Long Non-Coding RNA LINC00518 Induces Radioresistance by Regulating Glycolysis Through An miR-33a-3p/HIF-1α Negative Feedback Loop in Melanoma

doi:10.21203/rs.3.rs-37465/v1

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Long Non-Coding RNA LINC00518 Induces Radioresistance by Regulating Glycolysis Through An miR-33a-3p/HIF-1α Negative Feedback Loop in Melanoma

https://doi.org/10.21203/rs.3.rs-37465/v1

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Background: The long non-coding RNA (lncRNA),LINC00518, is highly expressed in many human cancers and is involved in cancer progression. However, the potential function and regulatory mechanism of LINC00518 in cutaneous malignant melanoma (CMM) remain unclear.

Methods:Short hairpin RNA (shRNA) was used to silence LINC00518 and HIF-1α, and real-time PCR was performed to determine mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of LINC00518 silencing on cellular radiosensitivity. Dual luciferase reporter system,CHIP and COIP was used to verify the target relationship between LINC00518,miR‐33a-5b and HIF-1α,.Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the expression of HIF-1α and LDHA. Finally, animal experiments were performed to demonstrate the effect of LINC00518 silencing on the radiosensitivity of melanoma in vivo.

Results: LINC00518 expression was significantly upregulated in CMM samples, and LINC00518 levels were associated with poor prognosis of patients with CMM. Knockdown of LINC00518 in CMM cells significantly inhibited cell invasion, migration, proliferation, and clonogenicity. LINC00518-mediated invasion, migration, proliferation, and clonogenicity were negatively regulated by the microRNA, miR-33a-3p, in vitro, which intensified sensitivity to radiotherapy via inhibition of the hypoxia-induced factor 1α (HIF-1α)/lactate dehydrogenase A (LDHA)-glycolysis axis. Additionally, HIF-1α recognized the miR-33a-3p promoter region and recruited histone deacetylase2 (HDAC2), which decreased the expression of miR-33a-3p and formed an LINC00518/miR-33a-3p/HIF-1α negative feedback loop. Furthermore, signalling initially activated glycolysis and radioresistance in CMM cells was recovered by Santacruzamate A (a histone deacetylase inhibitor) and 2-deoxy-D-glucose (a glycolytic inhibitor). Lastly, knockdown of LINC00518 expression sensitized CMM cancer cells to radiotherapy in an in vivo subcutaneously implanted tumour model.

Conclusion: LINC00518 was confirmed to be an oncogene in CMM, which induces radioresistance by regulating glycolysis through an miR-33a-3p/HIF-1α negative feedback loop. Our research may provide a potential strategy to improve the treatment outcome of radiotherapy in CMM.

Cancer Biology

Oncology

LINC00518

Negative feedback loop

Glycolysis

Radioresistance

Cutaneous malignant melanoma (CMM) is one of the commonest skin malignancies, with rapid progression and extremely poor prognosis, and is responsible for nearly 75% of all skin tumour mortalities [1,2]. Despite improvements in treatment, such as surgery, radiotherapy, chemotherapy, immunotherapy, and molecular-targeted treatment, CMM recurs in approximately 75% of patients a year after treatment, and the three-year overall survival rate of terminal CMM patients is no more than 30% [3-5]. The National Cancer Comprehensive Network guidelines (2018) point out that radiotherapy is the main available treatment for some primary CMMs, and adjuvant radiotherapy is necessary for infiltrating neutrophilic melanoma, metastatic encephaloma, and bone metastases [6,7]. In recent years, some studies have found that palliative radiotherapy significantly eased symptoms such as

bone pain and central nervous system dysfunction produced by MM metastases [8,9]. However, radioresistance limits the clinical application of radiotherapy to treat CMM, leading to poor prognosis.Thus, identification of approaches to overcome

radioresistance CMM could provide more effective therapeutic strategies and improve outcomes of patients with CMM.

Hypoxia, a common phenomenon in animal and human solid tumours, is an important contributor to radioresistance and can act as a prognostic indicator of the outcome of radiotherapy [10,11]. Conceptual progress in the last decade has indicated that reprogramming of energy metabolism is a newly emerging hallmark of cancer and that hypoxia plays a significant role in radioresistance because of reduced cell viability and fixation of DNA damage [12,13]. In a hypoxic microenvironment, cancer cells obtain energy mainly by inhibiting aerobic respiration and promoting glycolysis [14]. Many glycolysis- and metabolism-related proteins participate in the molecular mechanisms that regulate radioresistance. The hypoxia-induced factor 1α (HIF-1α), is an important factor that aggravates the transformation of normal melanocytes into melanoma, and HIF-1α activity in melanoma tissues and cell lines even exhibited an increase in atmospheric oxygen levels [15,16]. The HIF-1α pathway is involved in a tumour-protective response to radiotherapy, which is achieved mainly by increasing cancer antioxidation by launching glycolytic tumour metabolism [17]. Targeting tumour glucose metabolism and HIF-1α could change the tumour microenvironment, leading to metabolic alterations and sensitization of multiple solid cancers to radiotherapy [18]. Additionally, several studies have found that the levels of certain glycolysis-related proteins are closely connected to radiotherapy resistance; for instance, higher expression of glucose transporter 1(GLUT1) in breast and liver cancers indicated poor prognosis of patients treated with radiotherapy [19,20]. HK2 (Hexokinase 2) is a critical rate-limiting enzyme in the glycolytic pathway. Multiple regression analysis models implied that HK2, which facilitates glycolysis and inhibits cancer cell apoptosis, was an independent negative prognostic factor for cervical squamous carcinoma [21].

