Patients and specimens
Nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues were obtained from 10 NPC patients who received treatment at the Third Xiangya Hospital (Changsha, Hunan, China) from May 2017 to May 2019. The patients were diagnosed by pathological analysis and were not subjected to radiation therapy or chemotherapy. The Institutional Review Board of the Ethics Committee of Third Xiangya Hospital approved the consent procedure, and written informed consent was provided by all patients in the study.
Cell culture and transfection
Human NPC cells (CNE-1, CNE-2, HNE1, HNE2, 5-8F, and 6-10B) and nasopharyngeal epithelial cells (NP69) were obtained from American Type Culture Collection (ATCC). The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin liquid (Life Technologies) in a humidified atmosphere at 37°C with 5% CO2. ShRNAs and overexpression plasmids of FOXD1-AS1, FOXD1, and the negative control were designed by GeneChem Biotechnology Company (Shanghai, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the recombinant plasmid according to the manufacturer’s protocol as previously described . Transfection efficiency was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Quantitative real-time PCR (qRT-PCR)
A TRIzol reagent (Invitrogen) was used to extract total RNA from cells or tissues in accordance with the manufacturer’s instructions. Approximately 500 ng of total RNA was subjected to complementary DNA (cDNA) synthesis using a reverse transcription kit (Takara, Dalian, China). qRT-PCR was performed using an SYBR Green Premix Ex Taq kit (Takara, Dalian, China) on the ABI 7900HT qRT-PCR system (Life Technologies). β-actin was used as an internal control, and all experiments were performed at least three times. The 2−ΔΔCt method was used to calculate the relative expression levels of genes. The following primer sequences were used: FOXD1-AS1 -F: 5-CATAAATAGAGGG ACCCGCA-3; R: 5-GGAAGAAGGTAGGAATCCCG-3′; FOXD1 -F: 5-TGAGCA CTGAGATGTCCGATG-3; R: 5-CACCACGTCGATGTCT GTTTC-3′; β-actin -F: 5-CATGTACGTTGCTATCCAGGC-3; R: 5-CTCCTTAATGT CACGCACGAT-3′.
Approximately 1×104 NPC cells were seeded in a 96-well plate and incubated for 0, 12, 24, 48, and 72 h. After the incubation period, 50 μL MTT (Beyotime, Shanghai, China) was added into each well and re-incubated in a 5% CO2 atmosphere at 37°C for 4 h. Subsequently, the MTT was discarded and 150 μL of dimethyl sulfoxide (DMSO) was added into each well to dissolve the MTT crystals. Finally, the optical density at 570 nm was measured.
Wound healing assays
Wound healing assays were performed to assess the migration capacity of NPC cells as previously described. NPC cells (1×105/mL) were cultivated in 6-well plates. Once the cells were grown to 90% confluence, a wound was scratched using a 200-μl pipette tip, after which the cells were washed twice with phosphate-buffered saline (PBS) to dislodge any debris. DMEM without FBS was added to the 6-well plates. The photographs of wound healing were taken via a microscope at 0 h and 48 h.
Transwell assays were performed to assess the invasion of NPC cells. After transfection for 24 h, NPC cells were collected and washed twice with serum-free DMEM. A 200 μl (5×104 cells/mL) cell suspension was plated into the upper chamber, which was pre-coated with Matrigel Matrix (Corning, USA), while 600 μl DMEM containing 20% FBS was added to the lower transwell chamber. After incubation at 37°C for 24 h, cells that had invaded across the membrane were fixed with 95% ethanol for 20 min and then stained with hematoxylin for 10 min. Images of stained cells were randomly photographed and counted under an inverted microscope.
Apoptosis via flow cytometry
The apoptosis rate of NPC cells was detected using an Annexin V-PE/7-AAD apoptosis detection kit (KA3809, Abnova) following the manufacturer’s instructions. Briefly, approximately 3×105 cells were seeded into 6-well plates. After transfection with different shRNAs or plasmids for 48 h, the cells were fully digested and collected. Cells were suspended in a cold binding buffer to a concentration of 1×106 cells/mL, and then stained with 5 μl of Annexin V-PE and 5 μL of 7-AAD solution in the dark at room temperature. The results were observed via flow cytometry (Beckman Coulter, Inc.).
Measurement of lactate production and glucose consumption.
Lactate production and glucose consumption were measured as previously described . For this, 5×105 cells were cultured and incubated in 6-well plates for 10 h, after which the medium was discarded and the cells were incubated with a fresh medium for 8 h. The levels of lactate and glucose were detected using an automatic biochemical analyzer (HITACHI, Tokyo, Japan). Consistent amounts of protein were used to normalize the relative levels of lactate production and glucose consumption.
Cells and/or tissues were washed twice with PBS, and RIPA lysis buffer containing 10% protease inhibitor mixture (Roche, USA) was used to lyse cells and tissues. A bicinchoninic acid assay kit (Beyotime, Shanghai, China) was used to detect the protein concentration. For this, 30 μg of protein was separated on an SDS-PAGE gel, and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were blocked in 5% milk powder and then incubated overnight at 4°C with anti-FOXD1 (Omnimabs, OM203273), anti-LDHA (Ptgcn, 19987-1-AP), anti-PKM (Omnimabs, OM268791), anti-ENO1 (Ptgcn, 11204-1-AP), and anti-β-actin (Ptgcn, 66009-1-Ig). Membranes were incubated with secondary antibodies for 1 h. The signals of protein bands were visualized by enhanced chemiluminescence and normalized using β-actin as an internal reference.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) assays were performed to verify the location and expression of FOXD1-AS1 in NPC cells. Blue DAPI stain and red FISH Probe were purchased from RiboBio company (Guangzhou, China). A FISH kit (RiboBio, Guangzhou, China) was used to perform these assays in accordance with the manufacturer’s protocols.
