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. 2021 Jul 1;16(1):103–113. doi: 10.1007/s12079-021-00634-2

miR-137/ERRα axis mediates chemoresistance of nasopharyngeal carcinoma cells

Fei Liu 1, Chunsheng Gao 1, Wenjuan Wang 2, Jing Hu 1, Zuofeng Huang 1, Meng Liang 1, Shuo Li 1,✉
PMCID: PMC8688621  PMID: 34196940

Abstract

Nasopharyngeal carcinoma (NPC) is the most common malignant tumor of the head and neck region and is characterized by an increased risk of developing chemoresistance after treatment. The present study demonstrated that estrogen-related receptor α (ERRα) was upregulated in cisplatin- and fluorouracil-resistant NPC cells. In addition, ERRα knockdown or treatment of cells with the ERRα inverse agonist XCT-790 attenuated the chemoresistance of NPC cells. Mechanistically, the increased expression of ERRα in chemoresistant cells was associated with enhanced mRNA stability. Bioinformatics analysis for screening microRNAs (miRs) regulating the expression of ERRα revealed that miR-137 was downregulated in chemoresistant NPC cells. Additionally, transfection of cells with miR-137 mimics reduced ERRα mRNA stability and increased the chemosensitivity of NPC cells. Furthermore, ERRα knockdown reduced glucose consumption, and lactate and ATP production rates in chemoresistant cells. The aforementioned findings suggested that the miR-137/ERRα-mediated metabolic programming could be involved in the chemoresistance of NPC cells.

Keywords: Chemoresistance, NPC, ERRα, miR-137, Proliferation

Introduction

As the most common malignancy of the head and neck region, nasopharyngeal carcinoma (NPC) is more prevalent in Southeast Asia and Africa (Cao et al. 2011). It has been reported that Epstein-Barr virus infection, genetic susceptibility and lifestyle are the main risk factors associated with the tumorigenesis and development of NPC (Ablashi et al. 1983; Tsao et al. 2014). Adjuvant cisplatin (CDDP) chemotherapy combined with radiotherapy is the standard treatment for NPC (Lee et al. 2015). Although the primary NPC can be effectively treated with the above approach, NPC cells often develop chemoresistance after treatment. Additionally, the majority of metastatic NPCs are less sensitive to chemotherapy. Therefore, further investigations into the mechanisms underlying therapeutic resistance are urgently needed to improve therapeutic efficiency.

Estrogen-related receptor α/β/γ (ERRα/β/γ) are orphan nuclear receptors, which bind to ERR-response elements to induce gene transcription (Giguere et al. 1988). ERRα is widely expressed in various organs, particularly in tissues with high-energy demands such as the heart and kidneys (Luo et al. 2003). As a transcription factor and a master regulator, ERRα can regulate mitochondrial biogenesis and cellular energy metabolism via modulating the metabolism of fatty acids, tricarboxylic acid cycle and oxidative phosphorylation (Ranhotra, 2018). Previous studies have suggested that the expression of ERRα in human cancer tissues (Ariazi et al. 2002; Fujimoto et al. 2007; Fujimoto and Sato, 2009) is associated with poor prognosis. For example, increased expression of ERRα in a breast cancer xenograft model enhanced the metastatic ability of cancer cells (Ao et al. 2008; Fradet et al. 2011; Stein et al. 2009). In terms of therapy, ERRα overexpression could attenuate reactive oxygen species (ROS) production to confer methotrexate resistance (Chen et al. 2014b). Another study showed that ERRα regulated lactate production to induce lapatinib resistance in breast cancer cells (Deblois et al. 2016). In prostate cancer, ERRα could promote the expression of ATP-binding cassette subfamily C member 4 (ABCC4) to induce docetaxel resistance (Huang et al. 2020). All these findings suggested that ERRα could be a potent regulatory factor in mediating chemoresistance of cancer cells.

Thorough knowledge regarding the functional role of ERRα in NPC is currently lacking. Previous studies indicated that estrogen receptor α was involved in the NAG7-induced invasion of human NPC cells (Huang et al. 2009). To investigate the effects of ERRα/β/γ, their expression levels in CDDP-resistant NPC cells were first determined. Subsequently, the biological effects of ERRα on inducing CDDP resistance in NPC cells and the associated underlying mechanisms were investigated using gain- and loss-of-function experiments.