Long non-coding RNAs (lncRNAs) are an important class of non-coding RNAs of >200 bases in length that show limited protein coding capability but are closely correlated with the occurrence and development of human diseases [22,23]. lncRNAs participate in a series of modifications of the glycolysis pathway in cancer cells: LncRNA IDH1-AS1 links the functions of MYC proto-oncogene, BHLH transcription factor (c-MYC), and HIF-1α via isocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1), to regulate mitochondrial respiration and glycolysis in cervical carcinoma cells. Restoring IDH1-AS1 expression might provide a potential metabolic approach to treat cervical carcinoma [24]. LncRNA-p21 is a hypoxia-responsive lncRNA that causes HIF-1α accumulation by integrating HIF-1α and Von Hippel-Lindau tumour suppressor (VHL), thus destroying the VHL–HIF-1α interaction, which promotes glycolysis under hypoxic conditions in colorectal cancer cells [25]. Studies have shown that lnRNAs, such as LncRNA-TUG1 and LncRNA-MIF, participate in the glycolytic pathway and regulate glycometabolism via competing endogenous RNA (ceRNA) networks [26-29].

Although recent research has clarified that some lncRNAs participate in regulating radiotherapy resistance; till date, no studies have shown that lncRNAs play significant roles in regulating CMM radiosensitivity. In the present study, we analysed two CMM data sets, GSE46517 [30] and GSE4587 [31], which were published in the GEO database, to investigate dysregulated lncRNAs in CMM. Long intergenic noncoding RNA 518 (LINC00518) was significantly upregulated in CMM tissues compared with that in adjacent tissues. The Cancer Genome Database (TGCA) data set showed that high expression of LINC00518 indicated a worse relapse-free survival (RFS) and overall survival (OS) of patients with CMM. Furthermore, we verified that LINC00518 could form a negative feedback loop with HIF-1α and increase radiation resistance of CMM by modulating the glycolytic pathway. The results of the present study provide a new perspective concerning the role of lncRNAs in the radiation sensitivity of CMM and may assist in identifying new biomarkers or targeted therapies for CMM.

Data mining and analysis

To identify functional lncRNAs in CMM, the gene expression patterns of two different cohorts of CMM, derived from the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array, GSE46517 and GSE4587, were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). The Significant Analysis of Microarray (SAM) mode was used to analyse the different expression levels of lnRNAs between adjacent and CMM tissue samples in these two data sets. The critical value for the expressed lncRNAs was set at ≥ 2.0-fold change and a false discovery ratio (FDR) of < 0.05.

Cell lines and tissue

Human malignant melanoma cell lines (WM35, WM451, and A375) and human melanocytes (HM) were purchased from the ATCC (Rockville, MD, USA) and cultured in Dulbecco's modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin under a humidified atmosphere of 5% CO₂ at 37 °C. Human CMM tissues and corresponding adjacent non-cancerous skin tissues were collected from patients who underwent surgery and were pathologically diagnosed with MM at the Third Xiangya Hospital of the Central South University and Hunan Cancer Hospital from January 2015 to December 2017. Informed consent was obtained from all the participants.

Cell transfection

MiR-33a-3p-mimics,and-inhibitors, LINC00518-shRNAs,HIF_1α-shRNAs,HIF-1α overexpression plasmid, and negative controls(NC) were purchased from GeneChem Biotechnology Company. Cells were transfected as previously described [32] . CMM cells were cultured in 6-well plates and allowed to reach 70% confluence after 24 h. Lipofectamine™ 2000 (Invitrogen Life Technologies) was used to transfect cells with DNA complexes following the manufacturer's instructions. The cells were harvested after 24 h and 36 h, and RNA and protein were extracted after transfection, respectively. Real-time PCR was used to confirm the efficiency of transfection.

Quantitative real-time reverse transcription PCR

Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The PARIS kit (Ambion) was used to isolate RNA from the cytoplasm and nuclei of transfected cells. Real-time PCR was performed using RNA isolated from the cytoplasmic and nucleic fractions. RNA purity was assessed by spectrophotometry (A260/A280 > 1.8). To perform quantitative real-time reverse transcription PCR (qRT-PCR), 1 µg of RNA from the samples was reverse transcribed using a RevertAid™ H Minus First Strand cDNA Synthesis Kit (Fermentas #K1631; Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed to assess the expression of LINC00518 and miR-33a-3p using a SYBR Green PCR Master Mix (ABI 4309155) employing the CFX96 Real-Time PCR Detection System (ABI 7900; Applied Biosystems, Foster City, CA, USA). PCR amplifications were performed in triplicates. The sequences of the qRT-PCR primers were as follows:

LINC00518: forward:5′-TGCAATTCAGGTCGGTTGTA-3′;

reverse:5′-GTGGAGCTCCCTGAAGACAG-3′.

miR-33a-3p: forward:5′-ACACTCCAGCTGGGCAATGTTTCCACAGTG-3′;

reverse:5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGTGATGCA-3′.

