Long non‑coding RNA KCNQ1OT1 promotes nasopharyngeal carcinoma cell cisplatin resistance via the miR‑454/USP47 axis.

Long non‑coding RNAs serve an essential role in drug resistance in various types of cancer, including lung, breast and bladder cancer. The present study aimed to investigate whether KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) was associated with cisplatin (DDP) resistance in nasopharyngeal carcinoma (NPC). KCNQ1OT1, microRNA (miR)‑454 and ubiquitin specific peptidase 47 (USP47) expression levels were measured via reverse transcription‑quantitative PCR. 5‑8F/DDP and SUNE‑1/DDP cell viability and chemosensitivity were assessed by performing Cell Counting Kit‑8 assays. Colony forming and Transwell assays were conducted to assess the effect of the KCNQ1OT1/miR‑454/USP47 axis on DDP resistance in NPC cells. The association between miR‑454 and KCNQ1OT1 or USP47 was verified via bioinformatics analysis, dual‑luciferase reporter assays and RIP assays. KCNQ1OT1 and USP47 expression levels were significantly upregulated, whereas miR‑454 expression levels were significantly downregulated in DDP‑resistant NPC cells compared with parental NPC cells. KCNQ1OT1 knockdown promoted chemosensitivity in DDP‑resistant NPC cells (5‑8F/DDP and SUNE‑1/DDP), as indicated by significantly decreased cell proliferation, migration and invasion in the short hairpin RNA (sh)KCNQ1OT1 group compared with the sh‑negative control (NC) group. Moreover, miR‑454 was identified as a target of KCNQ1OT1. KCNQ1OT1 overexpression significantly reversed miR‑454 overexpression‑mediated effects on NPC cell viability and DDP resistance. Furthermore, the results indicated that miR‑454 directly targeted USP47. Compared with the shNC group, USP47 knockdown significantly suppressed NPC cell viability and DDP resistance, which was significantly reversed by co‑transfection with miR‑454 inhibitor. Furthermore, compared with the shNC group, KCNQ1OT1 knockdown significantly downregulated USP47 expression, which was significantly counteracted by miR‑454 knockdown. Collectively, the results of the present study indicated that KCNQ1OT1 enhanced DDP resistance in NPC cells via the miR‑454/USP47 axis, suggesting a potential therapeutic target for patients with DDP‑resistant NPC.

Introduction

Nasopharyngeal carcinoma (NPC) is one of the most common types of head and neck tumor (1,2). The incidence of NPC varies significantly worldwide, but is primarily prevalent in East Africa, North Africa, East Asia and Southeast Asia (3). At present, platinum chemotherapy is a widely used treatment strategy for NPC, and cisplatin (DDP) is a common platinum compound that has been reported to be effective in the treatment of cancer (4). However, drug resistance often leads to the failure of NPC chemotherapy (5). Therefore, understanding the mechanism underlying DDP resistance in NPC might aid with improving the prognosis of patients with NPC.

Long non-coding RNAs (lncRNAs), a class of transcripts >200 nucleotides in length, lack protein-coding capacity (6) and serve as regulatory factors in multiple biological processes, including apoptosis, metabolism and cell proliferation (7,8). Increasing evidence has demonstrated that lncRNAs confer chemoresistance in various types of cancer, such as renal and hepatocellular cancer (9,10). Several lncRNAs serve a vital role in drug resistance of NPC. For instance, lncRNA MAGI2 antisense RNA 3 conferred DDP resistance in NPC cells via regulating the microRNA (miRNA/miR)-218-5p/glycerophosphodiester phosphodiesterase domain containing 5 axis (11). lncRNA nuclear paraspeckle assembly transcript 1 promoted DDP resistance in NPC cells via the let-7a-5p/remodeling and spacing factor 1 axis (12). Testis associated oncogenic lncRNA knockdown inhibited tumorigenicity and attenuated DDP resistance in NPC cells (13). However, the function of KCNQ1OT1 in mediating chemoresistance in NPC is not completely understood.

miRNAs, small non-coding RNAs that are 20-22 nucleotides in length, serve important roles in multiple cellular processes, including cell proliferation and survival, of human tumors (14). Previous studies have revealed that dysregulated miRNAs could mediate the sensitivity of NPC cells to DDP by targeting mRNAs. For example, miR-19b served an important role in inhibiting cancer progression and promoting NPC sensitivity to DDP via inhibiting KRAS (15). Furthermore, miR-205-5p facilitated cell proliferation and DDP resistance in NPC by repressing PTEN (16). miR-139 overexpression suppressed DDP-induced progression and enhanced apoptosis in NPC cells (17). Nevertheless, the mechanism underlying miR-139 in tumorigenesis and chemosensitivity in NPC is not completely understood.