Materials and methods

Cells culture and treatment

The human NPC cell lines, HNE1 and CNE2, were purchased from the Cell Bank of Central South University, Changsha, China. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher Scientific, USA). Both cell lines were routinely checked for mycoplasma contamination. Cell line authentication was performed using short tandem repeat profiling. To establish chemoresistant NPC cells, CDDP and fluorouracil (5-FU; both from Qilu Pharmo Co. Ltd.) were diluted in PBS. Subsequently, cells were treated with increasing concentrations of CDDP or 5-FU (0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 μM in normal medium) for 6 months. The CDDP- or 5-FU-resistant cells were maintained in medium containing 0.5 μM CDDP or 5-FU, respectively. Finally, the established resistant cells were named as NPC/CDDP or NPC/5-FU cells.

Cell proliferation assay

The cell proliferation was assessed using a CCK-8 assay (Cell Counting Kit 8; CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. Briefly, cells were seeded into 96-well culture plates at a density of 2 × 103 cells/100 μl medium/well and treated under the indicated conditions. Following treatment, 10 μl CCK-8 solution was added into each well and cells were incubated at 37 °C for 4 h. The optical density value of each well was measured at a wavelength of 450 nm using a spectrophotometric plate reader (BioTek Instruments, Inc.; EL- × 800). In each case, three independent assays were performed. The concentration of CDDP or 5-FU resulting in cell growth inhibition of 50% (IC50) was finally determined.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA (cDNA) was synthesized from 500 ng total RNA using the PrimeScript RT reagent Kit (Takara Bio, Inc.). qPCR was carried out with the SYBR Premix ExTaq kit (Takara Bio, Inc.) on the ABI PRISM® 7900 HT Fast Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions used for qPCR were as follows: Pre-incubation at 95 °C for 30 s, 45 cycles consisting of 5 s at 95 °C and 30 s at 60 °C, followed by cooling at 40 °C for 30 s. The primer sequences used were as follows: GAPDH forward, 5′‐ATG GTG AAG GTC GGT GTG AAC‐3′ and reverse, 5′‐TGT AGT TGA GGT CAA TGA AGG‐3′; ERRα forward, 5′‐CCA CTA TGG TGT GGC ATC CTG T‐3′ and reverse, 5′‐GGT GAT CTC ACA CTC GTT GGA GG‐3′; ERRβ forward, 5′‐GAC ATT GCC TCT GGC TAC CAC T‐3′ and reverse, 5′‐CTC CGT TTG GTG ATC TCG CAC T‐3′; and ERRγ forward, 5′‐CGC AGG ATA GAT GCG GAG AAC A‐3′ and reverse, 5′‐TTC AGC CAC CAA CAA ATG TGA GAC‐3′. The relative fold expression and changes were calculated using the 2−ΔΔCq method. GAPDH served as the internal control for mRNA normalization.

The levels of ERRα precursor mRNA (pre-ERRα) were measured by RT-qPCR as the unspliced form of the ERRα gene transcript. The primers used could amplify exon 1 and the following intron. The primer sequences used for pre-ERRα were: Forward, 5′‐GCG ATG TCC TTT TGT GTC CT‐3′ and reverse, 5′‐CCT GAA CCC TGA CCA GTC C‐3′.

To evaluate the expression levels of microRNAs (miRNAs/miRs), the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was utilized to generate cDNA. The thermocycling conditions used were as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. The forward primer was the exact sequence of the mature miRNA (http://www.mirbase.org/search.shtml). The sequence of the forward primer for U6 was 5′-TGC GGG TGC TCG CTT CGC AGC-3′, while the reverse primer was supplied by the aforementioned kit. U6 served as the internal control for miRNA normalization. The gene expression levels were quantified using the 2−ΔΔCq method (Chen et al. 2019b). All experiments were performed in triplicate.