Paraffin-embedded NPC tissues were sliced, dewaxed, hydrated, and antigen-repaired. Endogenous peroxidase was blocked; anti-FOXD1 (Omnimabs, OM203273), anti-Ki67 (Genetex, GTX16667), and anti-Caspase3 were added to these sections and incubated together at 4 °C overnight. Polymer enhancers were incubated for 20 min at room temperature, then biotin-labeled secondary antibodies were added and incubated for 30 min at room temperature. Next, a diaminobenzidine staining solution was used to stain the sections, followed by counterstaining with hematoxylin, and then the sections were mounted in glycerol-vinyl-alcohol (GVA Mount, Zymed). Two independent pathologists, who were blinded to the data and histopathological characteristics of the patients, evaluated the IHC scores according to the scoring standards as previously described.
RNA immunoprecipitation (RIP)
The EZ-Magna RIP Kit (Millipore, USA) was used to perform the RIP assay. In brief, CNE-1 and 5-8F cells were collected and incubated with RIP lysis buffer. Subsequently, anti-FOXD1, anti-SNRNP70 antibody (positive control), and normal rabbit IgG (negative control) were added to the cell lysates. The acid phenol/chloroform method was used to isolate and purify RNA, followed by qRT-PCR for the detection of FOXD1-AS1 and FOXD1. The primers used were as follows: U1 -F: 5′-GGGAGATACCATGATCACGAAGGT-3′; R: 5′-CCACAA ATTA TGCAGTCGAGTTTCCC-3′; FOXD1-AS1 -F: 5′-CATAAATAGAGGGACCCGCA- 3′; R: 5′-GGAAGAAGGTAGGAATCCCG-3′; FOXD1 -F: 5′-TGAGCACTGAGATG TCCGATG-3′; R: 5′-CACCACGTCGATGTCTGTTTC-3′.
Stability and α-amanitin treatment
CNE-1 and 5-8F cells stably expressing shRNA against FOXD1-AS1 or NC were seeded into 6-well culture plates. Cells were then treated with 5 μg/ml actinomycin D and were harvested at the indicated time points. Total RNA was extracted for cDNA synthesis and subjected to qRT-PCR after 0, 2, 4, 6, 8, and 12 h of treatment.
RNA pull-down assay
Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher, Rockford, IL, USA) was used to perform RNA pull-down assays. Biotinylated bio-NC and bio-FOXD1-AS1 were transfected into CNE-1 and 5-8F cells. After being transfected for 48 h, the cell lysates were mixed with magnetic beads to form protein-bio/RNA–magnetic bead complexes, and high-salt elution was used to obtain protein-bio/RNA complexes. Subsequently, western blotting was performed to measure the relative expression of FOXD1 in the protein-bio/RNA mixture.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as previously described . For this, protein-DNA complexes were obtained with the anti-FOXD1 antibody (1:50; Omnimabs, OM203273), followed by analysis of the resulting retrieved RNA subjected to qRT-PCR. The primers for qRT-PCR quantification were designed according to the promoter regions of FOXD1-AS1, lactate dehydrogenase A (LDHA), pyruvate kinase M2 (PKM) and enolase 1 (ENO1), which contain FOXD1 binding sites. The primer sequences used were as follows: FOXD1-AS1 -F: 5-ACAGATGGCGGAAAACAAA C-3; R: 5-GGATAAC CACCCCATCCTCT-3; LDHA -F: 5-TTCACTGTGAGTGGG AGCTG-3; R: 5-CTCAGGAAGGCTT GGATCTG-3; PKM -F: 5-CCCAGCTCTGC GCTAATATC-3; R: 5-GGCCGTTTTCCTCTTAGGAC-3; ENO1 -F: 5-ACGGAATA TGACCCGTCTTG-3; R: 5-CGTGCTTCCCCAGTGTTAAT-3; GAPDH -F: 5-TACT AGCGGTTTTACGGGCG-3; R: 5-TCGAACAGGAGGAGCAGAGAGCGA-3′.
Xenograft mouse model
Female BALB/c nude mice, aged 6 weeks (20±2 g), were purchased from the Laboratory Animal Center of Central South University (Changsha, China) and subjected to tumor implantation. CNE-1 and 5-8F cells (5×106 cells/ml, 0.2 ml) were transfected with either shRNA targeting FOXD1-AS1, shRNA targeting FOXD1-AS1+FOXD1 overexpression plasmids, or vector plasmids, and were then subcutaneously injected into the mice. On day 25, the mice were euthanized to harvest tumors, and the volume of the tumors was calculated using the formula V= (L×W²)/2. Tumors obtained from the mice were examined using routine hematoxylin and eosin (H&E) staining and photographed under a light microscope. All experimental procedures were approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (Changsha, China).
Data are presented as mean ± SD, and all experiments were repeated independently three times. GraphPad Prism 8.0 Software (San Diego, CA, USA) was used to analyze the data. Student’s t-tests or one-way ANOVA were used to analyze differences between groups. P < 0.05 (two-sided) was considered statistically significant.