The present study investigated the regulatory role of KCNQ1OT1 in DDP resistance of NPC, and the results of the present study might provide a novel therapeutic strategy for DDP-resistant NPC.

Materials and methods

Patients

A total of 50 patients (31 male patients and 19 female patients; age range, 26-65 years; mean age, 46 years) with NPC (29 DDP-resistant and 21 DDP-sensitive) at Zhuji Central Hospital (Zhuji, China) were recruited between August 2017 and December 2019. The inclusion criteria were as follows: i) Diagnosed with NPC; and ii) had not received preoperative radiotherapy, chemotherapy or other adjuvant treatments. The exclusion criteria were as follows: i) Diagnosed with other diseases; and ii) failed to cooperate with researchers. All specimens were immediately frozen in liquid nitrogen and stored at -80°C. The present study was approved by the Ethical Committee of Zhuji Central Hospital. Written consent was obtained from all patients prior to starting the study.

Cell lines and culture

NPC cell lines (5-8F and SUNE-1) were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. 5-8F and SUNE-1 cells were cultured in RPMI-1640 (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific). To establish DDP-resistant NPC cells (5-8F/DDP and SUNE-1/DDP), cells were treated with 0.5 µg/ml DDP (Qilu Pharmaceutical Co., Ltd.) at 37°C for 3 weeks and then exposed to gradually increasing concentrations of DDP (2, 4, 6 or 8 µg/ml) every 3 weeks up to a final concentration of 10 µg/ml for a total 15 weeks. 5-8F/DDP and SUNE-1/DDP cells were cultured in RPMI-1640 supplemented with 5 µg/ml DDP and 10% FBS. All cells were cultured at 37°C with 5% CO2.

Cell transfection

Short hairpin (sh)RNA targeting KCNQ1OT1 (shKCNQ1OT1; 0.8 µg; 5′-GCA GAA CCA UCG AUG GUG CGU-3′), shRNA targeting USP47 (shUSP47; 0.8 µg; 5′-GCC UUU GCA GAC UCU CAU UUA-3′), shRNA-negative control (NC; shNC; 0.8 µg; scrambled; 5′-AGU GCU GCG CAC GUG UCU CAU-3′), miR-454 mimics (100 nM; 5′-UAG UGC AAU AUU GCU UAU AGG GU-3′), NC mimics (100 nM; 5′-UUG UAC UAC ACA AAA GUA CUG-3′), miR-454 inhibitor (100 nM; 5′-ACC CUA UAA GCA AUA UUG CAC UA-3′) and NC inhibitor (100 nM; 5′-CAG UAC UUU UGU GUA GUACAA-3′) were purchased from Shanghai GenePharma Co., Ltd. To overexpress KCNQ1OT1 or USP47, the full-length KCNQ1OT1 sequence was inserted into the pcDNA3.1 vector (Shanghai GenePharma Co., Ltd.). 5-8F/DDP and SUNE-1/DDP cells (1×105) were transfected with shRNA, miR-mimics, miR-inhibitors, overexpression vectors and the corresponding NCs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. At 48 h post-transfection, subsequent experiments were performed.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from tissues and cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. Subsequently, qPCR was performed using SYBR® Premix Ex Taq™ II (Takara Bio, Inc.) on the ABI 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 following primers were used for qPCR: KCNQ1OT1 forward, 5′-TTG GTA GGA TTT TGT TGA GG-3′ and reverse, 5′-CAA CCT TCC CCT ACT ACC-3′; miR-454 forward, 5′-TAG TGC AAU ATT GCT TAU AGG GT-3′ and reverse, 5′-CCU AUA AGC AAU ATT GCA CTA TT-3′; USP47 forward, 5′-GGC AGG ACG CTC ATT AGG T-3′ and reverse, 5′-GCA CAA CAT GAT TCC AAG TCA A-3′; GAPDH forward, 5′-TCA AGG CTG AGA ACG GGA AG-3′ and reverse, 5′-TGG ACT CCA CGA CGT ACT CA-3′; and U6 forward, 5′-CTC GCT TCG GCA GCA CAT ATA CT-3′ and reverse, 5′-CGC TTC ACG AAT TTG CGT GT-3′. miRNA and mRNA expression levels were quantified using the 2−ΔΔCq method (18) and normalized to the internal reference genes U6 and GAPDH, respectively.