Western blot analysis

Total proteins were extracted from cells using NP40 lysis buffer (Beyotime Institute of Biotechnology) supplemented with EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics). The lysates were then centrifuged at 13,000 × g for 10 min. The protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Subsequently, proteins were separated by 4–20% SDS-PAGE and electro-transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.). Following blocking with 5% w/v BSA (Thermo Fisher Scientific, USA), membranes were incubated with specific antibodies overnight at 4˚C. All primary antibodies used were purchased from Cell Signaling Technology, Inc. Subsequently, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit antibodies (dilution, 1:5,000; ProteinTech Group, Inc.) at room temperature for 1 h. The bands were visualized using an ECL detection system (Thermo Fisher Scientific, Inc.). GAPDH served as a loading control. Images were captured using the MiniChemi imaging system (Beijing Sage Creation Science Co., Ltd.).

Immunofluorescence

Cells were seeded onto chamber slides and fixed with methyl alcohol. Following permeabilization with 0.5% Triton X-100 for 20 min, the slides were blocked with 5% BSA for 1 h and then incubated with a primary antibody against ERRα at 4 °C in a humidified chamber overnight. Alexa Fluor 488 dye-labeled anti-mouse IgG was used as the secondary antibody. Nuclei were stained with DAPI (cat. no. P36935; Thermo Fisher Scientific, Inc.) and images were captured under a confocal scanning microscope (FV1000; Olympus Corporation).

Cell transfection

The small interfering (si)RNAs against ERRα (si-ERRα-1; sequence, 5′-GCG AGA GGA GUA UGU UCU A-3′; si-ERRα-2; sequence, 5′-GAG AGG AGU AUG UUC UAC UAA-3′) and negative control (si-NC; sequence, 5′-GCA CAA CAA GCC GAA UAC A-3′) were purchased from Qiagen, Inc. The miR-137 mimics (sequence, 5′-UAUUGCUUGAGAAUACACGUAG-3′) and control scramble oligonucleotides (miR; sequence, 5′-UUC UCC GAA CGU GUC ACG UTT-3′) were obtained from Guangzhou RiboBio Co., Ltd. For cell transfection, cells were plated into wells at a 60–70% confluency and transfected with siRNAs or miRNA mimics using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The working concentration of siRNAs/miRNA mimics was 20 nM. Following transfection for 6 h at 37˚C, the medium was replaced with fresh complete medium.

mRNA stability assay

To evaluate mRNA stability, cells were treated with 5 μg/ml actinomycin D (Act-D; cat. no. A9415; Sigma-Aldrich; Merck KGaA) to inhibit mRNA transcription. Following treatment with Act-D for 0–8 h, cells were harvested and the mRNA expression levels of ERRα were determined by RT-qPCR.

The cycloheximide (CHX) chase assay

A CHX chase assay was carried out to evaluate protein stability. Briefly, cells were treated with 50 μg/ml CHX for 0–12 h and harvested, and the extracted proteins were subjected to western blot analysis to measure the protein levels of ERRα.

Luciferase reporter assay

The potential binding sites of miR-137 on the 3'-untranslated region (UTR) of ERRα were predicted using the TargetScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de) and miRanda MiRBase (http://microrna.sanger.ac.uk) online available databases. The fragment of the human ERRα 3'-UTR encompassing both predicted complementary sequences was synthesized by by Shanghai Shenggong Biocompany (Shanghai, China) and subcloned into the pmiR-GLO luciferase reporter vector to construct the pmiR-GLO-ERRα-3'-UTR-wild-type (WT) plasmid with XhoI and SalI restriction sites. Both synthesized ERRα 3'-UTR and pmiR-GLO luciferase reporter vector were digested by XhoI and SalI. After purification, the both DNA fragments were ligated by T4 DNA ligase at 16˚C overnight. Site-directed mutagenesis was carried out to introduce miR-137 binding site mutations to generate a pmiR-GLO-ERRα-3'-UTR-mutant-type (MUT) plasmid using overlap extension-PCR (Ho et al. 1989). After validation by sequencing, cells were seeded into 24-well plates and were then co-transfected with the corresponding plasmids and miRNA mimics using Lipofectamine® 2000 in 100 μl Opti-MEM™ (Invitrogen; Thermo Fisher Scientific, Inc.). Firefly and Renilla luciferase activities were determined by a dual luciferase assay system (cat. no. E1910; Promega Corporation) using standardized protocols.

