1 INTRODUCTION

Melanoma is a type of aggressive cancer that originates from pigment cells and is the leading cause of skin cancer-related mortality1, 2; melanoma is characterized by rapid metastasis and progression.3, 4 Despite the adoption of diagnostic and therapeutic methods for melanoma, patients diagnosed with melanoma still have a relatively low survival rate with a poor prognosis.3 Thus, it is necessary to identify promising strategies for the treatment of melanoma.

Long noncoding RNAs (lncRNAs) are transcribed RNA molecules with no protein-coding potential that consist of more than 200 nucleotides.5 An increasing number of studies have confirmed that lncRNAs are actively involved in the regulation of many types of tumors.6 For example, lncRNA cancer susceptibility 9 was found to interact with cleaved and polyadenylation specific factor 3 to modulate the TGF-β pathway in colorectal cancer.7 LncRNA growth arrest specific 6 (GAS6) antisense RNA 1 (GAS6-AS1) promotes gastric cancer development by enhancing GAS6 expression.8 Upregulation of lncRNA X inactive specific transcript (Xist) facilitates osteosarcoma cell proliferation.9 Moreover, lncRNAs perform their functions in cancer progression through diverse mechanisms, including competing endogenous RNA (ceRNA) networks. Specifically, lncRNAs serve as molecular sponges to attenuate the inhibitory effects of microRNAs (miRNAs) on the expression of downstream messenger RNAs (mRNAs).10 Previous studies have shown that some lncRNAs participate in the development of melanoma by acting as ceRNAs. For example, lncRNA dopamine beta-hydroxylase AS1 (DBH-AS1) serves as a ceRNA for miR-233-3p to upregulate the expression of insulin growth factor receptor, thereby promoting the proliferation, migration, and invasion of melanoma cells.11 LICN00520 acts as a ceRNA by binding to miR-125b-5p to upregulate the expression of eukaryotic translation initiation factor 5A2, thus facilitating the progression of melanoma in vitro and in vivo.12 LncRNA activated by transforming growth factor-β (lncRNA) enhances the expression of Yes-associated protein 1 by interacting with miR-590-5p acting as a ceRNA, thereby contributing to melanoma cell proliferation and invasion.13 Importantly, previous research has revealed that lncRNA bladder cancer-associated transcript 1 (BLACAT1) exerts an oncogenic effect on some types of cancer and that its aberrant expression is closely related to tumor initiation and development. For example, BLACAT1 silencing prohibits the development of ovarian cancer by inhibiting Wnt/β-catenin signaling.14 BLACAT1 expression is upregulated in cervical squamous cell carcinoma, and its high expression predicts poor patient prognosis.15 BLACAT1 expression is upregulated in hepatocellular carcinoma, and its knockdown inhibits hepatocellular carcinoma progression by sponging hsa-miR-485-5p.16 BLACAT1 also participates in cancer progression via a ceRNA network. For example, BLACAT1 acts as a ceRNA for miR-608 to upregulate the expression of sex-determining region Y-box protein 12 (SOX12), which facilitates the migration and invasion of osteosarcoma cells.17 BLACAT1 facilitates cell proliferation, migration and invasion in gastric cancer by acting as a ceRNA for miR-149-5p, leading to the upregulation of kinesin family member 2A expression.18 BLACAT1 enhances cell viability, migration and invasion in prostate cancer by acting as a ceRNA to target the miR-29a-3p/disheveled segment polarity protein 3 axis.19 However, the biological role and underlying regulatory mechanism by which BLACAT1 functions in melanoma progression have not been investigated.

In this study, we hypothesized that BLACAT1 might function as an oncogene and affect cellular processes in melanoma cells via a ceRNA network. This study might provide a new direction for the development of targeted therapy for melanoma.

2 MATERIALS AND METHODS 2.1 Bioinformatics analysis

The starBase database (http://starbase.sysu.edu.cn/) is an open-source platform for studying miRNA–ncRNA, miRNA–mRNA, ncRNA–RNA, RNA–RNA, RBP–ncRNA, and RBP–mRNA interactions based on CLIP-seq, degradome-seq, and RNA–RNA interactome data.20 Potential miRNAs with binding sites in the BLACAT1 sequence were predicted with the starBase website with the screening condition of pan-cancer ≥10. The target genes of miR-374b-5p were searched in the starBase website with the criterion of pan-cancer ≥10, and the prediction programs that were used included miRmap and TargetScan.

