A total of 30 NPC and 30 adjacent normal tissue samples (distance from tumor margin, 2 cm) were obtained from patients with NPC (age range, 33–67 years) at Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine (Zhangjiagang, China) between March 2016 and September 2019. The inclusion criteria were as follows: Patients were diagnosed as NPC with no history of other tumor. The exclusion criteria were as follows: i) diagnosed with other diseases; and ii) patients who had received preoperative radiotherapy, chemotherapy or other adjuvant treatments prior to admission. All fresh specimens were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. Written informed consent was obtained from each participant. The protocol of the present study was approved by the ethics committee of Zhangjiagang Hospital of Traditional Chinese Medicine. The clinical information of patients with NPC is presented in Table I.

Human NPC cell lines (C666-1, SUNE1, 5–8F and 6–10B) and nasopharyngeal epithelial cells (NP69) were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂.

Short hairpin RNA (shRNA) targeting FBXL19-AS1 (sh-FBXL19-AS1; 5′-GCAGGCCCAUCUACGGUGUGU-3′) and control (sh-NC; 5′-AAUUAGCCGCAUGCGUCACAU-3′), miR-431 mimics (5′-UGCAAUGUUAGAUGGUGUGAGG-3′), negative control (NC mimics; 5′-GACUCCUUACUCGCUCUACUG-3′), miR-431 inhibitor (5′-UUCACCGGAUCUGUCACGUAU-3′) and control (NC inhibitor; 5′-CAGUAGAGAUUGAAAGUUGUC-3′) were obtained from Shanghai GenePharma Co., Ltd. To establish FBXL19-AS1 or PBOV1-overexpression plasmids (pcDNA3.1-FBXL19-AS1 or pcDNA3.1-PBOV1), the full-length FBXL19-AS1 or PBOV1 sequence was inserted into pcDNA3.1 vector (Thermo Fisher Scientific, Inc.), with pcDNA3.1 as a control. Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection of C666-1 and SUNE1 cells (1×10⁵) with sh-FBXL19-AS1 (10 nM), sh-NC (10 nM), miR-431 mimics (10 nM), NC mimics (10 nM), miR-431 inhibitor (10 nM), NC inhibitor (10 nM) pcDNA3.1-FBXL19-AS1 (10 nM), pcDNA3.1-PBOV1 (10 nM) or pcDNA3.1 (10 nM) at room temperature for ~30 min. At 48 h post-transfection, subsequent experiments were performed.

Total RNA was extracted from NPC tissues and cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, cDNA was established from total RNA using the PrimeScript™ 1st strand cDNA Synthesis Kit (cat. no. 6110A; Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol. qPCR was performed using SYBR Green Taq ReadyMix (Sigma-Aldrich; Merck KGaA) on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 15 sec; 40 cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 20 sec and extension at 72°C for 40 sec. The expression of genes was detected using the 2^−ΔΔCq method (28). GAPDH or U6 were used as the internal controls for FBXL19-AS1 and PBOV1 or miR-431, respectively. The primer sequences were as follows: FBXL19-AS1 forward, 5′-GGTACAACTACGGATATGA-3′ and reverse, 5′-TACGTCTCGACCATTACGCA-3′; miR-431 forward, 5′-TGTCTTGCAGGCCGTCATG-3′ and reverse, 5′-GCTGTCAACGATACGCTACCTA-3′; PBOV1 forward, 5′-TGAGTCCCCTCTCGGTAATG-3′ and reverse, 5′-GCCCCGAGTTAAGAACATCA-3′; GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-TCCACCACCCTGTTGCTGTA-3′; and U6 forward, 5′-ATTGGAACGATACAGAGAAGATT-3′ and reverse, 5′-GGAACGCTTCACGAATTTG-3′.

Cell viability was examined using a CCK-8 assay (Dojindo Molecular Technologies, Inc.). The transfected C666-1 and SUNE1 cells (1×10⁵ cells/well) were plated into 96-well plates for 48 h. Then, CCK-8 reagent was added to each well at 0, 24, 48 and 72 h and incubated at 37°C for 4 h. The absorbance at 450 nm was detected by a microplate reader (Olympus Corporation).

