Introduction
Malignant melanoma is the most dangerous skin tumor, which is the primary cause of death of skin cancer [
1‐
3]. In recent years, the global incidence of melanoma is growing rapidly each year [
2,
4]. Although melanoma patients are treated by the combination of surgery, chemotherapy, targeted therapy and immunotherapy, the therapeutic effect is still unsatisfactory, especially those with distant metastasis [
5]. The development of melanoma is closely related to the abnormal regulation of multiple genes and signaling pathways [
6]. Therefore, it is very important to explore the molecular mechanism of the malignant progression of melanoma and to find potential therapeutic targets for melanoma.
Long non-coding RNAs (lncRNAs), a kind of non-coding RNA over 200 nucleotides in length, play pivotal roles in various human tumors by modulating the malignant phenotype of tumor cells [
7‐
9]. A few lncRNAs has been found to be abnormally expressed in melanoma and involved in its malignant progress [
10,
11]. Long intergenic non-protein coding RNA 520 (LINC00520), located on chromosome 14, has been reported to overexpress and function as a oncogene in breast cancer, nasopharyngeal carcinoma and laryngeal squamous cell carcinoma [
12‐
14]. It has also been showed that LINC00520 inhibits the growth and metastasis of cutaneous squamous cell carcinoma [
15]. However, the exact role and molecular mechanism of LINC00520 in malignant melanoma has not been studied.
In the present study, we found that LINC00520 was highly expressed in melanoma by analyzing the lncRNAs expression profile of melanoma. LINC00520 promoted the proliferation and metastasis of melanoma. To date, the most widely studied mechanism of lncRNAs in tumor is that lncRNAs play the role of competitive endogenous RNAs (ceRNA) in tumor development [
16,
17]. Similarly, LINC00520 has been shown to play the same role in nasopharyngeal carcinoma [
14]. Here, we have established the ceRNA regulatory network of LINC00520 based on RNA-seq and miRNA-seq results and bioinformatics predictions. We demonstrated that LINC00520 exerts its oncogene effect in melanoma by regulating Eukaryotic initiation factor 5A2 (EIF5A2). EIF5A2, SUPPLlocated on human chromosome 3q25–27, function as a novel oncogene in many tumors [
18,
19]. Extensive studies have demonstrated that EIF5A2 participates in the proliferation, migration, invasion and chemotherapeutic resistance of hepatocellular carcinoma, esophageal cancer, gastric cancer, melanoma, etc. [
20‐
23]. We proved that miR-125b-5p exerts anti-cancer effects in melanoma by targeting EIF5A2. Furthermore, we showed that LICN00520 can remove the inhibition effect of miR-125b-5p on EIF5A2 through decoying miR-125b-5p, thus promoting the expression of EIF5A2. Therefore, LICN00520 can serve as a new special diagnostic indicator and therapeutic target in melanoma patients.
Materials and methods
Tissue samples
Forty-one primary malignant melanoma tissues and adjacent normal tissues (ANT) were collected from the melanoma patients in the Affiliated People’s Hospital of Jiangsu University, and informed consent was obtained from all patients.. The pathological diagnosis was made independently by two pathologists. None of the patients had undergone chemotherapy or radiotherapy. The study was approved by the Human Research Ethics Committee of the Affiliated People’s Hospital of Jiangsu University. The public database of melanoma from The Cancer Genome Atlas (TCGA), GEO#GSE15605, GEO#GSE34460 and GEO#GSE24996 were also included in this study.
RNA-seq, miRNA-seq and ceRNA analysis
Three melanoma tissues and adjacent normal tissues were stored in liquid nitrogen, and TRIzol (Invitrogen, USA) was used to extract RNA. Gminix (Shanghai, China) conducted the RNA-seq and miRNA-seq analysis. The network of LICN00520-miRNA-target gene was constructed by using Cytoscape software (v.3.6.0) based on the RNA-seq and miRNA-seq results. The interaction between LINC00520 and miRNAs was predicted through miRcode. TargetScan, miRDB and miRTarBase were used to find the target genes of miRNAs.
Cell lines and cell culture
Human malignant melanoma cell lines (A375, A2058, MeWo, CHL-1, SK-MEL-28) were obtained from the American Type Culture Collection (ATCC, USA), and growed in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) with 10% fetal bovine serum (Invitrogen, USA). Human epidermal melanocytes (HEMa-LP) was purchased from Invitrogen (USA), and maintained in medium 254 (Cascade Biologics, USA). These cell lines were incubated in the humidified incubator with the atmosphere of 37 °C containing 5% CO2.