2.2 Tissue samples

Melanoma tissue samples (n = 35) and noncancerous tissue samples (within 2 cm of the tumor site) (n = 35) were all provided by patients with melanoma (age range, 38–65 years) treated at Hubei Provincial Hospital of Traditional Chinese Medicine. The associations between BLACAT1 expression and the clinicopathological features of the patients are provided in Table 1. The data revealed that patients with high BLACAT1 expression always had a higher American Joint Committee on Cancer stage, lymphatic metastasis and a greater tumor tissue thickness. All the collected tissues were immediately stored at −80°C. None of these patients accepted treatment before the operation. Informed consent for this study was obtained from the patients. The research was conducted with the approval of the Ethics Committee of Hubei Provincial Hospital of Traditional Chinese Medicine (approval number: 2020-006; March 4, 2020).

TABLE 1. Association between BLACAT1 expression and clinicopathological characteristic (n = 35) Clinicopathological characteristics Low expression (n = 19) High expression (n = 16) p-Value Age 0.922 <45 11 9 ≥45 8 7 Gender 0.968 Male 12 10 Female 7 6 Histologic subtype 0.101 ALM 1 4 SSM 9 9 NM 9 3 AJCC stage 0.01* I-II 13 4 III-IV 6 12 Lymphatic metastasis 0.004* Yes 5 12 No 14 4 Thickness 0.001* <2 mm 14 3 ≥2 mm 5 13 Note: *p < 0.05. Abbreviations: AJCC, American Joint Committee on Cancer; ALM, acral lentiginous melanoma; BLACAT1, bladder cancer-associated transcript 1; NM, nodular melanoma; SSM, superficially spreading melanoma. 2.3 Cell lines and cell culture

Melanoma cell lines (A375, M21, and SK-MEL-2 cells) were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). A normal human epidermal melanocyte cell line (HEMa-LP cells, catalog number: C0245C; https://www.thermofisher.com/order/catalog/product/C0245C) was purchased from Thermo Fisher Scientific (Shanghai, China). The melanoma cell line MeWo was purchased from American Type Culture Collection (ATCC, Manassas, VA). All the cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA); 100 U/ml penicillin (Sigma-Aldrich, St. Louis, MO); and 100 μg/ml streptomycin (Sigma-Aldrich). The cells were maintained in a humidified incubator with 5% CO2 at 37°C.

2.4 Cell transfection

To knockdown BLACAT1 expression, short hairpin RNAs targeting BLACAT1 (5′-AGGCUGGUUUCUGCCCUCAUCCUUU-3′) were designed and synthesized by GenePharma (Shanghai, China). The 3′-UTR sequences of U2-associated factor homology motif kinase 1 (UHMK1) (length: 7215 nt) were synthesized and subcloned into the pcDNA3.1 vector (Invitrogen) to produce pcDNA3.1/UHMK1. MiR-374b-5p mimic, miR-374b-5p inhibitor, and the corresponding negative controls (NC mimic) were obtained from GenePharma. Then, A375 and M21 cells were seeded into six-well plates (5 × 105 cells per well) and incubated with 5 μl of Lipofectamine 2000 (Invitrogen), 50 nM sh-BLACAT1#1, 20 μg of pcDNA3.1/UHMK1, 50 nM miR-374b-5p mimic or 100 nM miR-374b-5p inhibitor at 37°C for 5 h. After 5 h of incubation, the transfection medium was replaced with 2 ml of standard growth medium. Forty-eight hours after transfection, the cells were harvested for subsequent assays.

2.5 Real-time quantitative polymerase chain reaction

Total RNA was extracted from melanoma tissues using TRIzol reagent (Invitrogen) and then reverse transcribed into cDNA using a reverse transcriptase kit (Takara, Tokyo, Japan) under the incubation conditions of 5 min at 25°C, 25 min at 52°C, and 10 min at 80°C. A SYBR Premix Ex Taq II reagent kit was used to conduct real-time quantitative polymerase chain reaction (RT-qPCR) with an ABI7500 RT qPCR system. U6 acted as the endogenous control for miRNAs. GAPDH acted as the internal control for lncRNAs and mRNAs. PCR was performed under the following conditions: 40 cycles of 95°C for 5 min, 95°C for 45 s, 55°C for 15 s, and 72°C for 50 s. The 2−ΔΔCt method was utilized for the quantification of relative gene expression.21 The primer sequences were as follows:

BLACAT1:

Forward: 5′-GATGGAAGTGGTATGGTGG-3′,

Reverse: 5′-TGACTTGCCTTTCCCTCTC-3′;

miR-374b-5p:

Forward: 5′-ATATAATACAACCTGCTAAGTGGCAGC-3′,

Reverse: 5′-CTCTACAGCTATATTGCCAGCCAC-3′;

UHMK1:

Forward: 5′-AATGGAAGGCAAACAGTTCTG-3′,

Reverse: 5′-GTGATAGGCTGGAATTGCG-3′;

GAPDH:

Forward: 5′-TCATTTCCTGGTATGACAACGA-3′,

Reverse: 5′-GTCTTACTCCTTGGAGGCC-3′;

U6:

Forward: 5′-AATACAGAGAAAGTTAGCACGG-3′,

Reverse: 5′-GAATGCTTCAAAGAGTTGTGC-3′.

2.6 RNA pull-down assays

Biotinylated BLACAT1 (Bio-BLACAT1) and the NC (Bio-NC) were purchased from Sangon (Shanghai, China) and transfected into A375 and M21 cells. The cells were lysed, and then, Bio-BLACAT1, Bio-NC, and cell lysates were cultured overnight. After Dynabeads M-280 Streptavidin (Invitrogen) were added to the cell lysates and cocultured for 10 min, the bound RNAs were subjected to RT-qPCR for quantification and analysis. This assay was repeated three times.

2.7 Western blotting analysis

Cells were transfected with 50 nM miR-374b-5p mimic or NC mimic, 50 nM sh-NC, 50 nM sh-BLACAT1#1, 50 nM sh-BLACAT1#1+ 100 nM miR-374b-5p inhibitor, or 50 nM sh-BLACAT1#1+ 20 μg pcDNA3.1/UHMK1. The cells were first lysed with RIPA lysis buffer (Beyotime, China) for half an hour. The protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher, Shanghai, China). Equal amounts of protein in each sample were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). After being blocked with 5% fat-free milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 60 min, the membrane was incubated at 4°C overnight with primary antibodies, including anti-GAPDH (#ab8245, 1/1000; Abcam, Cambridge, UK); anti-UHMK1 (#PA5-50622, 1/1000; Thermo Fisher); anti-PI3K (#ab133595, 1/1000; Abcam); anti-p-PI3K (#ab278545, 1/500; Abcam); anti-p-AKT (#ab38449, 1/1000; Abcam); and anti-AKT (#ab233755, 1/1000, Abcam) antibodies. After being washed with TBST and further incubated with secondary antibodies for 1 h, the blots were developed with enhanced chemiluminescence reagent. Western blotting analysis was performed in triplicate.

2.8 Luciferase reporter assay

The miR-374b-5p-Wt or miR-374b-5p-Mut vector was cotransfected with the sh-NC or sh-BLACAT1#1 into A375 and M21 cells. Then, 100 ng of UHMK1-Wt or UHMK1-Mut was cotransfected with 50 nM NC mimic or miR-374b-5p mimic into A375 and M21 cells. Lipofectamine 2000 was used for the transfections. After 2 days, the luciferase activity was evaluated using a luciferase reporter assay system. The luciferase activity of the reporters was determined by calculating the ratio of firefly luciferase activity to Renilla luciferase activity, and Renilla luciferase activity served as the reference for normalization. This assay was conducted in triplicate.

2.9 RNA immunoprecipitation assay

An EZ-Magna RNA immunoprecipitation (RIP) RNA-Binding Protein Immunoprecipitation Kit was used for the RIP assays. Cells from different groups were lysed in lysis buffer. Then, the lysates were incubated with RIP buffer containing magnetic beads conjugated with Ago2 antibodies (1/100) or NC IgG (1/100). After 2 h of incubation at 4°C, the coprecipitated RNA was isolated from the magnetic beads, and the enrichment of RNAs was measured by RT-qPCR analysis. The relative RNA enrichment in the RIP complex was determined by calculating the ratio of the relative RNA expression in the Ago2 complex to the relative RNA expression in the IgG complex. This assay was performed three times.

