Cell lines and cell culture
The human immortalized normal nasopharyngeal epithelial cell line NP69 and NPC cell lines (HNE1, HONE1) were purchased from the Advanced Research Center of Central South University (Changsha, Hunan, China). NPC cells were incubated in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) and NP69 cells were maintained in K-SFM medium (Invitrogen, Carlsbad, CA, USA), which were supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin–streptomycin (Gibco; Thermo Fisher Scientific, Inc.) in an incubator at 37 °C with 5% CO2.
684 newly diagnosed NPC patients and 823 cancer-free healthy control subjects were recruited from Hunan Provincial Cancer Hospital and Xiangya Hospital between 2015 and 2016. 3 ml of peripheral venous blood of each participant was collected and stored at − 80 °C. 92 pathologically confirmed NPC tissue biopsies and 10 chronic inflammation of nasopharyngeal mucosa tissue biopsies were collected from Hunan Provincial Cancer Hospital between 2015 and 2016. None of the patients have received any anticancer treatments before joining in the trial. Patients who had other malignancy or concomitant malignant diseases were excluded. All of the patients received intensity-modulated radiotherapy (IMRT) technique and were treated with platinum-based induction chemotherapy (IC) plus concurrent chemoradiotherapy (CCRT) regimen. The short-term efficacy of chemoradiotherapy was evaluated using the Response Evaluation Criteria in Solid Tumor (RECIST) guidelines according to the magnetic resonance imaging (MRI) 3 months after treatment. The survival status of patients was followed up by telephone once every 3 months.
This study was approved by the Independent Ethical Committee of Institute of Clinical Pharmacology, Central South University (CTXY-140007-2). All the participants signed informed consent at the time of enrollment. This study was performed in accordance with the Declaration of Helsinki.
DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood lymphocytes and NPC tissues using the QIAamp DNA Mini and Blood Mini Kit (Qiagen Inc., Valencia, CA, USA). The concentrations and purity of DNA were measured using NanoDrop TM 1000 spectrophotometer. Genotype of the candidate SNP was determined by Sequenom MassARRAY iPLEX (Sequenom, Inc., San Diego, CA, USA). The call rate threshold was at least 95%.
RNA isolation and real-time PCR
Total RNA was isolated from tissues or cells using RNAiso Plus reagent (Takara, Japan) and reverse-transcribed to complementary DNA using a PrimeScript RT Reagent kit (Takara, Japan). qPCR was performed using SYBR® Premix ExTaq™ (Takara, Japan) according to the manufacturer’s instructions. The primer sequences were as follows: GAPDH-F: 5′-ACAACTTTGGTATCGTGGAAGG-3′, GAPDH-R: 5′- GCCATCACGCCACAGTTTC-3′; FAS-AS1-F: 5′-GCGTGTGTGTGTGTATTCTTTC-3’, FAS-AS1-R: 5′-AGCTTGGAGCTATGCTTGTT-3′; FAS-F: 5′-CAAGGGATTGGAATTGAGGA-3′, FAS-R: 5′-CTGGAGGACAGGGCTTATGG-3′. The 2−ΔΔCt method was used to calculate the relative expression levels of target mRNA.
LncRNASNP and RegulomeDB was used to select functional SNP in FAS-AS1 and identify DNA features and regulatory elements at rs6586163. In silico prediction of the folding structure and mountain plot of FAS-AS1 rs6586163 wild type and mutant type were computed by RNAfold WebServer. The eQTL data of rs6586163 was downloaded from Ensembl. GO enrichment analysis was performed using the online The Gene Ontology Resource (http://geneontology.org/). KM Plotter (http://kmplot.com/analysis/index.php?p=background) was applied to evaluate the prognosis role of FAS-AS1 in other types of cancers. The PPI network of FAS-AS1 regulated mitochondria related genes were constructed with String database and analyzed by Cytoscape 3.9.1 software. The hub gene of the network was analyzed by cytoHubba tools.
Plasmid construction and cell transfection
The full length of human FAS-AS1 cDNA was directly synthesized by Genechem Company (Genechem, Shanghai, China) and cloned into pcDNA 3.1 plasmid to construct the FAS-AS1 overexpression vector. A length of 500 nt base covering wild-type of FAS-AS1 rs6586163 was directly synthesized by Genechem (Genechem, Shanghai, China) and cloned into KpnI/XhoI site of GV148 vector to get the wild-type luciferase reporter vector (WT). Site-directed mutagenesis at rs6586163 was utilized to get the mutant-type luciferase reporter vector (MUT). DNA sequencing was utilized to confirm the sequence of these vectors. Transfection was carried out using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA).
Dual luciferase reporter assay
The cells were co-transfected with Renilla vector and FAS-AS1-WT vector or FAS-AS1-MUT vector or control vector for 48 h. Then, cells were harvested and luciferase activities were assayed using the Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity in order to correct the transfection efficiency.
HNE1 cells were transfected with FAS-AS1 overexpression vector or control vector for 24 h. Then, total RNA was extracted and the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit. The transcriptome was sequenced by OE Biotech Co., Ltd. (Shanghai, China) using an Ilumina Novaseq 6000 platform. Q value < 0.05 and fold change > 2 was set as the threshold for significantly differential expression gene (DEGs). Gene Set Enrichment Analysis (GSEA) was performed using GSEA software. The heatmap and KEGG enrichment analysis was generated by online platform for data analysis and visualization named Metascape (https://metascape.org/gp/index.html#/main/step1) and Bioinformatics (http://www.bioinformatics.com.cn/).