Plasmids, oligonucleotides and transfection
The miR-125b-5p mimic, miR-125b-5p inhibitor and related negative control were obtained by GenePharma (Shanghai, China). The small interfering RNA (siRNA) and short hairpin RNA (shRNA) of LINC00520 were also chemically synthesized by GenePharma (Shanghai, China). The EIF5A2 plasmid was constructed by inserting the full length of EIF5A2 into pcDNA3.1 vector (Invitrogen, USA). The shRNA and the control were inserted into the lentivirus vector (GenePharma, Shanghai, China), and the stably expressing sh-LINC00520 shRNA A375 cells were constructed by infecting cells with the corresponding lentivirus. Lipofectamine 3000 (Invitrogen, USA) was used to transfect the related oligonucleotides into melanoma cells.
Quantitative RT-PCR
TRIzol reagent was used to extract RNA from cells and tissues according to the specified steps (Invitrogen, USA). Fermentas and microRNA reverse transcription kits (Applied Biosystems, CA) were used to conduct reverse transcription. The amplification reactions were conducted by using the ABI StepOnePlus System (Applied Biosystems, CA) according to the set reaction conditions. The special primer of miR-125b-5p was purchased from RiboBio (Guangzhou, China). GAPDH and U6 was used for normalization respectively. The following primers were used: LINC00520 forward 5′-CCTGCTCCTTCAGGGACATC-3′ and LINC00520 reverse 5′-TCCGCCCCTTGCTCAAATAG-3′; EIF5A2 forward 5′-TTCCAGCACTTACCCTT-3′ and EIF5A2 reverse 5′-TTCCCCTCTATTTTTG-3′; GAPDH forward 5′- GTCAACGGATTTGGTCTGTATT-3′ and GAPDH reverse 5′- AGTCTTCTGGGTGGCAGTGAT-3′. The method of 2–△△Ct was used to calculate the relative expression level.
Western blot
RIPA buffer (KenGEN, China) was used to extract the protein following the appropriate steps. BCA Protein Assay Kit (Beyotime, China) was used to measure the concentration of extracted protein. Western blotting is carried out as the previous described [
17]. Antibodies against EIF4A2 (Abcam, 1:1000, Cambridgeshire, UK), vimentin (Abcam, 1:2000, Cambridgeshire, UK), E-cadherin (Abcam, 1:500, Cambridgeshire, UK), N-cadherin (Abcam, 1:1000, Cambridgeshire, UK) was used to the related protein level. β-actin (1:1000, Abcam, UK) and GAPDH (1:2500, Abcam, UK) were used for normalization.
Cell proliferation assay
For cell counting kit-8 (CCK-8, Beyotime, Shanghai, China) assay, the transfected melanoma cells (5000 cells) were seeded in a 96-well plate, and the process is carried out as described previously [
24]. Microplate reader (Multiscan FC, Thermo Scientific) was used to measure the absorbance at an optical density of 450 nm. For EdU assay, the DNA synthesis of melanoma cells grown was measured by using a EdU imaging kit (life Technologies, USA). The assay were carried out according to the manufacturer’s instructions. Immunostaining were visualized by using Leica DMI3000B microscope, and the positive cells were counted.
Cell invasion and migration assays
Transwell assay was used to detect the invasiveness of melanoma cell. Transfected melanoma cells were digested and resuspended in serum-free DMEM, and were placed at the top of the Matrigel-coated chambers (BD Biosciences, USA). The culture medium with 10% fetal bovine serum was used as the chemical attractant and added to the lower chamber. After 24 h, the fixed invasive cells were stained with crystal violet, counted and photographed. Scratch wound assay was used to evaluate the migration of melanoma cells. Transfected melanoma cells were added into the 6-well plates, and the wound space was formed by the tip of a 200 μl pipette. The width of wound was recorded at 0 and 24 h respectively.