2.10 5-Ethynyl-2′-deoxyuridine assay

A 5-ethynyl-2′-deoxyuridine (EdU) assay was conducted with an EdU labeling/detection kit (RiboBio, China) to assess cell proliferation. A375 and M21 cells transfected with sh-NC, sh-BLACAT1#1, sh-BLACAT1#1+ miR-374b-5p inhibitor, or sh-BLACAT1#1+ pcDNA3.1/UHMK1 were treated with 50 μmol/L EdU for 2 h. Next, the cultured cells were fixed with 4% paraformaldehyde for 30 min. Anti-EdU working solution was used to stain the fixed cells. The cell nuclei were labeled with DAPI. Finally, the cells were visualized with fluorescence microscopy. The EdU assay was performed in triplicate.

2.11 Cell Counting Kit-8 assay

A Cell Counting Kit-8 (CCK-8) assay was used to assess cell proliferation. Cells transfected with sh-NC, sh-BLACAT1#1, sh-BLACAT1#1+ miR-374b-5p inhibitor, or sh-BLACAT1#1+ pcDNA3.1/UHMK1 were plated in 96-well plates (1 × 104 cells/well). After culture for 24, 48, 72, and 96 h, 10 μl of CCK-8 solution was added to each well, and then, the culture continued for another 4 h. A microplate reader was used to measure cell proliferation at a wavelength of 450 nm. All the experiments were conducted three times.

2.12 Transwell assays

Cells transfected with sh-NC, sh-BLACAT1#1, sh-BLACAT1#1+ miR-374b-5p inhibitor, or sh-BLACAT1#1+ pcDNA3.1/UHMK1 were seeded (1 × 105 cells/well) into the upper chambers of Transwell chambers (pore size: 8 μm, Corning Inc.), which had been precoated with Matrigel at 37°C for 6 h and contained 600 μl of serum-free RPMI 1640 medium. RPMI 1640 medium supplemented with 10% FBS was added to the lower chambers. The transfected cells were incubated for 2 days in a humidified atmosphere at 37°C in 5% CO2. A cotton swab was used to clear the noninvaded cells. Methanol (4%) was added and incubated for 20 min to fix the invaded cells. Then, the fixed cells were stained with crystal violet for 20 min, and the cells in five random fields were counted with optical light microscopy (Olympus, Tokyo, Japan). This assay was repeated three times.

2.13 Statistical analysis

All the data are presented as the mean ± SD. Student's t test or one-way ANOVA followed by Tukey's post hoc test was used to evaluate differences between groups. Chi-square test was utilized to determine p values in Table 1. A two-sided p < 0.05 value was considered statistically significant. Statistical analysis was carried out with SPSS 20.0 (Chicago, IL).

3 RESULTS 3.1 BLACAT1 expression is upregulated in melanoma tissues and cells

Aberrant expression of BLACAT1 has been detected in several types of cancer. Here, we examined the expression of BLACAT1 in melanoma. As shown by RT-qPCR analyses, BLACAT1 expression was significantly higher in melanoma tissues than in control benign nevus tissues (Figure 1(A)). Additionally, BLACAT1 was expressed at conspicuously higher levels in melanoma cells, especially in the M21 and A375 cell lines, than in normal HEMas (Figure 1(B)). Overall, the results suggested that BLACAT1 was highly expressed in melanoma tissues and cells.

Bladder cancer-associated transcript 1 (BLACAT1) expression is upregulated in melanoma cells and tissues. (A) The expression level of BLACAT1 in melanoma tissues and matched benign nevus tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR). (B) The expression levels of BLACAT1 in melanoma cell lines (A375, M21, MeWo, and SK-MEL-2 cells) and a normal human epidermal melanocyte cell line (HEMa-LP cells) were determined by RT-qPCR. *p < 0.05. Each biological sample was run in triplicate, and the experiments were repeated three independent times