Glucose consumption, and lactate and ATP production assays

Glucose consumption, and lactate and ATP production assays were carried out as previously described (Zhai et al. 2019). Briefly, the content of glucose and lactate in culture supernatants was measured using a glucose assay kit (cat. no. CBA086; Sigma‐Aldrich; Merck KGaA) or lactate assay kit (cat. no. K607-100; BioVision, Inc.), respectively. Additionally, a colorimetric ATP assay kit (cat. no. ab83355; Abcam) was utilized to measure the intracellular levels of ATP. The relative glucose consumption rate, lactate production rate and ATP levels were normalized to the total protein concentration of each sample.

Oxygen consumption rate (OCR)

Cells were transfected with si-NC or si-ERRα and were then seeded into a 96-well plate at a density of 10,000 cells/well. The OCR was measured as previously described (Lafargue et al. 2017). Briefly, the bicarbonate-free DMEM was supplemented with 25 mM glucose, 1 mM pyruvate and 2 mM glutamine to equilibrate cells. Following the addition of each chemical, OCR was measured using the XF24 Analyzer (Seahorse Bioscience). All experiments were repeated at least three times.

Statistical analysis

All data are presented as the mean ± SD. All data were analyzed using SPSS 16.0 software (SPSS, Inc.). Student's t-test (unpaired) was carried out for the comparison between two groups. One way ANOVA followed by a Bonferroni's post hoc test was used for multiple comparisons. p < 0.05 was considered to indicate a statistically significant difference.

Results

Establishment of CDDP- and 5-FU-resistant NPC cells

The chemoresistant NPC cells were established following treatment of cells with increasing concentrations of CDDP or 5-FU for 10 months. As shown in Fig. 1a and b, the IC50 values of CDDP in HNE1 and HNE1/CDDP cells were 0.43 and 8.34 μM, respectively. In addition, the IC50 values of 5-FU in HNE1 and HNE1/5-FU cells were 1.14 and 7.87 μM, respectively. Consistently, the IC50 values of CDDP in CNE2 and CNE2/CDDP cells were 0.95 and 11.4 μM, respectively, while those of 5-FU in the same cells were 0.93 and 9.45 μM, respectively. These findings verified the successful establishment of NPC chemoresistant cells.

Fig. 1.

Fig. 1

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The establishment of CDDP and 5-FU resistant NPC cells. Chemoresistant HNE1 or HNE1 cells were treated with increasing concentration of CDDP (a) or 5-FU (b) for 48 h; Chemoresistant HNE1 or HNE1 cells were treated with increasing concentration of 5-FU (c) or 5-FU (d) for 48 h

ERRα is upregulated in chemoresistant NPC cells

Subsequently, the expression levels of ERRα/β/γ were determined in parental and chemoresistant NPC cells. As shown in Fig. 2a and b, ERRα mRNA was increased in all chemoresistant NPC cells compared with the parental ones. Western blot analysis further confirmed ERRα upregulation in all chemoresistant NPC cells compared with the corresponding parental cells (Fig. 2c). Confocal microscopy demonstrated that the expression and nuclear localization of ERRα were increased in HNE1/CDDP cells compared with those in HNE1 cells.

Fig. 2.

Fig. 2

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ERRα was upregulated in chemoresistant NPC cells. The mRNA expression of ERRα/β/γ in chemoresistant HNE1 (a) or CNE2 (b) and their corresponding parental cells were measured; c The expression of ERRα in chemoresistant NPC cells and their corresponding parental cells were measured and quantitatively analyzed; d The expression of ERRα in HNE1 and HNE1/CDDP cells were checked by confocal. **p < 0.01

ERRα silencing reverses the chemoresistance of NPC cells

The current study also investigated whether ERRα was involved in the chemoresistance of NPC cells. Therefore, chemoresistant NPC cells were pre-treated with or without XCT-790, a potent and selective inverse agonist for ERRα. The results showed that XCT-790 could significantly increase the CDDP sensitivity of both HNE1/CDDP and CNE2/CDDP cells (Fig. 3a and b). Consistently, XCT-790 could enhance the 5-FU sensitivity of both HNE1/5-FU and CNE2/5-FU cells (Fig. 3c and d). Subsequently, the expression of ERRα was inhibited in both HNE1/CDDP and CNE2/CDDP cells following transfection with specific siRNA clones against ERRα (Fig. 3e). The results revealed that cell transfection with si-ERRα markedly increased the CDDP sensitivity of both HNE1/CDDP (Fig. 3f) and CNE2/CDDP (Fig. 3g) cells. The aforementioned findings indicated that ERRα silencing could reverse the chemoresistance of NPC cells.