The Transwell assay was performed using Transwell chambers (8.0-µm pore size; EMD Millipore). For the migration assay, transfected C666-1 and SUNE1 cells (1×10⁵) in serum-free culture RPMI-1640 medium (Thermo Fisher Scientific, Inc.) were added to the upper chamber. Subsequently, RPMI-1640 medium with 10% FBS was added to the lower chamber. After 24 h, 4% paraformaldehyde and 0.1% crystal violet solution was used to fix and stain the cells, respectively, that had migrated to the lower surface of the membrane for 20 min at room temperature, and the cells were counted under a light microscope (magnification, ×200). For cell invasion, the upper chamber was pre-coated with Matrigel for 1 h at room temperature. The remaining steps were consistent with those for the cell migration assay.

Starbase (version 2.0; starbase.sysu.edu.cn/) was used to predict the binding sites between FBXL19-AS1 and miR-431. TargetScan (www.targetscan.org/vert_72/) was used to predict the binding sites between miR-431 and PBOV1. The mutated 3′-UTR was generated using the QuikChange II Site Directed Mutagenesis kit (Agilent Technologies, Inc.). The 3′-untranslated region (UTR) of wild-type (WT) and mutant (Mut) FBXL19-AS1 (WT-FBXL19-AS1 and Mut-FBXL19-AS1) or PBOV1 (WT-PBOV1 and Mut-PBOV1) were inserted into the pmiR-GLO vector (Promega Corporation). Then, all plasmids (0.6 µg) were transfected into C666-1 and SUNE1 cells (1×10⁵) with 10 nM miR-431 mimics or 10 nM NC mimics using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). After 48 h, the luciferase activities were detected using the Dual-Luciferase Reporter System (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase gene activity.

A RIP assay was utilized with Magna RIP RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore). The transfected C666-1 and SUNE1 cells were dissolved in RIP lysis buffer (Beyotime Institute of Biotechnology), and then cell lysate was centrifuged at 40,000 × g at 4°C for 10 min and incubated with 50 µl magnetic beads bound with the 5 µg Ago2 antibody (cat. no. 2897; Cell Signaling Technology, Inc.). IgG (5 µg; cat. no. PP64B; EMD Millipore) was used as a control group. Then, immunoprecipitated RNA was detected via an RT-qPCR assay.

All experiments were repeated three times. All data were analyzed using GraphPad Prism 7 (GraphPad Software, Inc.) and presented as the mean ± SD. Paired/unpaired Student's t-test or one-way ANOVA, followed by Tukey's post hoc test were used to evaluate the differences. The correlation between FBXL19-AS1 and PBOV1 expression was assessed by a Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

To explore the function of FBXL19-AS1 and miR-431 (derived from 5′ of pre-miR-431) in NPC, RT-qPCR was performed to detect the levels of FBXL19-AS1 and miR-431 in NPC tissues. The data indicated that FBXL19-AS1 was increased and miR-431 was decreased in NPC tissues (n=30; Fig. 1A and B), and that FBXL19-AS1 expression was inversely correlated with miR-431 expression (Fig. 1C). Moreover, the levels of FBXL19-AS1 and miR-431 in NPC cell lines (C666-1, SUNE1, 5–8F and 6–10B) and NP69 cells were detected. Consistent with previous results, FBXL19-AS1 expression was upregulated in NPC cells, whereas miR-431 expression was downregulated, particularly in C666-1 and SUNE1 cell lines (Fig. 1D and E). These data suggested that FBXL19-AS1 may act as an oncogene and miR-431 as a tumor suppressor in the progression of NPC. Since C666-1 and SUNE1 exhibited the highest and lowest expression of FBXL19-AS1 and miR-431, respectively, these two cell lines were selected for subsequent experiments.

A growing body of evidence has demonstrated that lncRNAs can interact with miRNAs to modulate cancer development (29,30). In the present study, it was observed that FBXL19-AS1 acted as a molecular sponge for miR-431 (Fig. 2A). Moreover, the luciferase activity of FBXL19-AS1-WT was reduced by miR-431 mimics in C666-1 and SUNE1 cells, whereas the activity of FBXL19-AS1-Mut was not changed (Fig. 2B). Furthermore, the RIP assay revealed that FBXL19-AS1 and miR-431 were markedly enriched in the Ago2 group compared with the IgG group (Fig. 2C). To confirm whether FBXL19-AS1 could modulate the level of miR-431, pcDNA3.1-FBXL19-AS1 or sh-FBXL19-AS1 was transfected into C666-1 and SUNE1 cells to overexpress or suppress FBXL19-AS1, respectively (Fig. 2D), following which the expression of miR-431 was determined. The results indicated that overexpression of FBXL19-AS1 suppressed miR-431 expression and depletion of FBXL19-AS1 enhanced miR-431 expression (Fig. 2E). These results suggested that FBXL19-AS1 interacts with and negatively regulates the level of miR-431.