3.2 BLACAT1 binds to miR-374b-5p in melanoma cells

To verify the hypothesis that BLACAT1 might act as a ceRNA in melanoma, we searched BLACAT1-binding miRNAs on the starBase (http://starbase.sysu.edu.cn/) website. According to the screening criterion of a pan-cancer score ≥10, four potential miRNAs (miR-374b-5p, miR-185-5p, miR-378c, and miR-769-5p) were identified. Next, RNA pull-down assays showed that among the candidate miRNAs, only miR-374b-5p was significantly enriched in the complex pulled down by the Bio-BLACAT1 probe (Figure 2(A)). Thus, miR-374b-5p was chosen for subsequent analysis. The putative binding site between BLACAT1 and miR-374b-5p as well as the mutated binding sequence in miR-374b-5p are shown in Figure 2(B). RT-qPCR was performed to detect the knockdown efficiency of sh-BLACAT1#1/2 in A375 and M21 cells, suggesting that BLACAT1 expression was significantly decreased in cells (Figure 2(C)). Subsequently, a luciferase reporter assay was performed to explore the binding ability between miR-374b-5p and BLACAT1. The results revealed that the luciferase activity of the miR-374b-5p-Wt reporter was increased by BLACAT1 depletion, while the luciferase activity of the miR-374b-5p-Mut reporter was not significantly changed in A375 and M21 cells transfected with the sh-BLACAT1#1 (Figure 2(D)). In addition, RIP assays suggested that both BLACAT1 and miR-374b-5p were markedly enriched by Ago2-conjugated beads but not by NC IgG beads (Figure 2(E)). According to RT-qPCR analyses, miR-374b-5p expression was decreased in melanoma tissues and cells compared with control benign nevus tissues and normal HEMas (Figure 2(F,G)). In addition, miR-374b-5p expression was negatively correlated with BLACAT1 expression in melanoma tissues, as shown by Spearman correlation coefficient analyses (Figure 2(H)). In summary, BLACAT1 bound to miR-374b-5p in melanoma cells.

Bladder cancer-associated transcript 1 (BLACAT1) binds to miR-374b-5p in melanoma cells. (A) Four microRNAs (miRNAs) that may bind to BLACAT1 were predicted with starBase with the screening condition of pan-cancer ≥10. Since miR-374b-5p presented the most remarkable capacity to bind to BLACAT1, as shown by RNA pull-down assays, miR-374b-5p was identified for further exploration. (B) The binding site between BLACAT1 and miR-374b-5p was predicted with starBase, and the mutated binding sequence in miR-374b-5p is shown. (C) The knockdown efficiency of sh-BLACAT1#1/2 in A375 and M21 cells was determined by real-time quantitative polymerase chain reaction (RT-qPCR). (D,E) The ability of BLACAT1 to bind to miR-374b-5p was validated using luciferase reporter and RIP assays. (F,G) MiR-374b-5p expression levels in melanoma tissues and cells as well as in control benign nevus tissues and normal human epidermal melanocytes were examined by RT-qPCR analyses. (H) Spearman correlation coefficient analysis was used to identify the correlation between BLACAT1 expression and miR-374b-5p expression in melanoma tissues. All the experiments were performed in triplicate. *p < 0.05

3.3 UHMK1 is targeted by miR-374b-5p in melanoma cells

To identify the downstream targets of miR-374b-5p, we used starBase to predict four potential downstream targets of miR-374b-5p with the screening condition of pan-cancer ≥10, and the prediction programs used included miRmap and TargetScan. RT-qPCR analyses showed that only the expression levels of UHMK1 were significantly decreased after miR-374b-5p overexpression compared with the expression levels of other candidate mRNAs (Figure 3(A)). Thus, UHMK1 was selected for the following investigation. The predicted binding site between UHMK1 and miR-374b-5p and the mutant binding sequence of UHMK1 are shown in Figure 3(B). According to the luciferase reporter assay results, we discovered that miR-374b-5p overexpression exerted no significant effect on the luciferase activity of the cells transfected with the mutant UHMK1 sequence but markedly reduced the luciferase activity of the cells transfected with the wild-type UHMK1 sequence, suggesting that UHMK1 bound to miR-374b-5p at the predicted binding site in melanoma cells (Figure 3(B)). Subsequently, RT-qPCR and western blotting analyses revealed that the mRNA and protein levels of UHMK1 were decreased by miR-374b-5p overexpression in melanoma cells (Figure 3(C)). Moreover, UHMK1 expression in melanoma tissues and cell lines was assessed. The results revealed that UHMK1 expression was notably higher in melanoma cell lines than in noncancerous cell lines (Figure 3(D)). UHMK1 was also more highly expressed in melanoma tissues than in benign nevus tissues (Figure 3(E)). Based on Spearman correlation coefficient analyses, UHMK1 expression was negatively correlated with miR-374b-5p expression but positively correlated with BLACAT1 expression in melanoma tissues (Figure 3(F,G)). In summary, UHMK1 expression was upregulated in melanoma tissues and cells and targeted by miR-374b-5p in melanoma cells.