Colony forming assay

Transfected 5-8F/DDP and SUNE-1/DDP cells were trypsinized and seeded (1×103 cells/well) into 6-well plates. Subsequently, cells were cultured in RPMI-1640 supplemented with 10% FBS for 2-3 weeks. Visible colonies were fixed in 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature (Sigma-Aldrich; Merck KGaA). Stained colonies (>30 cells) were counted using a light microscope (magnification, ×40) and the colony formation rate was calculated.

Transwell assay

Transwell chambers (pore size, 8 µm; Corning, Inc.) were used to perform the Transwell assay. To assess cell migration, 5-8F/DDP and SUNE-1/DDP cells (1×105 cells/well) in serum-free RPMI-1640 were plated into the upper chamber. Medium supplemented with 10% FBS was plated into the lower chamber. Following incubation for 24 h at 37°C, migratory cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet at 37°C for 2 h. Stained cells were counted in five randomly selected fields of view using a light microscope (magnification, ×200). To assess cell invasion, the upper chambers were precoated with Matrigel for 30 min at 37°C and then the aforementioned protocol was performed.

Cell Counting Kit-8 (CCK-8) assay

Cells were seeded (5×103 cells/well) into 96-well plates. Cells were treated with different concentrations of DDP (0, 2, 4, 6, 8 or 10 µg/ml) for 24 h at 37°C. Subsequently, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and incubated for 2 h at 37°C with 5% CO2. Absorbance was measured at a wavelength of 450 nm using a microplate reader. IC50 values were calculated as the concentration of DDP resulting in 50% inhibition of cell viability, with higher IC50 values suggesting higher drug resistance potential.

Western blotting

Total protein was extracted from cells and tissues using RIPA buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Proteins (10 µg) were separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). After blocking with 5% non-fat dry milk at room temperature for 2 h, the membranes were incubated overnight at 4°C with primary antibodies targeted against: USP47 (1:1,000; cat. no. ab72143; Abcam) and GAPDH (1:1,000; cat. no. ab9485; Abcam). Subsequently, the membranes were incubated with a HRP-conjugated secondary antibody (1:1,000; cat. no. ab6721; Abcam) for 1.5 h at 37°C. Protein bands were visualized using an enhanced-chemiluminescence reagent (Beyotime Institute of Biotechnology). Protein expression levels were semi-quantified using Image Lab software (version 4.1; Bio-Rad Laboratories, Inc.) with GAPDH as the loading control.

Luciferase reporter assay

StarBase (starbase.sysu.edu.cn/) and TargetScan (www.targetscan.org/vert_72/) were used to predict the binding sites between miR-454 and KCNQ1OT1 or USP47. pmirGLO-KCNQ1OT1-wild-type (WT)/mutant (Mut) and pmirGLO-USP47-WT/Mut reporter plasmids were provided by Shanghai GenePharma Co., Ltd. 5-8F/DDP and SUNE-1/DDP cells (1×105) were co-transfected with 0.6 µg pmirGLO-KCNQ1OT1-Wt/Mut or pmirGLO-USP47-Wt/Mut plasmid and 100 nM NC mimics or miR-454 mimics using Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. At 48 h post-transfection, luciferase activities were assessed using the dual-luciferase reporter assay system (Promega Corporation). Firefly luciferase activities were normalized to Renilla luciferase activities.