Fig. 3.

Fig. 3

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Inhibition of ERRα can reverse the chemoresistance of NPC cells. a to d Cells were pretreated with or without XCT-790 for 6 h and then further treated with increasing concentrations of CDDP for 5-FU for 48 h; e Cells were transfected with si-NC or si-ERRα for 24 h, protein was measured; Cells pre-transfected si-NC or si- ERRα for the indicated 12 h and further traded with increasing concentrations of CDDP (f) or 5-FU (g) for 48 h

ERRα mRNA stability is increased in chemoresistant NPC cells

Subsequently, the mechanisms underlying ERRα upregulation in chemoresistant NPC cells were explored. Since ERRα mRNA was increased in chemoresistant NPC cells, the expression of pre-ERRα mRNA was then determined. There was no statistically significant difference in the expression of pre-ERRα mRNA between HNE1/CDDP and HNE1 cells, or between CNE2/CDDP and CNE2 cells (Fig. 4a). Furthermore, ERRα mRNA stability was significantly increased in both HNE1/CDDP and HNE1/5-FU cells compared with that in HNE1 cells (Fig. 4b). Consistently, the mRNA stability of ERRα was also notably enhanced in both CNE2/CDDP and CNE2/5-FU cells compared with that in CNE2 cells (Fig. 4c). However, the protein stability of ERRα was comparable between HNE1/CDDP and HNE1 cells (Fig. 4d). These results suggested that the mRNA stability of ERRα was elevated in chemoresistant NPC cells.

Fig. 4.

Fig. 4

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The mRNA stability of ERRα was increased in chemoresistant NPC cells. a The precursor mRNA levels of ERRα were checked in parental and CDDP resistant NPC cells; b Parental or chemoresistant HNE1 cells were treated with Act-D and incubated for 0 to 8 h, the mRNA levels of ERRα were checked; c Parental or chemoresistant CNE2 cells were treated with Act-D and incubated for 0 to 8 h, the mRNA levels of ERRα were checked; d HNE1 and HNE1/CDDP cells were treated with CHX and incubated for 0 to 12 h, the protein levels of ERRα were checked and quantitatively analyzed. **p < 0.01

miR-137 regulates ERRα mRNA stability in NPC cells

It is well known that miRNAs can bind with the 3'-UTR of target mRNAs to trigger their degradation (Vishnoi and Rani, 2017). Previous studies indicated that miR-125a (Ji et al. 2014), miR-135a (Tribollet et al. 2016), miR-137 (Zhao et al. 2012) and miR-497 (Han et al. 2016) could directly bind with the 3'-UTR of ERRα to reduce mRNA stability. Therefore, the expression levels of miR-125a, miR-135a, miR-137 and miR-497 were determined in parental and chemoresistant cells. RT-qPCR analysis showed that miR-137 was downregulated in HNE1/CDDP cells compared with HNE1 cells, while the expression of miR-125a, miR-135a, and miR-497 was not altered (Fig. 5a). Consistently, decreased expression of miR-137 was observed in CNE2/CDDP cells compared with CNE2 cells (Fig. 5b). Subsequently, the effect of miR-137 on ERRα mRNA stability in 5-FU resistant NPC cells was investigated.

Fig. 5.

