Clinical samples
This study received approval from the Institutional Review Board of Sun Yat-Sen University Cancer Center (SYSUCC). All patients provided written informed consent. For the microarray analysis, 18 pairs of freshly frozen NPC tissues and normal nasopharyngeal epithelial tissues were obtained by biopsy at the SYSUCC. For the prognostic analysis, 220 formalin-fixed NPC paraffin specimens were collected from SYSUCC between January 2006 and December 2009. The following inclusion criteria were used: NPC that was confirmed pathologically, previously untreated, and nonmetastatic; patients did not have other previous malignancies or serious liver, kidney, heart or lung disease; patients received radical radiotherapy, with or without platinumbased chemotherapy; with regular follow-up. The 8th edition of the American Joint Committee on Cancer (AJCC) guidelines were used to restage all the patients, and clinical characteristics and long-term follow-up data were collected.
Cell culture
The human immortalized nasopharyngeal epithelial cell line (NP69) was cultured in keratinocyte serum/free medium (Invitrogen, Waltham, MA, USA) supplemented with bovine pituitary extract (BD Biosciences, San Jose, CA, USA) at 37°C under 5% CO2. Seven human NPC cell lines (SUNE-1, HONE-1, CNE-1, CNE-2, HNE1, HK-1, and 5-8F) were maintained in Roswell Park Memorial Institute (RPMI)1640 medium or Dulbecco’s modified Eagle’s medium (DMEM) (both Invitrogen) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA). The cell lines were a generous gift from M. Chen (SYSUCC, Guangzhou, China) and were subjected to authentication.
RNA extraction and quantitative real-time reverse transcription PCR (qRT-PCR)
Total RNA from NPC cell lines and NPC paraffin specimens was extracted using TRIzol (Invitrogen), and reversed transcribed to cDNA using HiScript III RT SuperMix (Vazyme, Nanjing, China) according to the manufacturer's instructions. The qPCR step of the qRT-PCR protocol was performed using Platinum SYBR Green qPCR SuperMix-UDG reagents (Vazyme)) on the CFX96 Touch sequence detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Primers for microRNA 615-5p, microRNA − 1293, and U6 were obtained from GeneCopoeia (Rockville, MD, USA). The level of GAPDH mRNA (encoding gGlyceraldehyde-3-phosphate dehydrogenase) was employed for normalization and the 2−ΔΔCT method50 was used to calculate the relative expression levels. Table S1 shows the specific primers used for qPCR.
Oligonucleotide transfection and generation of stably transfected cell lines
The small interfering RNAs (siRNAs) targeting LINC01770 and TEAD1, miR-615-5p mimics, miRNA-1293 mimics, the miR-615-5p inhibitor, the miRNA-1293 inhibitor, and the scrambled negative control siRNA (si-NC) were purchased from GenePharma (Shanghai, China) (Supplementary Table S2). Plasmid vectors (pCDNA-LINC01770, sh-LINC01770, pcDNA-3.1-TEAD1, and empty vectors) for transfection were prepared by GeneCopoeia.
shRNA was transfected into NPC cells using RNAmate (Invitrogen) or Lipofectamine 3000 reagent (Invitrogen). Cells were harvested and assayed 48 hours after transfection. shLINC01770 was inserted into vector pLKO.1 vector and the psPAX2 packaging plasmid (Addgene, Watertown, MA, USA) and pMD2.G envelope plasmid (Addgene) were co-transfected into 293T cells using polyethyleneimine (PEI; Polysciences, Warrington, PA, USA). After transfection, the medium was changed at 12 hours and the supernatant was collected at 48 hours, and then the virus contents the indicated shRNA was using for the infection of NPC cells. Puromycin (1 mg/mL) was used to screen for stably transfected cells and qRT-PCR was used to determine whether a stable knockdown or high expression NPC cell line of LINC01770 was successfully established. Transfection with lentiviral plasmids overexpressing TEAD1 was carried out to gain NPC cells stably coexpressing shRNA-LINC01770 and TEAD1.