β-actin:forward:5′-GTGGGGCGCCCCAGGCACCA-3′;

reverse:5′-CTCCTTAATGTCACGCACGATTTC-3′.

ACTB (encoding β-actin) was used as an internal reference.

Western blotting

Phosphate-buffered saline (PBS) was used to wash the cancer biopsy specimens and cancer cells, and later the cells were lysed on ice using the radioimmunoprecipitation assay (RIPA) buffer containing 10% protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland) for 30 min. Proteins were quantified using a Total Protein Extraction Kit (ProMab, Richmond, CA, USA). The proteins were separated using a 10% SDS-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. The membranes were incubated with the following primary antibodies: anti-HIF-1α (1:1000, Bioss, bs-20398R), anti-lactate dehydrogenase A (LDHA) (1:50, Proteintech, 19987-1-AP), anti-Histone H3 (acetyl K4) (1:500, Abcam, ab232931), anti-Histone H3 (acetyl K9) (1:2000, Abcam, ab4441), anti-Histone H3 (acetyl K27) (1:200, Abcam, ab177178), anti-Histone H3 (1:1000, CST,#4499), and anti-β-Actin (1:2000, CST, # 3700S) at 4 °C overnight, followed by the addition of HRP-conjugated secondary antibodies at 37 °C for 60 min. The signals of the immunoreactive protein bands were assessed using densitometric analysis software and quantified by densitometry using the ImageJ system. β-actin served as a loading control.

Invasion and migration assays

The migration capacity of CMM cells was assessed using a wound-healing assay. Cells (1×10⁵per ml) were cultivated in 6-well plates and grown to 85–90 % confluence. A wound was scratched in the middle of the monolayer using a 10-µl pipette tip, and the cells were washed twice with 10% PBS to dislodge cellular debris. A photograph was taken at 0 h under a microscope. The plates were placed in a humidified atmosphere of 5% CO₂ at 37 °C, and the wound was photographed again at 48 h. The difference between the two measured widths on the images represented the extent of cell migration.

CMM cell invasion capacity was detected using a Transwell assay. Cells (1×10⁵) in 100 μL of serum-free medium were added to the apical chamber of a Transwell plate that was pre-coated with Matrigel matrix, and 500 μL of 10% FBS-containing medium was added to the lower chamber. After incubation for 48 h at 37 °C, the invading cells that traversed the membrane were fixed with methanol and stained with 0.1% crystal violet. Cells that remained on the upper chamber surface of the Transwell membrane were wiped off using a cotton bud. Six visual fields at 20 × magnification were randomly captured and the numbers of invaded tumour cells were counted.

Cell proliferation bioassay

CMM cells treated with a short interfering RNA (siRNA) or plasmid were cultivated in 96-well plates (500 cells per well). Cell proliferation after irradiation or transfection was determined using a commercial 3-(4,5-dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium bromide (MTT) assay kit (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. First, 50 μL of MTT was added to the cells in the wells and incubated for 4 h. Later, 150 μL of dimethyl sulfoxide was added to dissolve the MTT crystals. The light absorption value of each well was measured using a spectrometer at 570 nm. All the assays were repeated no less than three times in triplicate wells.

Cell cycle and apoptosis assays

Cell cycle and apoptosis assays were performed using flow cytometry. CMM cells transfected with siRNA or vector were cultivated in 6-well plates. Cells were treated with or without irradiation or additional processing for 48 h, and then harvested by centrifugation. An annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit (Mbchem) and cell cycle detection kit (Sigma) were used to stain the cells, according to the manufacturers’ protocols. All the tests were performed in triplicates.

Clonogenic assay

A clone formation assay was used to assess the proliferation activity and cellular radiosensitivity of MM cells. Cells transfected with the siRNA or plasmid were seeded in 6-well plates at 1000 cells per well. The cells were treated with or without irradiation or additional processing and then incubated for 14 days. The culture medium was replaced at intervals of 2–3 days. MM cells were washed twice with 10% PBS before being harvested and immobilized using 1 mL of poly-formaldehyde for 45 min. The surviving fractions (>50 cells) were stained with haematoxylin and counted under a microscope. All the clone formation assays were performed in triplicates.

Dual-luciferase experiment

For the luciferase reporter experiment, the LINC00518-WT (wild-type), LINC00518-MUT (mutant), HIF-1α-3′-UTR-WT, and HIF-1α-3′-UTR-MUT vectors were used. WM451 and A375 cells were seeded in 6-well plates and co-transfected with hsamiR-33a-3p mimics or empty plasmids for 48 h. The dual luciferase reporter system (Promega) was used to assess firefly and sea pansy luciferase activities. The relative luciferase activities were calculated, and control cells were used for normalization.

Chromatin Immunoprecipitation (ChIP)

WM451 and A375 cells were treated with or without Santacruzamate A (an HDAC inhibitor) and underwent two rounds of dual crosslinking. The protein-DNA complexes were precipitated using anti-H3K27AC antibodies (ab177178). Signal intensity of the promoter region of the target gene was determined from PCR results. The signal strength of the miR-33a-3p promoter was measured by PCR using a SYBR Green PCR Master Mix Kit (ABI 4309155). According to the promoter region of miR-33a-3p, containing HIF-1α-binding sites (AGACGTGA and GGGCGTGG), primers were designed to analyse the purified DNA. The following primer sequences for qRT-PCR were used:

miR-33a-3p forward: 5′-CTTAGCAGCAGACGTGATGG-3′;

reverse: 5′-GAGTCGAGAGGCAGGTCACT-3′.