Fig. 5

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miR-137 can regulate the mRNA stability of ERRα in NPC cells. The relative miRNA levels in HNE1/CDDP (a), CNE2/CDDP (b) and their parental cells were checked; c The levels of miR-137 in parental and 5-FU resistant NPC cells were checked; d The potential binding site between miR-137 and 3’UTR of ERRα and the mutation of 3’UTR of ERRα; e HNE1 cells were transfected with ERRα 3’UTR-wild type or mutant for 6 h and further transfected with scrambled control or miR-137 mimics for 24 h, the luciferase assay was conducted; HNE1/CDDP (f) and CNE2/CDDP (g) cells were pre-transfected with scrambled control or miR-137 mimics for 24 h and further treated with Act-D and incubated for 0 to 8 h, the mRNA levels of ERRα were checked. **p < 0.01; NS, no significant

miR-137 was also downregulated in 5-FU-resistant NPC cells compared with parental cells (Fig. 5c). Dual luciferase reporter assay demonstrated that transfection of cells with miR-137 mimics could significantly attenuate the luciferase activity of pmiR-GLO-ERRα-3'-UTR-WT, while co-transfection of cells with pmiR-GLO-ERRα-3'-UTR-MUT and miR-137 mimics had no effect on the luciferase activity (Fig. 5d and e). Furthermore, cell transfection with miR-137 mimics notably attenuated ERRα mRNA stability in both HNE1/CDDP (Fig. 5f) and CNE2/CDDP (Fig. 5g) cells.

miR-137/ERRα axis regulates the chemoresistance of NPC cells

Western blot analysis confirmed that miR-137 overexpression could significantly downregulate ERRα protein expression in both HNE1/CDDP and CNE2/CDDP cells (Fig. 6a). Furthermore, miR-137 overexpression markedly increased CDDP sensitivity of both HNE1/CDDP (Fig. 6b) and CNE2/CDDP (Fig. 6c) cells. Consistently, transfection with miR-137 mimics significantly elevated the 5-FU sensitivity of both HNE1/5-FU (Fig. 6d) and CNE2/5-FU (Fig. 6e) cells. The aforementioned findings verified that the miR-137/ERRα axis could be involved in the chemoresistance of NPC cells.

Fig. 6.

Fig. 6

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miR-137/ ERRα axis regulated the chemoresistance of NPC cells. a Cells were transfected with scrambled control or miR-137 mimics for 24 h, the expression of ERRα was checked; HNE1/CDDP (b) and CNE2/CDDP (c) cells were pre-transfected with scrambled control or miR-137 mimics for 12 h and further treated with increasing concentrations of CDDP for 48 h; HNE1/5-FU (d) and CNE2/5-FU (e) cells were pre-transfected with scrambled control or miR-137 mimics for 12 h and further treated with increasing concentrations of 5-FU for 48 h

ERRα regulates the metabolic phenotype of chemoresistant NPC cells

Emerging evidence has suggested that ERRα can regulate mitochondrial biogenesis and cellular energy metabolism (Ranhotra 2018). The present study demonstrated that ERRα silencing could significantly attenuate glucose consumption (Fig. 7a), lactate generation (Fig. 7b) and ATP production (Fig. 7c) in both HNE1/CDDP and CNE2/CDDP cells. Additionally, ERRα knockdown reduced OCR and proton ionophore (uncoupler) p-trifluoromethoxyphenylhydrazone-induced OCR, indicating maximal respiration capacity, in both HNE1/CDDP (Fig. 7d) and CNE2/CDDP (Fig. 7e) cells. Therefore, these findings suggested that ERRα could regulate the metabolic phenotype of chemoresistant NPC cells.

Fig. 7.

Fig. 7

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ERRα regulated the glycolysis of chemoresistant NPC cells. Cells were transfected with si-NC or si-ERRα for 24 h, and then glucose consumption (a), lactate production (b), and ATP levels (c) were checked; HNE1/CDDP (d) or CNE2/CDDP (e) cells were transfected with si-NC or si-ERRα for 24 h, the cellular OCR was measured. **p < 0.01