Microarray analysis, RNA sequencing, and bioinformatic analysis
An Arraystar LncRNA Microarray 3.0 V3.0 platform (Agilent Technologies, Santa Clara, CA, USA) was used the determine lncRNA expression in NPC and normal tissues. We isolated total RNA from SUNE-1 transiently transfected with shLINC01770, control shRNA, LINC01770OE (LINC01770 overexpression), and control OERNA, purified, and subjected it to RNA sequencing (RNA-seq), carried out by BGI Genomics (Beijing, China). Differentially expressed genes were screened using a threshold of p < 0.05 and fold change ≥ 1.5. The DAVID software was used to carry out Gene Ontology (GO) Enrichment Analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes (DEGs). In addition, we determined the biological functions enriched in NPC in response to LINC01770 knockdown GO and gene set enrichment analysis (GSEA). Significant terms were selected based on a threshold of p < 0.05. The RNA-seq data were deposited in the Gene Expression Omnibus (GEO) at the National Center for Biotechnology Information (accession number GSE236418).
CCK8 and colony formation assays
Cells at 1 × 103 cells per well were seeded in triplicate in 96-well plates and their viability was measured daily for 7 days using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan). In the assay, 10 µL of CCK-8 solution was added to each well. After 2 h of incubation, a spectrophotometric plate reader (ELX800, BioTek, Winooski, VT, USA) was used to measure the absorbance at 450 nm. To determine their colony formation ability, 500 cells were cultured in 2 mL of medium in a 6-well plate for one or two weeks. The formed cell colonies were washed using phosphate-buffered saline (PBS), fixed in paraformaldehyde, crystal violet stained, and counted using ImageJ software (NIH, Bethesda, MD, USA).
Flow cytometry
Cells stained with propidium iodide (PI) or SYTOX Green (both Invitrogen) were analyzed by microscopy or flow cytometry to observe cell death. Cells were fixed with 70% ice-cold ethanol overnight and treated with RNase. Finally, cells were stained with Propidium Iodide (PI) for 30 min at 4°C and submitted for cell cycle analysis on Flow Cytometry (Ex = 488 nm).
Lipid peroxidation measurement
Cells at 1×105 cells per well were seeded in twelve-well plates. One day later, the indicated compounds were used to treat the cells, which were then collected, stained using 5 µM BODIPY 581/591 C11 (Invitrogen, D3861) for 30 min at 37°C, and then subjected to flow cytometry analysis. For BODIPY 581/591 C11 staining, the signals from both oxidized C11 (FITC channel) and non-oxidized C11 (PE channel) were monitored. The ratio of the mean fluorescent intensity (MFI) of FITC to the MFI of PE was calculated for each sample. The data were normalized to control samples to determine the relative lipid peroxidation. The experiments were carried out at least three times, with 5,000 cells being analyzed in each group.
In vitro assays of invasion and migration
Cell invasion and migration were determined using Transwell chambers (Corning Inc., Corning, NY, USA). Cells were cultured in 200 mL of medium without serum and then added the upper chamber, which did or did not contain Matrigel (BD Biosciences); the lower chamber contained medium with 10% FBS. After incubation for 12 or 24 h, cells under the membrane were fixed and stained to observe their migration or invasion.
RNA pull-down assay
Full-length sense and antisense sequences of LINC01770 were transcribed in vitro using a MEGAscript T7 Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). A Pierce RNA 30 End Desthiobiotinylation Kit (Thermo Fisher Scientific) was used to purify and biotinylate the resultant RNA. Cell under test were lysed and combined with the transcribed RNA for pull-down experiments, performed using a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific), followed by analysis using western blotting.
Western blotting
NPC cell protein extracts were obtained and subjected to 10–14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Blocking was performed using 5% skim milk for 1 h, followed by overnight incubation with primary antibodies and then with horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h. The primary antibodies were as follows: anti-ACSL4 (Abcam, Cambridge, MA, USA, 1:1000), anti-GPX4 (Abcam, 1:1000), anti-SLC7A11 (CST, Danvers, MA, USA, 1:500), anti-TEAD1 (CST, 1:500), TFRC (also known as CD71; CST, 1:1000). Between incubations, three washes were performed using Trisbuffered saline-Tween20 (TBST) for 5 min each time. Immunoreactivity was detected using Clarity Western ECL substrate (Bio-Rad) followed by chemiluminescence detection using the ChemiDoc MP imaging system (Bio-Rad).