GAPDH: forward: 5′-TACTAGCGGTTTTACGGGCG-3′;

reverse: 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′.

RNA pull-downassay

An RNA pull-down assay was performed using the Magnetic RNA-Protein Pull-Down Kit (Pierce™). In vitro biotin-labelled (Bio)-miR-NC, Bio-miR-33a-3p, and Bio-miR-33a-3p-Mut were transfected into WM451 and A375 cells, and the cells were for 48 h. The cell lysates were collected and incubated with streptavidin magnetic beads, forming protein-Bio/RNA-magnetic bead complexes. High salt elution was used to obtain the protein-bio/RNA from the magnetic bead-protein-bio/RNA mixture. The TRIzol reagent (Invitrogen, Carlsbad, CA, USA)was used to purify the protein-bio/RNA, and PCR was used to measure the relative expression of LINC00518.

Co-immunoprecipitation assay

WM451 and A375 cells transfected with an siRNA or a plasmid overexpressing HIF-1α were seeded in a 10 cm petri dish. The cells were washed twice with pre-chilled PBS and disrupted using RIPA lysis buffer (1 mL for 10⁷cells). Protein A+G Agarose (Bioss, P1012) was used to precipitate protein complexes. The collected complexes were subjected to SDS-PAGE followed by western blotting. Anti-histone deacetylase 1 (HDAC1) (1:100, SAB, 32034), anti-HDAC2 (1:100, Abcam, ab12169), and anti-HIF-1α (1:200, Bioss, bs-20398R) were used as detection antibodies; anti-IgG (1:150, Bioss, bs-0297P) was used as a loading control.

Measurement of glucose consumption and lactate production

Glucose consumption and lactate production were estimated as previously described [33]. Briefly, cells (5 × 10⁵) were recovered and cultured in 6-well plates. After incubation for 10 h, the cell culture medium was removed and the cells further incubated with fresh medium for 8 h. Glucose and lactate levels were measured (Automatic Biochemical Analyzer, 7170A, HITACHI, Tokyo, Japan) at the Clinical Biochemical Laboratory of Third Xiangya Hospital. The relative rates of glucose consumption and lactate production were normalized using consistent amounts of protein.

X-ray irradiation

X-ray irradiation was performed using an X-ray generator (Varian, USA), emitting at a fixed dose rate of 4 Gy/min. The energy of the X-rays used to irradiate

the cells was graded as 0, 2, 4, 6, and 8 Gy.

Xenograft Mouse Model

Male BALB/c nude mice (5 weeks old, 18±0.75 g) were purchased from the Laboratory Animal Center of Central South University (Changsha, China) and were maintained under specific pathogen-free conditions (IVC) in the Experimental Animal-feeding Centre of the Xiangya Medical College of Central South University (Changsha, China). To generate tumours in vivo, WM451 and A375 cells (1×10⁶, 0.2 ml) were treated with shRNA-mock1, shRNA-mock2, shRNA-LINC00518-transfected, or Santacruzamate A, co-cultured, and injected into mice that were divided into four groups: A, B, C, and D (n = 3 animals per group, 5 weeks old); knockdown assays were performed as previously described [32]. The cells were injected subcutaneously on the superior border of the right upper limb, and isoflurane anaesthesia machine was used to administer inhalational anaesthesia to the animals. The procedure was simple and painless (Induction: 2-4%, ZS, Beijing, ZS-MV-HR). When the mice developed a 100-mm³ tumour, a single 2 Gy dose of irradiation was delivered to the B, C, and D groups. The tumour volume was estimated every 2 days by measuring the tumour length (L) and width (W). The tumour volume (V) was calculated using the formula V = 1/2(L×W²). After 40 days, all the mice were sacrificed by cervical dislocation at the Laboratory Animal Center of the Xiangya Medical College of Central South University， and the tumours were collected. All the animal experiments were approved by the Ethics Committee of the Third Xiangya Hospital of Central South University.

Statistical analysis

The statistical software package GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) was used for all statistical analyses. All experiments were performed at least in triplicates. All data are shown as mean ± SD, and P < 0.05 was considered to be statistically significant. Differences between the groups were analysed using analysis of variance (ANOVA) followed by comparison between specific groups using Student’s t test.

LINC00518 is overexpressed in CMM and indicates poor prognosis in patients with CMM

To identify novel lncRNAs active in CMM, we downloaded two cohorts of gene expression data sets for CMM from the GEO database (GSE46517 and GSE4587). The SAM software was used to analyse differences in lncRNA expression in biopsy samples of CMM and adjacent non-cancerous skin tissues. We identified 12 lncRNAs that showed increased expression in CMM compared with normal skin tissues (Fig. 1A). Among the differentially expressed lncRNAs, LINC00518 was overexpressed in the CMM samples from both the data sets (Fig. 1B) and had never been reported in human CMM. Based on data from the TCGA database, we found that the expression level of LINC00518 was higher in CMM than in non-CMM samples (Fig. 1C). Patients with CMM who showed high expression of LINC00518 had a shorter OS (P = 0.0009, Fig. 1C) and RFS (P = 0.038, Fig. 1C). In conclusion, LINC00518 shows increased expression in patients with CMM, and this high expression is associated with poor survival of these patients.