RNA immunoprecipitation (RIP) assay

RIP assays were performed to investigate whether KCNQ1OT1 and miR-454 were in the same RNA-induced silencing complex. The RIP assay was performed using the Magna RIP RNA-binding protein immunoprecipitation kit (EMD Millipore). 5-8F/DDP and SUNE-1/DDP cells were lysed using RIP lysis buffer (Beyotime Institute of Biotechnology) and incubated with 50 µl A/G magnetic beads conjugated with argonaute RISC catalytic component 2 (Ago2) (EMD Millipore) and IgG (EMD Millipore). Subsequently, immunoprecipitated RNA was extracted. KCNQ1OT1 and miR-454 expression levels were measured via RT-qPCR.

Statistical analysis

Statistical analyses were performed using SPSS software (version 16.0; SPSS, Inc.). Data are presented as the mean ± SD. Each experiment was performed in triplicate. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. Comparisons between two groups were analyzed using an unpaired Student's t-test. Pearson's correlation analysis was used to evaluate the correlation between the expression levels of miR-454 and KCNQ1OT1 or USP47. P<0.05 was considered to indicate a statistically significant difference.

Results

High expression of KCNQ1OT1 in DDP-resistant NPC tissues and cells

The RT-qPCR results demonstrated that KCNQ1OT1 expression was significantly upregulated in the 29 DDP-resistant NPC tissues compared with the 21 DDP-sensitive NPC tissues (Fig. 1A). Subsequently, DDP-resistant NPC cells (5-8F/DDP and SUNE-1/DDP) were established. Cell viability and the IC50 values of DDP in 5-8F/DDP and SUNE-1/DDP cells were significantly higher compared with 5-8F and SUNE-1 cells, respectively (Fig. 1B and C). Furthermore, KCNQ1OT1 expression levels were significantly upregulated in 5-8F/DDP and SUNE-1/DDP cells compared with parental NPC cells (Fig. 1D). The results indicated that KCNQ1OT1 might be involved in DDP sensitivity and resistance in NPC cells.

Figure 1

High expression of KCNQ1OT1 in DDP-resistant NPC tissues and cells. (A) KCNQ1OT1 expression levels in DDP-sensitive and DDP-resistant NPC tissues. Cell Counting Kit-8 assays were performed to detect cell viability and calculate the IC50 values of DDP in (B) 5-8F/DDP, 5-8F, (C) SUNE-1/DDP and SUNE-1 cells. (D) KCNQ1OT1 expression levels in 5-8F/DDP, 5-8F, SUNE-1/DDP and SUNE-1 cells. Data are presented as the mean ± SD. *P<0.05. KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; DDP, cisplatin; NPC, nasopharyngeal carcinoma; RT-qPCR, reverse transcription-quantitative PCR; Cis, cisplatin; OD, optical density.

KCNQ1OT1 knockdown increases DDP sensitivity in NPC cells

To explore the effect of KCNQ1OT1 knockdown on DDP resistance in NPC cells, shKCNQ1OT1 was transfected into 5-8F/DDP and SUNE-1/DDP cells to knock down KCNQ1OT1 expression (Fig. 2A). Compared with the shNC group, KCNQ1OT1 knockdown significantly decreased the IC50 value of DDP in 5-8F/DDP and SUNE-1/DDP cells (Fig. 2B). The CCK-8 assay results indicated that KCNQ1OT1 knockdown also significantly attenuated 5-8F/DDP and SUNE-1/DDP cell viability compared with the shNC group (Fig. 2C). In addition, the colony forming and Transwell assay results indicated that 5-8F/DDP and SUNE-1/DDP cell proliferation, migration and invasion were significantly inhibited by KCNQ1OT1 knockdown compared with the shNC group (Fig. 2D-F). Therefore, the results indicated that KCNQ1OT1 knockdown decreased DDP resistance in NPC cells.

