western blot. (F) The effects of LINC01224 knockdown on the mRNA expression and the protein level of NR1D2 were assessed by RT-qPCR and western blot analyses. (G) The binding of LINC01224 or NR1D2 to miR-193a-5p was determined by an RNA pull-down assay. (H) The overexpression efficiency of LINC01224 was examined by RT-qPCR. (I and J) NR1D2 mRNA and protein expression in cells transfected with pcDNA3.1, pcDNA3.1/LINC01224, or pcDNA3.1/LINC01224 + miR-193a-5p mimics was measured by RT-qPCR and western blot. (K) Kaplan–Meier analysis was used to assess the correlation between LINC01224 expression and NR1D2 expression in melanoma tissues. **p < 0.01; ***p < 0.01

3.4 NR1D2 overexpression reversed the effects of silencing LINC01224 in melanoma cells

To verify whether NR1D2 expression is critical for the functions of LINC01224 in melanoma, rescue experiments were carried out. First, M21 cells were transfected with pcDNA3.1/NR1D2 to overexpress NR1D2 (Figure 4A). The MTT assay results revealed that overexpression of NR1D2 abolished the inhibitory effect of LINC01224 depletion on cell viability in melanoma (Figure 4B). Similarly, overexpression of NR1D2 restored the cell proliferation that had been inhibited by LINC01224 knockdown (Figure 4C). Additionally, flow cytometry and western blot analysis demonstrated that LINC01224 depletion promoted melanoma cell apoptosis, but this effect was reversed by the overexpression of NR1D2 (Figure 4D,E). Moreover, increased expression of NR1D2 attenuated the suppressive effect of LINC01224 knockdown on the survival of M21 cells (Figure 4F). In summary, these results indicate that NR1D2 is involved in the regulation of LINC01224 in melanoma.

Nuclear receptor subfamily 1 group D member 2 (NR1D2) overexpression reverses the effect of silencing LINC01224 on melanoma cell proliferation and radiosensitivity. (A) pcDNA3.1 or pcDNA3.1/NR1D2 was transfected into M21 cells, and reverse transcription–quantitative polymerase chain reaction was used to evaluate the transfection efficiency. (B) An MTT assay was used to assess the viability of M21 cells with sh-NC, sh-LINC01224#1, or sh-LINC01224#1 + pcDNA3.1/NR1D2. (C) A colony formation assay was used to detect M21 cell proliferation after transfection with sh-NC, sh-LINC01224 #1, or sh-LINC01224 #1 + pcDNA3.1/NR1D2. (D) Flow cytometry was adopted to examine the apoptosis of M21 cells transfected with sh-NC, sh-LINC01224#1, or sh-LINC01224#1 + pcDNA3.1/NR1D2. (E) The levels of proteins associated with cell apoptosis in M21 cells after transfection with the indicated plasmids were assessed by western blot. (F) The survival fraction of M21 cells transfected with sh-NC, sh-LINC01224#1, or sh-LINC01224#1 + pcDNA3.1/NR1D2 was detected by a colony formation assay. OD, Optical density. ***p < 0.01

4 DISCUSSION

In general, patients with advanced melanoma have a poor survival rate.7 Generally, radiotherapy has been regarded as a preferred treatment for patients at an advanced stage.21 Recently, multiple studies have explored the radiosensitivity of melanoma.22-24 However, there is still ample opportunity to obtain a more profound understanding of the regulatory mechanism behind the radiosensitivity of melanoma.

Previous studies have established that lncRNAs perform vital functions in many cellular biological processes.25 Recently, significant roles of numerous lncRNAs in the regulation of melanoma development have been revealed.26-28 However, the biological role and regulatory mechanisms of LINC01224 in melanoma remain unclear. Additionally, whether LINC01224 exerts a substantial effect on radiotherapy in melanoma is unclear. Here, we measured the LINC01224 level in melanoma tissues and cells. We found that LINC01224 was upregulated, implying that it may serve as an oncogene in melanoma. As previously reported, overexpression of LINC01224 promotes cell proliferation, migration, and invasion; accelerates tumor formation; and attenuates apoptosis in gastric cancer.29 LINC01224 silencing has also been shown to inhibit colorectal cancer cell proliferation and invasion.30 LINC01224 depletion in epithelial ovarian cells suppresses cell proliferation, migration, and invasion and facilitates cell apoptosis in vitro.31 These studies show the oncogenic properties of LINC01224 in cancer progression. Thus, we explored the functional role of LINC01224 in melanoma and found that LINC01224 promoted the viability and proliferation but inhibited the apoptosis of melanoma cells. Importantly, we discovered that LINC01224 knockdown inhibited cell proliferation after exposure to radiation, revealing that LINC01224 depletion promoted the radiosensitivity of melanoma. Therefore, LINC01224 acts as an oncogene in melanoma.

Similar to lncRNAs, miRNAs have also been widely researched due to their essential roles in the regulation of multiple cancers. LncRNAs can function as ceRNAs and competitively bind to miRNAs to regulate messenger RNAs (mRNAs) in numerous cancers, including melanomas.26, 32, 33 We found that LINC01224 was preferentially located in the cytoplasm of melanoma cells. Hence, we predict that LINC01224 acts as a ceRNA in melanoma. With the help of online tools, miR-193a-5p was shown to have a binding site for LINC01224. We further confirmed the binding of miR-193a-5p to LINC01224. MiR-193a-5p was found to be downregulated and act as a tumor inhibitor in several cancer types, such as pancreatic cancer,34 hepatoblastoma,35 and esophageal cancer.36 In this study, consistent with previous studies, miR-193a-5p was found to exhibit a low expression level in melanoma tissues and cells. Overall, LINC01224 served as a ceRNA to sponge miR-193a-5p in melanoma cells.

Following the ceRNA network, we identified the targets of miR-193a-5p. NR1D2 (nuclear receptor subfamily 1 group D member 2) is a widely studied mRNA and has been verified to be an oncogene in certain cancer types. Silencing of NR1D2 inhibits cell proliferation and motility in glioblastomas.37 NR1D2 accelerates hepatocellular carcinoma progression by driving the epithelial-to-mesenchymal transition.38 However, whether NR1D2 exerts an oncogenic effect in melanoma cells is unknown. In this study, NR1D2 was upregulated in melanoma tissues and cells. Mechanistically, NR1D2 was targeted by miR-193a-5p in melanoma cells. We further demonstrated that LINC01224 positively regulated NR1D2 expression. In addition, increased NR1D2 antagonized the effects of LINC01224 suppression on melanoma cell proliferation and apoptosis. Moreover, the overexpression of NR1D2 abolished the inhibitory effect of LINC01224 silencing on cell proliferation after exposure to radiation, implying that NR1D2 overexpression restored the radiosensitivity of melanomas.

In conclusion, we investigated the effect of LINC01224 on melanoma cell proliferation, apoptosis, and radiosensitivity. The results suggest that LINC01224 facilitates cell proliferation and inhibits the radiosensitivity of melanomas via the miR-193a-5p/NR1D2 axis. These findings further reveal the pathogenesis of melanoma, which may contribute to the development of therapeutic methods for this disease.

CONFLICT OF INTEREST

All authors declare no conflict of interest.