LINC00518 knockdown suppressed cell proliferation, colony formation, migration and invasion, and induced apoptosis.

LINC00518 was upregulated in CMM tissues compared with normal tissues in GSE46517 and GSE 4587 data sets (Fig. 2A).Subsequently, LINC00518 expression was validated in 12 paired CMM tissues and their corresponding adjacent non-cancerous skin tissues using qRT-PCR (Fig. 2B). To investigate the function of LINC00518 in CMM, we analysed LINC00518 expression in several CMM cell lines (HM, WM35, WM451, and A375). LINC00518 expression was higher in WM451 and A375 cells, which have a high invasive and metastatic tendency, and lower in HM and WM35 cells, which are poorly invasive (Fig. 2C). Therefore, we knocked down LINC00518 expression using a LINC00518-targeting small hairpin RNA (shRNA) in CMM cell lines, WM451 and A375 (Fig. 2D). Next, we conducted phenotypic and functional analyses of the effects of LINC00518 knockdown in WM451 and A375 cell lines after establishing shRNA efficacy. MTT assay showed that the proliferation of LINC00518-silenced cells was significantly decreased compared to that of control CMM cells (Fig. 2E). Flow cytometry demonstrated that silencing of LINC00518 expression in WM451 and A375 cells increased apoptosis (Fig. 2F). Colony formation assays indicated that the cell survival capability of LINC00518-silenced cells decreased significantly (Fig. 2G). The effect of LINC00518 on the migration and invasion of CMM cells were measured by wound healing assays and Transwell Matrigel invasion assays, respectively. LINC00518 shRNA-transfected CMM cells displayed lower migratory and invasive capabilities than the control cells (Fig. 2H, Fig. 2I).

LINC00518 directly targets miR-33a-3p and promotes HIF-1αexpression

To explore the mechanisms of LINC00518 in CMM tumorigenesis, we initially investigated the intracellular location of LINC00518. Using qRT-PCR assays, we found that LINC00518 was mainly localized in the cytoplasm (Fig. 3A). Subsequently, miRNA target prediction databases (RegRNA and TargetScan) were used to predict miRNAs that LINC00518 could sponge and the target genes that miRNAs could regulate (Fig. 3B). We observed that LINC00518 could directly target miR-33a-3p, which in turn regulated HIF-1α expression. HIF-1α is a known oncogenic gene that we have previously described to be involved in the occurrence and development of CMM[34]. Next, presumptive binding sites for miR-33a-3p were identified in the LINC00518 sequence and in HIF1α 3′ UTR by using RegRNA 2.0 website (Fig. 3C). Results of RNA pull-down demonstrated that the bio-miR-33a-3p group was enriched for larger amounts of LINC00518 than bio-miR-NC and bio-miR-33a-3p-MUT groups (Fig. 3D). Expression of miR-33a-3p in CMM biopsy tissues compared with that in adjacent non-tumour tissues was assessed, and an inverse correlation between miR-33a-3p and LINC00518 expression was observed (Fig. 3E). We employed dual-luciferase experiments to explore whether LINC00518 could directly target miR-33a-3p. These assays demonstrated that miR-33a-3p mimics significantly inhibited the relative dual-luciferase activity of the LINC00518-WT group compared with that of control group, while they did not affect the reporter activity of LINC00518-MUT group, (Fig. 3F). These results indicated that LINC00518 could directly target miR-33a-3p and reduce its expression. In addition, correlation analysis showed a negative correlation between miR-33a-3p and HIF-1α expressions in CMM biopsy tissues (Fig. 3G). The significant inhibition of the relative dual-luciferase activity of the HIF-1α-WT group by miR-33a-3p mimics indicated that miR-33a-3p could regulate HIF-1α expression (Fig. 3H). To explore the effect of LINC00518 knockdown and miR-33a-3p on glycolysis, we examined changes in the abundance of key proteins of the glycolysis pathway in CMM cell line. Western blotting analysis indicated that HIF-1α and LDHA protein levels were regulated by LINC00518 and miR-33a-3p (Fig. 3I and Fig. 3J). These results indicated that LINC00518 increased the expression of HIF-1α in CMM by targeting miR-33a-3p.