Discussion

The development of chemoresistance is a major cause of cancer-associated mortality in NPC patients (Holohan et al. 2013). The present study revealed that ERRα was upregulated in NPC chemoresistant cells, while targeted inhibition of ERRα could reverse cell chemosensitivity. ERRα acts as an oncogene in several types of cancer via regulating cellular energy metabolism (Ranhotra 2015). For example, a study demonstrated that the ERRα/carnitine palmitoyltransferase 1C axis could promote cancer cell proliferation, metabolism and tumorigenesis (Chen et al. 2020). In terms of chemosensitivity, reprogramming of the ERRα target gene landscape could trigger tamoxifen resistance of breast cancer cells (Thewes et al. 2015), while XCT-790 could promote cell death in chemotherapy-resistant cancer cells (Wu et al. 2009). Additionally, ERRα could confer methotrexate resistance in osteosarcoma cells via attenuating ROS production and P53-mediated apoptosis (Chen et al. 2014a). In addition, ERRα could positively regulate the expression of ABCB1 to mediate chemotherapy resistance of osteosarcoma cells (Chen et al. 2019a). These findings supported that ERRα could be a potential mediator for overcoming NPC chemoresistance and targeted silencing of ERRα could improve the efficiency of chemotherapy in NPC patients.

Furthermore, the results of the current study demonstrated that the miR-137-regulated mRNA stability could be responsible for ERRα upregulation in chemoresistant NPC cells. Decreased expression of miR-137 and increased mRNA stability was observed in chemoresistant NPC cells. In addition, miR-137 could directly bind with the 3'-UTR of ERRα to regulate mRNA stability and chemoresistance of NPC cells. In breast cancer cells, miR-137 downregulated ERRα at both protein and mRNA levels, whereas the miR-137-mediated ERRα downregulation was associated with the impaired proliferative and migratory capacities of cancer cells (Zhao et al. 2012). It has been reported that the expression of miR-137 is frequently downregulated in several types of cancer, including colorectal, gastric and oral cancer (Balaguer et al. 2010; Chen et al. 2011; Yao et al. 2013). This could be due to the fact that this miRNA is located on chromosome 1p22, a region embedded in a CpG island that is often silenced by methylation (Kozaki et al. 2008). Therefore, the miR-137/follistatin like-1/integrin β3/Wnt/β-catenin signaling axis could maintain stemness and induce chemoresistance in breast cancer cells (Cheng et al. 2019). Additionally, miR-137 could alleviate doxorubicin resistance in breast cancer cells via attenuating epithelial-mesenchymal transition (Du et al. 2019). Consistently, miR-137 was involved in CDDP resistance via inhibiting caspase-3 in lung adenocarcinoma (Su et al. 2016). The aforementioned results supported the results of the current study, suggesting that the miR-137/ERRα axis could serve a critical role in regulating chemoresistance in NPC cells.

Herein, knockdown of ERRα, a regulator of cell energy metabolism (Ranhotra 2015), suppressed glucose consumption and lactate and ATP generation in chemoresistant NPC cells. It has been reported that aerobic glycolysis plays an important role in cancer cell survival and resistance of cancer cells to therapy (Martinez-Outschoorn et al. 2011). It has been also suggested that increased glucose consumption and lactate production are critical for promoting chemoresistance, while chemoresistant cells can reprogram metabolic pathways via increasing ATP generation and OCR (Lukey et al. 2018; Wicki et al. 2016). Therefore, the changes in metabolism could be involved in the miR-137/ERRα-mediated CDDP and 5-FU resistance of NPC cells.

Collectively, the present study suggested that ERRα upregulation could promote chemoresistance in NPC cells via regulating cell energy metabolism. Furthermore, the miR-137 downregulation-mediated increased mRNA stability could be involved in ERRα upregulation in chemoresistant NPC cells. Therefore, targeted inhibition of the miR-137/ERRα axis could be useful for overcoming chemosensitivity and improving therapy efficiency in patients with NPC.

Funding

This study was funded by the Science and Technology Foundation of Shenzhen (Grant No. JCYJ20180302144624391).

Availability of data and material

All data and material are available.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Consent for publication

All authors give the consent for the publish of this study.

Footnotes

Publisher's Note

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Contributor Information

Fei Liu, Email: liufei2004phil@126.com.

Chunsheng Gao, Email: szgaocs@qq.com.

Wenjuan Wang, Email: 1249422475@qq.com.

Jing Hu, Email: hujingstella@163.com.

Zuofeng Huang, Email: 513131404@qq.com.

Meng Liang, Email: 33106304@qq.com.

Shuo Li, Email: drshuoli@protonmail.com.

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