CCK-8 cell viability assay
The cells transfected with FAS-AS1 overexpression vector or control vector were seeded into 96-well plates at a density of 2000 cells/well. After being cultured for 24 h, 48 h, 72 h, 96 h, the cell culture medium was removed, and 100 µL cell culture medium containing 10 µL CCK-8 reagent (BioGLP, Montclair, CA, USA) was added into each well. Then, the cells were incubated in the dark for 1 h at 37 °C. The absorbance was measured at 450 nm using a spectrophotometer for analyzing cell viability.
CellTiter luminescent cell viability assay
NPC cells were seeded into 96-well plate and transfected with FAS-AS1 overexpression vector or control vector. The next day, take out the cell culture plate and balance it at room temperature for 10 min. Add 100 μL CellTiter-Lumi to each well and shake it at room temperature for 2 min to lysis cell. Then, incubate the cells at room temperature for 10 min to stabilize the luminous signal. Chemiluminescence was detected by a multifunctional microplate reader.
Cell cycle progression
The cells were transfected with FAS-AS1 overexpression vector or control vector for 24 h. Then, they were carefully harvested, resuspended in pre-cooled 70% ethanol, and stored at − 20 °C overnight. After centrifugation and washing by PBS, the cells were resuspended in 0.5 mL propidium staining buffer (Beyotime, China) containing RNase and incubated at 37 °C for 30 min. Finally, cell cycle analysis was performed using BD FACSDiva and FlowJo software.
Mito-Tracker red CMXRos staining
NPC cells were seeded into 24-well plate and transfected with FAS-AS1 overexpression vector or control vector. 24 h after transfection, remove the cell culture medium, add Mito Tracker Red CMXRos working solution, and incubate at 37 °C for 15–30 min. Then, remove Mito Tracker Red CMXRos working solution, and add fresh cell culture medium. Observe and capture representative images with fluorescence microscope.
Annexin V-FITC cell apoptosis assay
NPC cells were seeded into 6-well plate and transfected with FAS-AS1 overexpression vector or control vector for 48 h. Then, the cells were harvested and resuspended in the binding buffer and stained with FITC-Annexin V (AV) and propidium iodide (PI) according to the protocols (Beyotime, China). Cell apoptosis was immediately analyzed with a flow cytometry (BD Biosciences, San Jose, CA, USA).
NPC cells were seeded into 24-well plate and transfected with FAS-AS1 overexpression vector or control vector. 24 h after transfection, the cells were washed by PBS, fixed by paraformaldehyde for 10 min, and stained with Hoechst33342 (Beyotime, China) for 15 min in darkness. Then, remove the staining solution and wash the cells with PBS. The EVOS M7000 Imaging System was used to capture representative images.
NPC cells were seeded into 24-well plate and transfected with FAS-AS1 overexpression vector or control vector. 24 h after transfection, the cells were washed by PBS and co-stained with PI and SYTO 9 (Invitrogen, Carlsbad, CA, USA) for 15 min in darkness. SYTO 9 can enter all cells regardless of their membrane integrity, bind to DNA and RNA and emit green fluorescence while PI can only enter cells with compromised membranes, bind to DNA and RNA and emit a red fluorescent signal. Then, remove the staining solution and wash the cells with PBS. The EVOS M7000 Imaging System was used to capture representative images.
Transmission electron microscopy (TEM)
The morphology and ultrastructure of mitochondrial in NPC cells were captured by TEM. Cells were fixed in TEM fixative at 4 °C for 4 h and pre-embedded in 1% agarose. The samples were post fixed with 1% OsO4 in 0.1 MPB (pH 7.4) for 2 h at room temperature and dehydrated using a gradient series of ethyl alcohol. Samples were then embedded in Embed 812 resin and cut to 60–80 nm thin on the ultra microtome. The sections were stained with 2% uranyl acetate and lead citrate and observed using a transmission electron microscope (Hitachi, Japan).
NPC cells were transfected with FAS-AS1 overexpression vector or control vector using Lipofectamine 3000. After 48 h, the cells were collected and lysed in RIPA buffer (Beyotime, China) with 1% PMSF and phosphatase inhibitor cocktail on ice for 30 min. The concentration of protein was detected by BCA kit (Beyotime, China). A total of 10 μg protein was loaded into a SDS-PAGE gel and separated by electrophoresis. The blots were transferred to PVDF membranes at 250 mA for 90 min. The membranes were blocked with 5% skimmed milk for 1 h and incubated with primary antibodies against cleaved caspase 3 (cat: A11021, ABclonal), bax (cat: A0207, ABclonal), bcl2 (cat: A19693, ABclonal) and β-Actin (cat: AC026, ABclonal) at 4 °C overnight. Then, the blots were incubated with secondary antibodies at room temperature for 1 h. After washing three times with TBST, the blots were detected by ECL kit and visualized by ChemiDoc MP Imaging System.
Multivariate logistic regression analysis was used to assess the association between the selected SNP and NPC susceptibility. Kaplan–Meier analysis and Cox proportional hazards regression analysis were applied to evaluate the overall survival of NPC patients. All in vitro experiments were performed at least three times and the data were represented as means ± standard deviations. A two-tailed Student’s t-test was used to compare significant difference between two groups. Statistical analyses were carried out using SPSS software (SPSS, Chicago, IL, USA) and GraphPad Prism software (GraphPad, San Diego, CA, USA). P value < 0.05 was considered as statistically significant, ns means no significance, *P < 0.05, **P < 0.01.