Isolation of RISC-associated RNA
We used 1% formaldehyde to fix miR-125b-5p overexpressed melanoma cells. We did the chromatin fragmentation. NETN buffer was used to dissolve the cells, the cells were then cultured with Dynabeads protein A (Invitrogen, USA) plus IgG or anti-Pan-Ago, clone 2A8 antibody (Millipore, USA). We used proteinase K digestion to release immunoprecipitated RNA. The extracted RNA was purified by glycogen ethanol precipitation and treated with DNase I.
Luciferase reporter assay
The fragment of EIF4A2 3′-UTR and LINC00520 containing the miR-125b-5p binding site were inserted into pMIR-REPORT plasmid, and the mutated plasmid the used as the control. The corresponding oligonucleotides and luciferase reporter plasmids were co-transfected into melanoma cells. The luciferase activity of luciferase reporter plasmids was measured by Dual Luciferase Reporter Assay System (Promega, USA) .
Fluorescence in situ hybridization (FISH)
RiboTM Fluorescent In Situ Hybridization Kit (RiboBio, Guangzhou, China) was used to for FISH. The procedure was carried out according to the previous study [
25]. The probe of LINC00520 was synthesized by RiboBio (Guangzhou, China). and the cell nucleus were stained with DAPI. Representative images were obtained by using a confocal microscopy, and the image J software was used to collect signals.
MS2-RIP assay
Maltose-binding protein (MBP)-affinity purification was used to detect miRNAs that binding to LINC00520. According to the Steitz laboratory steps, MS2-MBP was purified from E. colicoli. 3 bacteriophage MS2 coat protein binding sites were inserted in the downstream of LINC00520 by using Stratagene Quik Change Site Directed Mutagenesis Kit. The MS2-tagged LINC00520 was transfected into the melanoma cell to obtain miRNAs that associated with LINC00520. The RIP analysis was performed on the cells as previously described after 48 h [
17], and the miR-125b-5p level was detected by qRT-PCR.
RNA pull-down assay
The Biotinylated of miR-125b-5p was chemical synthesized by GenePharma (Shanghai, China), and the biotinylated mutant and NC were used as control. The related oligonucleotides were transfected into melanoma cells. The lysates of cells were cultivated with M-280 streptavidin magnetic beads (Invitrogen, USA) [
26]. QRT-PCR was used to detect the LINC00520 level in the bound RNA.
Xenograft tumor and in vivo lung metastasis assay
10 nude mice were obtained from the Beijing Laboratory Animal Center (Beijing, China), and these mice were subcutaneously injected with A375 cells stably expressing LINC00520 siRNA. The volume of tumour was measured every 4 days according to the formula (0.5 × length × width2). After 28 days, mice were sacrificed, and tumour tissues were stripped and weighed. We injected A375 cells stably expressing sh-LINC00520 into the tail vein of mice. 10ul/g sterile D-Luciferin firefly potassium salt (Beyotime, China) were intraperitoneal injected into 8 nude mice, and the PerkinElmer IVIS Spectrum (Xenogen, CA) was used for in vivo imaging. The results were quantified by using the Living Image software (Xenogen, CA). After 20 days, the lung was dissected and the metastatic nodules were counted. The study was approved by the Experimental Animal Ethics Committee of the Affiliated People’s Hospital of Jiangsu University.
Immunohistochemistry staining and HE staining
Immunohistochemistry is performed as described previously [
27] using the antibody EIF4A2. The optical density of the image was analyzed by image J software. For HE staining, the sections were deparaffinizated and rehydrated. Then, the sections were incubated with hematoxylin and stained in acid ethanol and eosin. The sections were dehydrated with alcohol and cleared with xylene. Representative images were taken with a microscope.
Statistical analysis
Data expressed as mean ± SD. SPSS13.0 was used to analyse the data. Data was evaluated by t-test or one-way ANOVA, and spearman correlation analysis was analysed by using the MATLAB. Kaplan-Meier survival curves was used to evaluate the relationship between LINC00520 expression and melanoma patient survival. Melanoma tissues were separated into two groups according to the expression of LINC00520, the differences between the curves were tested by the log-rank test. GraphPad Prism was used to plot the Kaplan-Meier survival curves. P value < 0.05 is statistically significant.