HIF-1αnegativelyregulates miR-33a-3p expression in CMM cells

HIF-1α is a recognized transcription factor and the JASPAR server predicted presumptive binding sites at positions 632 bp to 639 bp in a sequence upstream of the origin of miR-33a, with a score of 7.1 out of 10 (Fig. 4A). In addition, HDACs are highly expressed in CMM tissues and cell lines (Fig. 4B); therefore, we speculated that HIF-1α might inhibit the transcription of miR-33a and decrease the relative expression level of miR-33a-3p by binding to the miR-33a promoter and recruiting HDACs. PCR assays demonstrated that miR-33a-3p expression was elevated after LINC00518 and HIF-1α knockdown in WM451 and A375 cell lines. We observed that enhanced HIF-1α expression in WM451 and A375 cells could rescue the effect on miR-33a-3p expression by LINC00518 knockdown (Fig. 4C,D). Furthermore, LINC00518 expression was increased when HIF-1α expression was enhanced in LINC00518 knockdown CMM cells (Fig. 4E). The miR-33a-3p expression level was upregulated in WM451 and A375 cells after HDAC1-3 knockdown (Fig. 4F). Moreover, ChIP PCR was performed to verify that Santacruzamate A (an inhibitor of HDACs) could recover the relative expression levels of miR-33a-3p in WM451 and A375 cells (Fig. 4G). A co-immunoprecipitation assay showed that the HIF-1α–HDAC immunoprecipitate was decreased after HIF-1α knockdown (Fig. 4H). Western blotting analysis indicated that expressions of H3K4ac, H3K9ac, H3K27ac, and total Histone H3 increased after CMM cells were treated with Santacruzamate A (Fig. 4I). LINC00518 knockdown decreased HIF-1α and LDHA protein levels, while overexpression of HIF-1α in LINC00518 knockdown cells enhanced HIF-1α and LDHA expression, suggesting that a LINC00518-HIF-1α axis exists in CMM cells (Fig. 4J). These results indicated that HIF-1α could provide a negative feedback to regulate miR-33a-3p expression. Specifically, it is probable that HIF-1α binds to the miR-33a promoter and represses the transcription of miR-33a by accelerating histone deacetylation of miR-33a.

Overexpression HIF-1α could reverse the regulatory effect of miR-33a-3p mimics on cell proliferation, apoptosis, and metastasis in CMM cells

LINC00518 regulates proliferation, apoptosis, autophagy, colony formation, migration, and invasion in CMM cells, and directly regulates the expression of miR-33a-3p; therefore, we determined whether miR-33a-3p could also regulate these phenotypes in CMM cells. The expression of miR-33a-3p assessed in CMM cell lines (HM, WM35, WM451, and A375) showed that miR-33a-3p expression was higher in HM and WM35 cells, which are poorly invasive, and lower in WM451 and A375 cells, which have high invasive and metastatic tendencies (Fig. 5A). Transfection of WM451 and A375 cells with miR-33a-3p mimics reduced cell proliferation and increased apoptosis, while enhancing miR-33a-3p overexpression of HIF-1α in WM451 and A375 cells increased proliferation and reduced apoptosis (Fig. 5B,C). Additionally, colony formation assays demonstrated that the cell survival capability of miR-33a-3p – overexpressing cells was significantly decreased, while overexpression of HIF-1α and miR-33a-3p reversed this negative effect on cell survival (Fig. 5D). Wound healing assays and Transwell Matrigel invasion assays suggested that miR-33a-3p is involved in regulating migration and invasion in CMM cells, and that HIF-1α could reverse the regulatory effects of miR-33a-3p (Fig. 5E,F). The experimental outcome of enhancing miR-33a-3p expression in WM451 and A375 cells also demonstrated that miR-33a-3p participates in the regulation of proliferation, apoptosis, colony formation, migration, and invasion in CMM cells by targeting HIF-1α.

Involvement of the LINC00518/miR-33a-3p/HIF-1αnegative feedback loop in glycolysis-mediated radiotherapy resistance of CMM cells

HIF-1α has been reported to participate in regulating radiotherapy resistance by initiating glycolytic tumour metabolism in a variety of cancers [35] . Therefore, we explored whether the LINC00518/HIF-1α signalling loop affects the radiosensitivity of CMM. To further study the function of the LINC00518/HIF-1α signalling loop in CMM cell radioresistance, WM451 and A375 cells were treated with doses of 0, 1, 2, 4, and 6 Gy, and MTT and flow cytometry assays were performed to determine their radiosensitivity. The results indicated that after exposure to a radiation dose of 2 Gy, the proliferation capacity of CMM cell lines decreased significantly and the levels of glycolysis increased simultaneously (Supplementary Fig. 1A). Furthermore, the protein levels of HIF-1α and LDHA were increased in cells treated with 2 Gy of radiation (Supplementary Fig. 1B). These results indicated that treatment of CMM cells with 2 Gy of radiation could inhibit their cell proliferation abilities while maintaining their radioresistance. Therefore, we selected a single dose of 2 Gy as the radiation dose for subsequent experiments.

Western blotting assays confirmed that knockdown of LINC00518 expression in WM451 and A375 cells decreased HIF-1α and LDHA protein levels, while overexpression of HIF-1α could reverse this effect. To identify the role of the LINC00518/HIF-1α signalling loop in mediating radiosensitivity of CMM cells, the effects of the glycolytic inhibitor, 2-deoxyglucose (2DG), and HDAC inhibitor, Santacruzamate A, were investigated in WM451 and A375 cells. Both 2DG and Santacruzamate A decreased HIF-1α and LDHA protein levels, which were inhibited by HIF-1α overexpression or by upregulation of LINC00518 and HIF-1α in CMM cells (Fig. 1C).