Figure 2

KCNQ1OT1 knockdown increases nasopharyngeal carcinoma cell DDP sensitivity. (A) Transfection efficiency of shKCNQ1OT1 in 5-8F/DDP and SUNE-1/DDP cells. (B) CCK-8 assays were performed to detect cell viability and calculate the IC50 values of DDP in 5-8F/DDP and SUNE-1/DDP cells transfected with shNC or shKCNQ1OT1. (C) CCK-8 assays were performed to detect the cell viability of 5-8F/DDP and SUNE-1/DDP cells transfected with shKCNQ1OT1. (D) Colony forming (magnification, ×40), (E) Transwell migration and (F) Transwell invasion assays were performed to assess cell proliferation, migration and invasion in 5-8F/DDP and SUNE-1/DDP cells transfected with shNC or shKCNQ1OT1 (scale bar, 100 µm). Data are presented as the mean ± SD. *P<0.05. KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; DDP, cisplatin; sh, short hairpin RNA; CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density.

KCNQ1OT1 directly interacts with miR-454

Increasing evidence has suggested that lncRNAs mediate their effects via competitively binding to miRNAs (19-22). Therefore, star-Base was used to predict the miRNA targets of KCNQ1OT1. The results suggested that KCNQ1OT1 contained binding sites complementary to miR-454 (Fig. 3A). To verify the interaction, luciferase reporter and RIP assays were conducted. Compared with the corresponding NC groups, miR-454 mimics and miR-454 inhibitor significantly reduced and increased the luciferase activity of the KCNQ1OT1-WT reporter in 5-8F/DDP and SUNE-1/DDP cells, respectively, whereas the luciferase activity of the KCNQ1OT1-Mut reporter was not significantly altered (Fig. 3B). Moreover, the RIP assay results indicated that KCNQ1OT1 and miR-454 expression levels were significantly enriched in Ago2 compared with IgG (Fig. 3C). miR-454 expression was significantly higher in DDP-sensitive NPC tissues compared with DDP-resistant NPC tissues (Fig. 3D). Similarly, miR-454 expression was significantly downregulated in 5-8F/DDP and SUNE-1/DDP cells compared with parental NPC cells (Fig. 3E). The expression levels of KCNQ1OT1 were negatively correlated with miR-454 expression levels in NPC tissues (Fig. 3F). To explore whether KCNQ1OT1 regulated miR-454 expression, pcDNA3.1-KCNQ1OT1 and shKC-NQ1OT1 were transfected into 5-8F/DDP and SUNE-1/DDP cells to overexpress or knock down KCNQ1OT1 expression, respectively (Fig. 3G), and then miR-454 expression levels were measured. The results demonstrated that KCNQ1OT1 overexpression significantly downregulated miR-454 expression compared with the pcDNA3.1 group, whereas KCNQ1OT1 knockdown significantly increased miR-454 expression levels compared with the shNC group (Fig. 3H). Collectively, the results indicated that KCNQ1OT1 served as a molecular sponge for miR-454.

Figure 3

KCNQ1OT1 directly interacts with miR-454. (A) Binding sites between miR-454 and KCNQ1OT1. (B) Luciferase activity of KCNQ1OT1-WT and KCNQ1OT1-Mut in 5-8F/DDP and SUNE-1/DDP cells following transfection with NC mimics, miR-454 mimics, NC inhibitor or miR-454 inhibitor. (C) RNA immunoprecipitation assays were performed to assess the interaction between KCNQ1OT1 and miR-454. (D) miR-454 expression levels in DDP-sensitive and DDP-resistant NPC tissues. (E) miR-454 expression levels in 5-8F/DDP, 5-8F, SUNE-1/DDP and SUNE-1 cells. (F) Correlation between KCNQ1OT1 expression and miR-454 expression in NPC tissues. (G) Transfection efficiency of pcDNA3.1-KCNQ1OT1 and shKCNQ1OT1. (H) miR-454 expression levels in 5-8F/DDP and SUNE-1/DDP cells transfected with pcDNA3.1, pcDNA3.1-KCNQ1OT1, shNC or shKCNQ1OT1. Data are presented as the mean ± SD. *P<0.05. KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; miR, microRNA; WT, wild-type; Mut, mutant; NC, negative control; DDP, cisplatin; NPC, nasopharyngeal carcinoma; sh, short hairpin RNA; Ago2, argonaute RISC catalytic component 2; Cis, cisplatin.