Discussion
Recently, numerous studies have revealed that some lncRNAs play crucial role in the development and progression of many human tumors [
24,
31,
32]. LINC00520, located on chromosome 14, is a novel identified lncRNA. LINC00520 has been shown to up-regulate and modulate the malignant phenotype of tumor cells in some malignant tumors [
12,
14,
33]. LINC00520 promotes the proliferation, migration and invasion of glioma cells, but inhibits its apoptosis [
34]. Wu et al. reported that LINC00520 contribute to the metastasis of laryngeal squamous cell carcinoma [
13]. However, the role of LINC00520 in malignant melanoma has not been studied until now. In this study, we first analyzed the lncRNAs expression profile of melanoma tissue, and found that LINC00520 was increased in melanoma. We verified the results of lncRNAs expression profile using more samples, and found that high LINC00520 level conferred a poorer prognosis to melanoma patients based on the analysis of our samples and public database. LINC00520 has also been demonstrated to promote the proliferation, invasion and migration of melanoma cell.
We further explored the mechanism of LINC00520 in melanoma. A handle of studies have been proved that some lncRNAs can act as ceRNAs in the malignant progression of many tumors [
35,
36]. ceRNAs reduce the binding of miRNAs to target genes by decoying miRNAs, thus regulating the expression of specific genes [
16,
37]. It is reported that LINC00520 exhibits pro-oncogenic function in nasopharyngeal carcinoma by regulating the miR-26b-3p/USP39 axis [
14]. We subsequently constructed the LINC00520-miRNA-target gene network based on our miRNA-seq and RNA-seq data and bioinformatics predictions. LINC00520, miR-125b-5p and EIF5A2 were found to have a potential ceRNA correlation in melanoma. Our melanoma samples and public database further confirmed the network of LINC00520, miR-125b-5p and EIF5A2. We demonstrated that LINC00520 directly binds to miR-125b-5p by using Luciferase reporter assay, MS2-RIP assay and RNA pull-down assay. EIF5A2 has also been proved to be the target gene of miR-125b-5p in melanoma. LINC00520 siRNA reperssed the expression of EIF5A2 and the luciferase activity of wild type EIF5A2 3’UTR luciferase vectors, and this repression were attenuated by miR-125b-5p inhibitor. All results suggested that LINC00520 promotes EIF5A2 expression by decoying miR-125b-5p in melanoma.
It has been proved that miR-125b-5p acts as a tumor suppressor in the malignant progress of many human tumors [
38,
39]. In particular, miR-125b-5p is an independent predictor of survival in melanoma patients, and miR-125b-5p is down-regulated and suppresses the proliferation and invasion of melanoma [
40,
41]. miR-125b-5p were shown to be involved in the vemurafenib resistance of resistant BRAF-mutant melanoma cell [
42]. EIF5A2, the member of the EIF family, is a novel oncogene and up-regulated in ovarian cancer, esophageal cancer, gastric cancer, etc. [
22,
43‐
45]. EIF5A2 participates in many biological processes of tumor cells, including growth, metastasis and EMT [
19,
22,
45]. It was found that EIF5A2 also plays a role of oncogene in melanoma [
23]. Here, we confirmed that miR-125b-5p exert its anti-proliferation and anti-metastasis effects by targeting EIF5A2 in melanoma. Moreover, we demonstrated that the effect of LINC00520 siRNA on the proliferation, EMT, invasion and migration of melanoma cells were reversed by miR-125b-5p inhibitor. LINC00520 also promotes melanoma growth and metastasis in vivo by regulating miR-125b-5p/EIF5A2 axis. We also domnostrated that the role of LINC00520 in melanoma cells is independent of BRAF mutation. Take together, our research reveal the influence of LINC00520/miR-125b-5p/EIF5A2 on the biological progression of melanoma. Huber et al. have reported that miR-125b-5p is released in the circulation and associated with immunotherapy of melanoma [
46]. Therefore, to detect the expression level of LINC00520 in blood circulation and explore its value in the clinical diagnosis and treatment of patients with melanoma is our future research direction.
Conclusion
In conclusion, all results indicated that LINC00520 plays the pivotal role in the development of melanoma. LINC00520 facilitates the growth and metastasis of malignant melanoma by competitively binding to miR-125b-5p to liberate EIF5A2 mRNA transcripts, thereby promotes the EIF5A2 expression. Understanding the molecular mechanism of LINC00520 in melanoma is important to improve our knowledge of the molecular biological of malignant progression of melanoma. The deep study of LINC00520/miR-125b-5p/EIF5A2 axis is helpful for us to identify new biomarkers or therapeutic target for melanoma patients.
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