To investigate whether LINC00518/HIF-1α plays a role in anaerobic glycolysis of CMM, we transfected WM451 and A375 cells with a LINC00518 expression-silencing plasmid or a LINC00518 expression-silencing plasmid together with an HIF-1α– overexpression plasmid, and biochemically tested glucose consumption, ATP levels, and lactic acid production. Knockdown of LINC00518 expression in WM451 and A375 cells decreased glucose consumption, ATP levels, and lactic acid production in the cells, while overexpression of HIF-1α significantly reversed the effects of LINC00518 silencing of glycolytic tumour metabolism in WM451 and A375 cells (Fig. 6A–C). These results suggest that the LINC00518/HIF-1α signalling loop could increase CMM cell glycolytic metabolism.

MTT, flow cytometry, colony formation, and immunofluorescence assays revealed that knockdown of LINC00518 expression in WM451 and A375 cells increased radiosensitivity, while overexpression of HIF-1α reversed this effect. Both 2DG and Santacruzamate A increased radiosensitivity, which was inhibited by HIF-1α overexpression or by upregulation of LINC00518 and HIF-1α in CMM cells (Fig. 6D,E; Fig. 7A,B) .The results demonstrated that the LINC00518/HIF-1α signalling loop could regulate the HIF-1α-LDHA pathway and promote glycolytic metabolism, thereby increasing the radioresistance of CMM cells.

Effect of the LINC00518/miR-33a-3p/HIF-1αnegative feedback loop on radiosensitivity in CMM cells in vivo

We employed a xenograft mouse model to validate the effect of LINC00518/HIF-1α on the radiosensitivity of CMM cells in vivo. Repression of LINC00518 expression using an sh-LINC00518 plasmid or treating the cells with Santacruzamate A reduced the tumorigenic ability of WM451 and A375 cells, and increased their radiosensitivity in the subcutaneous sarcoma model (Fig. 8A).

To analyse the relationship between radiosensitivity and glycolysis, immunohistochemistry and western blotting analysis were performed to measure HIF-1α and LDHA protein levels in the xenograft tumours. Immunohistochemistry showed that HIF-1α, Ki67, and LDHA proteins were significantly downregulated after knockdown of LINC00518 in WM451 and A375 cells or in cells treated with Santacruzamate-A (Fig. 8B). Western blotting assays also confirmed the above-mentioned results (Fig. 8C). The subcutaneous sarcoma model further confirmed that the LINC00518/miR-33a-3p/HIF-1α negative feedback loop could increase radiotherapy resistance, accompanied by accelerated glycolysis. Schematic of this search (Fig. 8D)

The long intergenic non-protein coding RNA 518 (LINC00518) is located on human chromosome 6p24.3 and shows low expression in a majority of normal and malignant human tissues. However, its expression is markedly upregulated in some tumour tissues [36-38]. LINC00518 has been shown to regulate chemoresistance and promote epithelial cell growth and metastasis in breast cancer [37], promote cell proliferation, and to affect the invasion and metastasis of cervical cancer [38], indicating its significant role in the development and progression of malignant tumours. LINC00518 overexpression as poor prognosis factor for melanoma have been reported previously[39], but its effect on cancer cell radioresistance is a relatively recent observation.

Recently, growing evidence has suggested that lncRNAs play an important role in the complex etiology and mechanism of carcinogenesis progression [40,41]. In the present study, we noted that LINC00518 expression was significantly upregulated in CMM tissue compared with that in normal skin tissue, and that patients with CMM with high expression of LINC00518 have a remarkably poor prognosis in terms of both RFS and OS. This indicates that LINC00518 may be a molecular target in CMM cells and a potential biomarker for the diagnosis and prognosis of patients with CMM. Additionally, LINC00518 was shown to increase the proliferation and clonogenicity of CMM cells, induce their migration and invasion, and decrease their apoptosis, suggesting that LINC00518 acts as an oncogene in CMM cells.

LINC00518 is mainly localized in the cytoplasm, and this kind of lncRNA mainly functions by regulating the target genes of miRNAs by acting as miRNA sponges, thereby inhibiting their functions [42,43]. Therefore, we identified the miRNAs that bind to LINC00518. Further studies confirmed that LINC00518 directly targets miR-33a-3p; miR-33a-3p targets HIF-1α, a finding that was demonstrated in our previous studies[34]. LINC00518 upregulates HIF-1α expression by binding competitively to miR-33a-3p. An increasing number of studies have shown that transcription factors could promote lncRNA expression by binding to the promoter region of the gene [44-46]; however, the influence of transcription factors on the regulation of miRNA expression is rarely reported. Our study is the first to demonstrate that HIF-1α could bind to the miR-33a-3p promoter region and recruit histone deacetylase in CMM cells and could restrain miR-33a-3p expression by promoting miR-33a histone deacetylation. HDAC1/2/3 are overexpressed in melanoma cell lines than in primary melanocytes. HDACs deacetylate histones and non-histone proteins, which are conducive to regulating gene transcription [47]. In cancer cells, inhibition of HDACs has been shown to influence the execution of extrinsic and intrinsic apoptosis pathways as well as DNA repair [48]. Our results also provide a new aspect and method to explore the interaction model between histone deacetylation and transcription factors. Besides, LINC00518, miR-33a-3p and HIF-1α could comprise a negative feedback loop, providing the first evidence for the presence of a LINC00518–miR-33a-3p–HIF-1α regulatory axis in CMM cells.