KCNQ1OT1 modulates the sensitivity of DDP-resistant NPC cells by downregulating miR-454

To further investigate whether KCNQ1OT1 exerted its function in the DDP resistance of NPC cells by regulating miR-454 expression, miR-454 was overexpressed in 5-8F/DDP and SUNE-1/DDP cells by transfection with miR-454 mimics (Fig. 4A). Subsequently, 5-8F/DDP and SUNE-1/DDP cells were transfected with NC mimics, miR-454 mimics, miR-454 mimics + pcDNA3.1 or miR-454 mimics + pcDNA3.1-KCNQ1OT1. The CCK-8 assay results indicated that compared with the NC mimics group, miR-454 overexpression significantly suppressed cell viability, which was significantly reversed by co-transfection with pcDNA3.1-KCNQ1OT1 (Fig. 4B). Furthermore, the colony forming and Transwell assay results demonstrated that pcDNA3.1-KCNQ1OT1 significantly reversed miR-454 overexpression-induced inhibition of 5-8F/DDP and SUNE-1/DDP cell proliferation, migration and invasion (Fig. 4C-E). Collectively, the results indicated that KCNQ1OT1 enhanced DDP resistance in NPC cells by downregulating miR-454 expression.

Figure 4

KCNQ1OT1 modulates the sensitivity of DDP-resistant nasopharyngeal carcinoma cells by downregulating miR-454. (A) Transfection efficiency of miR-454 mimics. (B) Cell Counting Kit-8 assays were performed to detected the cell viability of 5-8F/DDP and SUNE-1/DDP cells transfected with NC mimics, miR-454 mimics, miR-454 mimics + pcDNA3.1 or miR-454+ pcDNA3.1-KCNQ1OT1. (C) Colony forming (magnification, ×40), (D) Transwell migration and (E) Transwell invasion assays were performed to assess cell proliferation, migration and invasion in 5-8F/DDP and SUNE-1/DDP cells transfected with NC mimics, miR-454 mimics, miR-454 mimics + pcDNA3.1 or miR-454+ pcDNA3.1-KCNQ1OT1 (scale bar, 100 µm). Data are presented as the mean ± SD. *P<0.05. KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; DDP, cisplatin; miR, microRNA; NC, negative control; OD, optical density.

USP47 is directly targeted by miR-454

The binding sites of miR-454 on the 3′-UTR of USP47 were predicted using TargetScan software (Fig. 5A). Compared with the corresponding NC groups, miR-454 mimics significantly inhibited the luciferase activity of USP47-WT, whereas miR-454 inhibitor significantly increased the luciferase activity of USP47-WT in 5-8F/DDP and SUNE-1/DDP cells (Fig. 5B). The RIP assay results demonstrated that miR-454 and USP47 expression levels were significantly enriched in Ago2 compared with IgG (Fig. 5C). In addition, USP47 expression was significantly upregulated in DDP-resistant NPC tissues compared with DDP-sensitive NPC tissues (Fig. 5D). The RT-qPCR and western blotting results indicated that USP47 mRNA and protein expression levels were increased in DDP-resistant NPC cells compared with parental NPC cells (Fig. 5E and F). Pearson's correlation analysis suggested that USP47 expression was negatively correlated with miR-454 expression in NPC tissues (Fig. 5G). The aforementioned results indicated that USP47 was a target of miR-454.

Figure 5

USP47 is directly targeted by miR-454. (A) Binding sites between miR-454 and the 3′-untranslated region of USP47. (B) Luciferase activity of USP47-WT and USP47-Mut in 5-8F/DDP and SUNE-1/DDP cells following transfection with NC mimics, miR-454 mimics, NC inhibitor or miR-454 inhibitor. (C) RNA immunoprecipitation assays were performed to assess the interaction between USP47 and miR-454. (D) USP47 expression levels in DDP-resistant and DDP-sensitive NPC tissues. USP47 (E) mRNA and (F) protein expression levels in DDP-resistant NPC cells. (G) Correlation between USP47 expression and miR-454 expression in NPC tissues. Data are presented as the mean ± SD. *P<0.05. USP47, ubiquitin specific peptidase 47; miR, microRNA; WT, wild-type; Mut, mutant; DDP, cisplatin; NC, negative control; NPC, nasopharyngeal carcinoma; Ago2, argonaute RISC catalytic component 2.