U2-associated factor homology motif kinase 1 (UHMK1) is targeted by miR-374b-5p in melanoma cells. (A) The messenger RNAs (mRNAs) that bind to miR-374b-5p were predicted with the starBase website with the condition of pan-cancer ≥10 and the prediction programs used included miRmap and TargetScan. The screening process is shown by a Venn diagram. MiR-374b-5p overexpression triggered the most significant decrease in UHMK1 expression, and UHMK1 was selected for further study. (B) The binding site between UHMK1 and miR-374b-5p was predicted with starBase, and the mutant binding sequence in UHMK1 is shown. The capacity of UHMK1 to bind to miR-374b-5p was confirmed by luciferase reporter assays. (C) The mRNA and protein levels of UHMK1 in A375 and M21 cells transfected with the miR-374b-5p mimic were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting analyses. (D) The expression levels of UHMK1 in melanoma cell lines (A375, M21, MeWo, and SK-MEL-2 cells) and the normal human epidermal melanocyte cell line (HEMa-LP cells) were examined by RT-qPCR. (E) RT-qPCR was performed to measure UHMK1 expression in melanoma tissues and matched benign nevus tissues. (F,G) Spearman correlation coefficient analyses were used to identify the correlation between UHMK1 and miR-374b-5p (or BLACAT1) expression in melanoma tissues. Each biological sample was run in triplicate, and the experiments were repeated three independent times. *p < 0.05

3.4 BLACAT1 activates the PI3K/AKT signaling pathway by upregulating the expression of UHMK1

To further explore the relationship among BLACAT1, miR-374b-5p and UHMK1 and the downstream signaling pathway of UHMK1, RT-qPCR and western blotting analyses were carried out. We found that silencing BLACAT1 expression upregulated miR-374b-5p expression but downregulated UHMK1 expression in M21 cells (Figure 4(A)). The western blotting analysis results suggested that BLACAT1 silencing decreased the protein levels of UHMK1, p-PI3K/PI3K and p-AKT/AKT, while miR-374b-5p knockdown or UHMK1 overexpression partially reversed these effects of BLACAT1 knockdown on these protein levels (Figure 4(B,C)). Overall, BLACAT1 knockdown downregulated the protein level of UHMK1, and BLACAT1 activated the PI3K/AKT pathway by upregulating the expression of UHMK1 via miR-374-5p.

Bladder cancer-associated transcript 1 (BLACAT1) activates the PI3K/AKT signaling pathway by upregulating the expression of U2-associated factor homology motif kinase 1 (UHMK1). (A) The expression of miR-374b-5p was markedly upregulated while the messenger RNA (mRNA) expression of UHMK1 was significantly downregulated in melanoma cells transfected with sh-BLACAT1#1, as indicated by real-time quantitative polymerase chain reaction (RT-qPCR). (B,C) The protein levels of UHMK1, p-PI3K, PI3K, p-AKT, and AKT and the ratios of p-PI3K/PI3K and p-AKT/AKT in melanoma cells transfected with sh-NC, sh-BLACAT1#1, sh-BLACAT1#1 + miR-374b-5p inhibitor, or sh-BLACAT1#1 + pcDNA3.1/UHMK1 were evaluated by western blotting analyses. All the experiments were performed in triplicate. *p < 0.05, #p < 0.05

3.5 BLACAT1 promotes the proliferation and invasion of melanoma cells via the miR-374b-5p/UHMK1 axis

To further determine the biological roles of BLACAT1, miR-374b-5p, and UHMK1 in melanoma, loss-of-function assays and rescue experiments were conducted. EdU and CCK-8 assays were carried out to evaluate melanoma cell proliferation, and the results showed that BLACAT1 silencing markedly inhibited melanoma cell proliferation, while miR-374b-5p knockdown or UHMK1 overexpression partially reversed the suppressive effects caused by BLACAT1 knockdown (Figure 5(A,B)). Moreover, the invasive ability of melanoma cells was distinctly reduced by BLACAT1 silencing, while miR-374b-5p knockdown or UHMK1 overexpression reversed this inhibitory effect of BLACAT1 silencing (Figure 5(C)). In summary, BLACAT1 promoted the proliferation and invasion of melanoma cells by upregulating UHMK1 expression via miR-374b-5p.