Next, we verified whether LINC00518 and HIF-1α could specifically inhibit miR-33a-3p expression. In some studies, miR-33a-3p is a recognized antioncogene that suppresses a malignant phenotype by inhibiting the expression of pre–B-cell leukaemia homeobox in hepatocellular cancer [49], and increased miR-33a-3p expression can promote the malignant phenotype of gastric carcinoma [50]. Metastasis is a multistep process that includes de-adhesion, migration, adhesion, and invasion; however, the influence of miR-33a-3p on the CMM phenotype has not been studied. To investigate the role of miR-33a-3p in CMM in vitro, malignant phenotype assays were performed after overexpressing miR-33a-3p in CMM cells. We found that miR-33a-3p acts as an antioncogene by inhibiting the activity of HIF-1α in CMM. Our findings indicated that miR-33a-3p might be a tumour suppressor, which has an enormous value in diagnostic and prognostic evaluation and in the development of new targeted therapy of CMM.

Glycolysis is an important mechanism that confers a selective advantage to cancer cells by supporting uninterrupted growth, and a higher glycolytic rate in tumour cells is considered the main cause of radiotherapy failure due to radioresistance [51,52]. Thus, interrupting or disrupting tumour glycolysis will impact tumour growth via energy depletion as well as sensitization to therapeutics. Hypoxia and nutrient deficiency are common phenomena in advanced solid human tumours. As a hypoxia-induced factor, HIF-1α can be stimulated by these stress conditions [53,54]. Our study found that the HIF-1α-LDHA pathway regulates the activation of anaerobic glycolysis in tumour cells and suppresses cells that are sensitive to radiotherapy and chemotherapy. However, a significant correlation has not been established between HIF-1α and the prognosis of malignant melanoma in public databases. Research has revealed two possible reasons for this: the first one is that HIF-1α acts as a transcription factor to increase the transcription level of tumour suppressor genes, and the second is that HIF-1α promotes transcription of oncogenes. Biochemical tests of glucose consumption, ATP levels, and lactic acid production suggested that the LINC00518–miR-33a-3p–HIF-1α negative feedback loop plays a major role in the glycolysis of CMM cells. Thus, we hypothesized that this negative feedback loop might inhibit the radiotherapy sensitivity of CMM cells. To test this hypothesis, in vitro radio response assays were performed after knockdown of LINC00518 in CMM cells. Considering that the endogenous expression of LINC00518 was very high in these cells, overexpression of LINC00518 was not performed. We found that the LINC00518–miR-33a-3p–HIF-1α negative feedback loop could increase radiation resistance in CMM by regulating the HIF-1α-LDHA glycolysis signalling pathway. Therefore, anticancer therapies that target the LINC00518–miR-33a-3p–HIF-1α negative feedback loop might represent a potential therapeutic approach to enhance CMM cell radiosensitivity.

In summary, LINC00518 is highly expressed in CMM tissues, and high expression of LINC00518 is an indicator of poor prognosis in patients with CMM . The LINC00518–HIF-1α–glycolysis axis can promote oncogenesis and the development of CMM cells by causing CMM cells to adapt to a state of tumour hypoxia. The LINC00518–HIF-1α–glycolysis axis can also significantly upregulate HIF-1α and LDHA, thereby inhibiting apoptosis and inducing proliferation of CMM cells in response to radiation, ultimately causing radioresistance Our results demonstrated that LINC00518 might be a prognostic biomarker and an actionable target for radiosensitization of CMM.

HIF-1α ：hypoxia-induced factor 1α；LDHA：lactate dehydrogenase A；HDAC2：histone deacetylase2；Santacruzamate A：a histone deacetylase inhibitor；2DG：2-deoxy-D-glucose，a glycolytic inhibitor；CMM：Cutaneous malignant melanoma.

Ethical approval and consent to participate

The design and methods of the research are in accordance with the requirements of related regulations and procedures (such as Ethical Review of Biomedical Research Involving Human Subject,ICH-GCP) as well as the ethical principles.The IRB has approved the research to be conducted in The Third Xiangya Hospital,Central South University.

Consent for publication

Not applicable.

Availability of data and material

Previously reported GSE4587 and GSE46517 data were used to support this study and are available at GEO datasets,the prognostic analysis of LINC00518 were used to support this study and are available in Gene Expression Profiling Interactive Analysis datasets. These prior studies (and datasets) are cited at relevant places within the text as references.

Competing interests

The authors declare no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China (81874137).the Outstanding Youth Foundation of Hunan Province (2018JJ1047), the Huxiang Young Talent Project (2016RS3022).

Authors contributions

Yan Liu and Ke Cao designed the study,analyzed and interpreted the data,and wrote the manuscript.Dong He contribute to data acquisition and interpretation.Mengqin Xiao and Liang Xiang performed all bioinformatics analysis and carried out the experiments.Yuxing Zhu provide technical expertise and support.All aythors have seen and approved the final version of the manuscript.

Acknowledgements

We thank our colleagues in the department of laboratory medicine for helpful discussions and valuable assistance.

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Long Non-Coding RNA LINC00518 Induces Radioresistance by Regulating Glycolysis Through An miR-33a-3p/HIF-1α Negative Feedback Loop in Melanoma

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