KCNQ1OT1 confers DDP resistance in NPC cells via upregulating USP47 expression by sponging miR-454

Subsequently, the RT-qPCR results demonstrated that miR-454 and USP47 expression levels were significantly decreased in 5-8F/DDP and SUNE-1/DDP cells following transfection with miR-454 inhibitor or shUSP47 compared with the NC inhibitor and shNC groups, respectively, which suggested that miR-454 inhibitor and shUSP47 downregulated miR-454 and USP47 expression levels in DDP-resistant NPC cells, respectively (Fig. 6A and B). In addition, 5-8F/DDP cells were transfected with shNC, shUSP47, shUSP47 + NC inhibitor or shUSP47 + miR-454 inhibitor. The CCK-8 assay results demonstrated that USP47 knockdown significantly reduced cell viability compared with the shNC group, which was significantly reversed by co-transfection with miR-454 knockdown in 5-8F/DDP and SUNE-1/DDP cells (Fig. 6C). The colony forming and Transwell assay results demonstrated that miR-454 knockdown significantly reversed shUSP47-induced inhibitory effects on DDP-resistant NPC cell proliferation, migration and invasion (Fig. 6D-F). Collectively, the results demonstrated that miR-454 participated in DDP resistance in NPC via USP47.

Figure 6

KCNQ1OT1 confers NPC cell DDP resistance via upregulating USP47 expression in NPC cells by sponging miR-454. (A) Transfection efficiency of miR-454 inhibitor. (B) USP47 expression levels in 5-8F/DDP and SUNE-1/DDP cells transfected with shNC or shUSP47. (C) Cell Counting Kit-8 assays were performed to detect the cell viability of 5-8F/DDP and SUNE-1/DDP cells transfected with shNC, shUSP47, shUSP47 + NC inhibitor or shUSP47 + miR-454 inhibitor. (D) Colony forming (magnification, ×40), (E) Transwell migration and (F) Transwell invasion assays were performed to assess cell proliferation, migration and invasion in 5-8F/DDP and SUNE-1/DDP cells transfected with shNC, shUSP47, shUSP47 + NC inhibitor or shUSP47 + miR-454 inhibitor (scale bar, 100 µm). Data are presented as the mean ± SD. *P<0.05. KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; NPC, nasopharyngeal carcinoma; DDP, cisplatin; USP47, ubiquitin specific peptidase 47; miR, microRNA: sh, short hairpin RNA; NC, negative control.

USP47 expression is regulated by KCNQ1OT1 and miR-454

Pearson's correlation analysis suggested that USP47 expression was positively correlated with KCNQ1OT1 expression in NPC tissues (Fig. 7A). To further determine whether KCNQ1OT1 mediated its effects via regulating the miR-454/USP47 axis, 5-8F/DDP and SUNE-1/DDP cells were transfected with shNC, shKCNQ1OT1, shKCNQ1OT1+ NC inhibitor or shKC-NQ1OT1 + miR-454 inhibitor. The RT-qPCR results suggested that USP47 expression was significantly downregulated in KCNQ1OT1-knockdown DDP-resistant NPC cells compared with the shNC group, and miR-454 knockdown significantly reversed KCNQ1OT1 knockdown-mediated downregulation of USP47 expression levels (Fig. 7B). The results indicated that KCNQ1OT1 contributed to DDP resistance in NPC cells via regulating the miR-454/USP47 axis.

Figure 7

USP47 expression is regulated by KCNQ1OT1 and miR-454. (A) Correlation between USP47 expression and KCNQ1OT1 expression in nasopharyngeal carcinoma tissues. (B) USP47 expression levels in 5-8F/DDP and SUNE-1/DDP cells transfected with shNC, shKCNQ1OT1, shKCNQ1OT1 + NC inhibitor or shKCNQ1OT1 + miR-454 inhibitor. Data are presented as the mean ± SD. *P<0.05. USP47, ubiquitin specific peptidase 47; KCNQ1OT1, KCNQ1 opposite strand/antisense transcript 1; miR, microRNA; DDP, cisplatin; sh, short hairpin RNA; NC, negative control.