Bladder cancer-associated transcript 1 (BLACAT1) promotes the proliferation and invasion of melanoma cells via the miR-374b-5p/U2-associated factor homology motif kinase 1 (UHMK1) axis. (A,B) Melanoma cell proliferation was assessed using EdU and Cell Counting Kit-8 (CCK-8) assays after transfection with sh-NC, sh-BLACAT1#1, sh-BLACAT1#1 + miR-374b-5p inhibitor, or sh-BLACAT1#1 + pcDNA3.1/UHMK1. (C) The invasion ability of melanoma cells was assessed using Transwell assays after the transfections described above. All the experiments were performed in triplicate. *p < 0.05, #p < 0.05

4 DISCUSSION

Due to the strong metastatic potential of melanoma cells, melanoma is extremely aggressive.22 Accumulating evidence has verified that lncRNAs exert significant effects on the metastasis and progression of various types of cancer, including melanoma.23-25 It has been reported that BLACAT1 performs an oncogenic function in some types of cancer, including prostate cancer,26 breast cancer,27 ovarian cancer,14 and pancreatic cancer.28 However, the biological role of BLACAT1 in melanoma remains unclear. In this study, our results showed that BLACAT1 expression in melanoma tissues and cell lines was distinctly higher than that in matched nontumor tissues and cells. Additionally, depletion of BLACAT1 inhibited the proliferation and invasion of melanoma cells. Since a lncRNA-mediated ceRNA network has been verified in melanoma and the ceRNA role of BLACAT1 in other types of cancer has been reported,13, 17, 29 we explored the role of BLACAT1 in melanoma in the context of this ceRNA hypothesis. To perform ceRNA functions, lncRNAs bind to miRNAs to prevent the degradation of the targets of these miRNAs, that is, mRNAs.30, 31

miRNAs are noncoding RNA molecules that are 20–24 nucleotides in length and play a significant role in regulating gene expression at the posttranscriptional level.32 In this study, we identified miR-374b-5p according to bioinformatics analysis and the results of RNA pull-down assays. Previous studies have demonstrated that miR-374b-5p can act as a tumor suppressor or tumor enhancer in the progression of different types of cancer. For example, miR-374b-5p inhibits the migration and invasion of bladder cancer cells by targeting zinc finger E-box binding homeobox 2.33 The upregulation of miR-374b-5p expression represses cell proliferation, migration, and invasion and induces cell apoptosis in non-small cell lung cancer.34 MiR-374b-5p enhances cell invasion and metastasis in gastric cancer.35 Nevertheless, the biological role of miR-374b-5p in melanoma still needs to be explored. In the present study, we discovered that miR-374b-5p expression was negatively correlated with BLACAT1 expression in melanoma. Additionally, the ability of BLACAT1 to bind to miR-374b-5p in melanoma was validated. Subsequently, target genes of miR-374b-5p in melanoma were explored, and UHMK1 was identified by bioinformatics analysis and experiments.

The oncogenic role of UHMK1 in some types of cancer has been previously reported in many studies. For example, UHMK1 facilitates gastric cancer development by reprogramming nucleotide metabolism.36 UHMK1 expression is related to a poor prognosis in patients with pancreatic ductal adenocarcinoma.37 In this study, UHMK1 was validated as a target gene of miR-374b-5p in melanoma. Additionally, UHMK1 expression was upregulated in melanoma cells and tissues, and its expression was negatively correlated with miR-374b-5p expression while positively associated with BLACAT1 expression in melanoma. Mechanistically, BLACAT1 upregulated UHMK1 expression by interacting with miR-374b-5p. Functionally, BLACAT1 promoted cell proliferation and invasion in melanoma via the miR-374b-5p/UHMK1 axis. Furthermore, we found that BLACAT1 activates the PI3K/AKT pathway by upregulating UHMK1 via miR-374b-5p.

In conclusion, BLACAT1 plays an oncogenic role and promotes cell proliferation and invasion in melanoma via miR-374b-5p and the inhibition of the PI3K/AKT signaling pathway. BLACAT1 is suggested to be a diagnostic or therapeutic target in the treatment of melanoma, and chemically synthesized BLACAT1 might be used to inhibit melanoma progression. However, there were also some limitations of our study. First, the results acquired from different cell lines were not compared. Second, pharmacological inhibitors were not used in this study. Third, an in vivo experiment is needed for further exploration in the future.

CONFLICT OF INTEREST

The authors declare no potential conflict of interest.