RNA immunoprecipitation (RIP) assay
To determine if LINC01770 could interact with or bind to argonaute 2 (AGO2)/TEAD1 in HONE-1 and SUNE-1 cells, we used an EZ-Magna RIP Kit (Millipore). NPC cell lysates were co-incubated with anti-TEAD1, anti-Ago2 or anti-IgG antibody-conjugated magnetic beads (68 h, 4°C). The magnetic beads were then collected and washed with RIP buffer. Finally, the beads were resuspended in the TRIzol regent to isolate the coprecipitated RNA for qRT-PCR analysis.
Induction of ferroptosis
The compounds Erastin (E7781, Sigma-Aldrich,) and RSL3 (HY-100218A, MCE, Monmouth Junction, NJ, USA) were used to induce ferroptosis in NPC cells, followed by flow cytometry and CCK-8 assays to detect cell viability.
Fluorescence in-situ hydration (FISH) and immunofluorescence (IF)
Probes for LINC01770 were synthesized by RiboBio (Guangzhou, China) for FISH detection of the co-localization and interaction of LINC01770 and TEAD1. Cells were grown to 30% confluence and seeded on glass coverslips, with or without RSL3 treatment. The coverslips were washed briefly with PBS, fixed using 4% paraformaldehyde for 30 min at room temperature, blocked using 4% bovine serum albumin in PBS for 1 h, and then incubated with anti-TEAD1 antibodies overnight at 4°C. Alexa Fluor 647-labeled goat anti-rabbit IgG antibody was then incubated with the cells on the coverslips for 1 h at room temperature, stained for 5 min using 1 µg/ml 4′,6-diamidino-2-phenylindole (DAPI), rinsed using PBS, and dried. An Olympus FV-1000 confocal microscope (Tokyo, Japan) was used to observe the cells (100× magnification).
Models of in vivo tumor growth and metastasis
The Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center (approval number L025501202208011) approved all the animal procedures. Nude mice were purchased from Gempharmatech-GD (Nanjing, China). For the tumor growth model, LINC01770 stable knockdown or overexpression NPC cells suspended in PBS were injected subcutaneously into the nude mice to establish a subcutaneous transplantation tumor model. When the tumors had grown to a palpable size, the mice were grouped randomly into four or six groups as indicated in the figure legends, and injected intraperitoneally with Sulfasalazine (50 mg/kg), the mice were treated 6Gy. On day 30, the mice were dissected to observe the size and weight of the tumors. To construct a tumor lung metastasis model, NPC cells were injected via the tail vein. To construct a model of lymph node metastasis in the popliteal fossa of the foot of nude mice, NPC cells were injected via their footpads. At the end of the experiment, excised tissues were fixed in paraffin and made into sections. The sections were then deparaffinized, rehydrated, and the antigen was retrieved. For IHC, the sections were incubated with primary antibodies recognizing ACSL4 (Abcam), GPX4(Abcam), SLC7A1(CST), TEAD1 (CST), CD71(CST), followed by incubation with secondary antibodies. Finally, the sections were stained using diaminobenzidine (Sigma) and counterstained using hematoxylin (Sigma).
Statistical analysis
Statistical analyses were performed using SPSS 23.0 (IBM Corp., Armonk, NY, USA) or GraphPad Prism (version 9.0, GraphPad Software, Inc., La Jolla, CA, USA). Each experiment was repeated at least three times and the data were expressed as the mean ± SD. For quantitative variables, groups were compared using analysis of variance (ANOVA) or Student's t-test. For categorical variables, the chi-squared test or Fisher's exact test were used to compare the groups. The Kaplan–Meier method was used to plot the survival curves, and the log-rank test was used for group comparisons. The effects of variables on survival outcomes were analyzed using univariate and multivariate Cox regression analysis. All tests were two-tailed and p < 0.05 was considered statistically significant.