To further explore the function of FBXL19-AS1 in the development of NPC, C666-1 and SUNE1 cells were transfected with sh-FBXL19-AS1 or sh-NC. The CCK-8 assay indicated that the interference of FBXL19-AS1 led to the inhibition of C666-1 and SUNE1 cell viability (Fig. 3A). Moreover, FBXL19-AS1 depletion markedly suppressed the migration and invasion of NPC cells (Fig. 3B and C). Thus, FBXL19-AS1 knockdown may inhibit the occurrence of NPC.

To investigate whether pcDNA3.1-FBXL19-AS1 can abolish the inhibition of NPC progression mediated by miR-431 mimics, the expression of miR-431 was assessed in C666-1 and SUNE1 cells transfected with NC mimics, miR-431 mimics, miR-431 mimics + pcDNA3.1, and miR-431 mimics + pcDNA3.1-FBXL19-AS1. The results indicated that the addition of FBXL19-AS1 diminished miR-431 expression (Fig. 4A). Further studies revealed that the overexpression of miR-431 inhibited the viability, migration and invasion of C666-1 and SUNE1 cells, which was partially reversed by pcDNA3.1-FBXL19-AS1 transfection (Fig. 4B-D). These results suggested that the FBXL19-AS1/miR-431 axis may be implicated in NPC progression.

A putative miR-431 binding site located in the 3′-UTR of PBOV1 mRNA was predicted by TargetScan (Fig. 5A). As shown in Fig. 5B, miR-431 mimics led to a significant reduction in relative luciferase activity of PBOV1-WT, but not in PBOV1-Mut. miR-431 and PBOV1 were markedly enriched by Ago2, while IgG enrichment was not obvious (Fig. 5C). Next, RT-qPCR results showed that miR-431 expression was reduced by transfecting miR-431 inhibitor in NPC cells (Fig. 5D). Moreover, the overexpression of miR-431 inhibited PBOV1 expression and the knockdown of miR-431 enhanced PBOV1 expression (Fig. 5E). Furthermore, PBOV1 was markedly enhanced in NPC tissues compared with normal tissues (Fig. 5F). In addition, PBOV1 expression was negatively correlated with miR-431 expression (Fig. 5G). These results suggested that PBOV1 is a direct target of miR-431.

To further study the association between PBOV1 and miR-431 in NPC, the expression of PBOV1 was detected in C666-1 cells transfected with NC mimics, miR-431 mimics, miR-431 mimics + pcDNA3.1 and miR-431 mimics + pcDNA3.1-PBOV1. The transfection efficiency of PBOV1-overexpression plasmid was confirmed by observing the upregulation of PBOV1 in C666-1 cells (Fig. 6A). pcDNA3.1-PBOV1 partially reversed the suppressive effects of miR-431 mimics on PBOV1 expression (Fig. 6B). Moreover, the overexpression of miR-431 suppressed the viability, migration and invasion of C666-1 cells, which was counteracted following pcDNA3.1-PBOV1 transfection (Fig. 6C-E). FBXL19-AS1 expression was positively correlated with PBOV1 expression (Fig. 6F). Furthermore, C666-1 cells were treated with sh-NC, sh-FBXL19-AS1, sh-FBXL19-AS1 + NC inhibitor and sh-FBXL19-AS1 + miR-431 inhibitor, and the expression of PBOV1 was detected in transfected cells. The results demonstrated that FBXL19-AS1 knockdown decreased the expression of PBOV1, whereas these effects could be abolished by miR-431 inhibitor transfection in NPC cells (Fig. 6G). Taken together, the aforementioned results indicated that FBXL19-AS1 may drive the progression of NPC via modulation of the miR-431/PBOV1 axis.