Discussion

Increasing evidence has indicated that abnormal lncRNAs might lead to drug resistance in various types of cancer (23-25), including NPC (26,27). The present study demonstrated that compared with the shNC group, KCNQ1OT1 knockdown significantly suppressed cell viability and promoted DDP sensitivity in DDP-resistant NPC cells by sponging miR-454 via downregulating USP47. Moreover, the results suggested that the KCNQ1OT1/miR-454/USP47 axis participated in the regulation of DDP resistance in NPC.

KCNQ1OT1 is an imprinted antisense lncRNA located at 11p15.5 (28,29). Recent studies have suggested that KCNQ1OT1 is closely associated with the drug resistance of various tumors. For example, DDP-induced KCNQ1OT1 regulated the chemoresistance of tongue cancer via regulating the miR-124-3p/tripartite motif containing 14 axis (30). The lncRNA KCNQ1OT1/miR-34a axis increased the chemoresistance of colon cancer cells by targeting autophagy related 4B cysteine peptidase (31). Moreover, KCNQ1OT1 knockdown sensitized osteosarcoma cells to DDP and inhibited cell invasion via the potassium voltage-gated channel subfamily Q member 1/DNA methyltransferase 1 axis (32). In the present study, the functions and mechanisms underying KCNQ1OT1-induced DDP resistance in NPC were investigated. The results indicated that KCNQ1OT1 expression was significantly increased in DDP-resistant NPC compared with DDP-sensitive NPC, and KCNQ1OT1 knockdown significantly suppressed NPC cell viability and DDP resistance compared with the shNC group.

Previous studies have revealed that lncRNAs might serve as competitive endogenous RNAs for miRNAs to alter the binding of miRNAs and miRNA target gene expression levels (33-36). The present study demonstrated that KCNQ1OT1 directly interacted with miR-454. miR-454 has been reported to serve vital roles in tumor proliferation, apoptosis and metastasis in various types of cancer. For example, miR-454 remarkably inhibited bladder cancer cell invasion and migration via targeting ZEB2 antisense RNA 1 (37). miR-454 overexpression accelerated apoptosis and suppressed proliferation of glioblastoma cells by downregulating nuclear factor of activated T cells 2 (38). Moreover, miR-454 inhibition decreased the repressive effects of HOXA11 antisense RNA knockdown on DDP-resistant non-small cell lung cancer cells (39). In the present study, compared with the NC mimics group, miR-454 overexpression significantly inhibited cell viability and promoted sensitivty to DDP in DDP-resistant NPC cells, but KCNQ1OT1 overexpression reversed the effects of miR-454 overexpression on cell viability and DDP sensitivity.

USP47 is a member of the deubiquitinating enzyme family (40). Previous studies have revealed that USP47 was abnormally expressed in multiple types of cancer (41-43). For example, USP47 overexpression accelerated ovarian cancer cell development (44), whereas USP47 knockdown inhibited gastric cancer cell proliferation (45). USP47 facilitated osteosarcoma cell invasion and migration, and suppressed apoptosis (46). In the present study, USP47 was identified as a target of miR-454, and USP47 knockdown significantly inhibited cell viability and DDP resistance in NPC cells compared with the shNC group. Furthermore, miR-454 knockdown significantly reversed shUSP47-mediated effects in DDP-resistant NPC cells. Compared with the shNC group, KCNQ1OT1 knockdown significantly downregulated USP47 expression, and miR-454 knockdown significantly reversed shKCNQ1OT1-mediated effects on USP47 expression.

However, the present study had a number of limitations. Firstly, other mRNAs should be explored to detemine the downstream regulatory mechanism underlying the KCNQ1OT1/miR-454 axis. Secondly, the present study lacked in vivo experiments, which should be performed in future studies to further the understanding of the mechanism under-lying NPC.

In conclusion, the results of the present study suggested that KCNQ1OT1 facilitated cell viability and DDP resistance in NPC cells via regulating the miR-454/USP47 axis. Therefore, KCNQ1OT1 might serve as a potential target for overcoming DDP resistance in the chemotherapy of NPC.