Tissues collection
Fifty-five patients who were diagnosed with nasopharyngeal carcinoma in West China Hospital of Sichuan University were enrolled in our study. The ethics committee of West China Hospital of Sichuan University approved our study protocol. NPC tissues and adjacent healthy tissues were frozen until experiments and clinical characteristics were recorded. The collection and the use of tissues followed the ethical standards built by the Helsinki Declaration.
Cell culture
The human NPC cell lines SUNE-1, 5-8F, and C666-1 as well as normal nasal mucosal epithelial cell NP-69 were all bought from BeNa Culture Collection (Beijing, China). SUNE-1, 5-8F, and C666-1 were cultured in RPMI-1640 with 10% FBS.
RNA was detected by real-time quantification PCR.
Total RNA was disintegrated by Trizol (DP501, Tiangen Biochemical, China). The RNA was reverse transcribed into cDNA using a cDNA synthesis kit (KR211, Tiangen Biochemical, China) after checking the purity of RNA. Next, the expression of lncRNA ZFAS1, miR-7-5p and ENO2 mRNA were analyzed by ABI 7500 using a SYBR Green PCR kit (FP411, Tiangen Biochemical, China). U6 acted as a reference gene for miR-7-5p while GAPDH acted as a reference gene for lncRNA ZFAS1 and ENO2 mRNA.
Cell transfection
Si-ZFAS1-1, si-ZFAS1-2, miR-7-5p inhibitor, ENO2 siRNA (si-ENO2), and si-NC were designed and synthesized by Tiangen Biochemical (Beijing, China). Lipofectamine 2000 reagent (ThermoFisher, 11668027, USA) was used to transfect plasmids into target cells (SUNE-1 and C666-1 cell lines) on the basis of the protocol.
Cell fractionation
The Invitrogen PARIS Kit (ThermoFisher, AM1921, USA) was employed for separating and purifying cytoplasmic and nuclear RNA based on specifications. Expression levels of lncRNA ZFAS1, GAPDH (cytoplasmic control) and U6 (nuclear control) in cytoplasm and nucleus were examined using qRT-PCR.
Luciferase reporter assay
We purchased constructed plasmids containing wild type or mutant type of lncRNA ZFAS1 and ENO2 mRNA from Tiangen Biochemical (Beijing, China). Next, we transfected these plasmids into SUNE-1 and C666-1 cell lines by Lipofectamine 2000 reagent (ThermoFisher, 11668027, USA). Then, miR-892b mimics were co-transfected into the same cells by the same method. After 48 hours, we gathered cells to lyse by lysis buffer. The dual-luciferase reporter assay system (GeneCopoeia, LF031, China) was used to analyze the relative luciferase activity.
RNA pull-down assay
RNA pull-down experiment was performed as the instruction of the kit (ThermoFisher, 20164, USA). First, miR-7-5p, antisense oligo, and miR-7-5p mutant were labeled with biotin, which can bind to the streptavidin magnetic beads. Cell lysates were incubated with biotin-labeled miR-7-5p, antisense oligo, and miR-7-5p mutant. Only biotin labeled miR-7-5p could bind to the target in a RISC dependent manner. Then, the incubated lysate samples went through the streptavidin magnetic beads. The elusion was done using non-denaturing Biotin Elution Buffer or SDS-PAGE Loading Buffer. At last, qRT-PCR was undertaken to measure the expression of lncRNA ZFAS1 or miR-7-5p in the elution.
CCK-8 assay
CCK-8 kit was purchased from Dojindo Laboratories (Kumamoto, Japan) to determine the cell viability. In the irradiation dose-dependent assay, cell viability of every group was detected at 0Gy, 4Gy, and 8Gy doses of irradiation respectively. In the irradiation time-dependent assay, cell viability of every group was detected at 24h, 48h, 72h and 96h, respectively. 10 μL CCK-8 solution was added to every well to incubate with the cells for 2h. The absorbance of cells in every well was read at 450 nm using an ELISA plate reader.
Edu assay
The proliferation of NPC cells under irradiation was measured by Edu assay which was conducted using Edu Apollo DNA in vitro kit bought from RIBOBIO (C10341-3, Guangzhou, China). All manipulates were done following the protocol from the kit. Three random fields under a fluorescence microscope were selected and the Edu positive cells were counted. The Edu positive rate (=Edu positive cell number/DAPI positive cell number) represented the proliferation condition of the cells. Representative images of every group were given.
Flow cytometric apoptosis assay
Flow cytometry was employed for the apoptosis detection. Briefly, cell suspension with 104 cells were put in every tube, and went through centrifugation to lose the culture media. The cells were then washed with cold PBS twice. Then, the cold PBS was removed. 100 µl 1× binding buffer was added to the cells and the cells were re-suspended. 5 µl Annexin V and 5 µl PI (Beyotime, Beijing, China) were added to the cells in dark and incubated for 15 min. 300 µl 1× binding buffer was added to the cells and the cells were re-suspended again. The cell suspension was moved to 5 ml flow tubes. Within the next one hour, the cells went through flow cytometry analysis.
Statistical analyses
All the data were analyzed using Graphpad Prism 8.0. All results represented three independent experiments. Values were expressed as mean±SD. Two-tailed t-test was used for analyzing differences between two groups, while one-way ANOVA with Dunnett’s post hoc test was used for analyzing differences in multiple groups. P-values less than 0.05 were regarded as statistically significant.