Research in contextEvidence before this studyInfection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Previously, EBV-miR-BART22 has been found highly expressed in NPC tissues. However, how the NPC-associated EBV miRNA precisely regulate the tumor progression and chemoresistance remains obscure in NPC.Added value of this studyThis study identifies EBV-miR-BART22 as an oncogene that promotes tumor stemness, metastasis and DDP chemotherapy resistance in NPC. The role and detailed molecular basis of EBV-miR-BART22 was examined by transwell, boyden, sphere formation assay, western blotting and mouse xenograft tumor model et al. The expression levels of EBV-miR-BART22 and its targeted gene mitogen-activated protein kinase kinase 4 (*MAP2K4*) were evaluated in NPC and nasopharynx tissues. EBV-miR-BART22 expression was negatively correlated with *MAP2K4* protein expression in NPC tissues, and NPC patients with high miR-BART22 and low *MAP2K4* protein expression showed the worst survival prognosis. Furthermore, we found that EBV-miR-BART22 targets *MAP2K4* and further stimulates *MYH9*-mediated *GSK3β* ubiquitin protein degradation, which thus activates *β-catenin* and its downstream tumor stemness and EMT signaling pathways. Finally, the anti-tumor activity of cinobufotalin in reversing EBV-miR-BART22-induced DDP chemoresistance was shown by inducing *MAP2K4* to antagonize *MYH9/GSK3β/β-catenin* signaling pathway in NPC.Implications of all the available evidenceTogether,our study not only highlights the key role of EBV-miR-BART-22 in the pathogenesis of NPC, but also demonstrates the significance of cinobufotalin in reversing EBV-miR-BART-22-stimulated DDP chemoresistance in NPC.Alt-text: Unlabelled Box

1. Introduction {#s0020}
===============

NPC is endemic in Southern China and Southeast Asia with high incidences seen \[[@bb0005],[@bb0010]\]. It is a malignant tumor that is characterized by a high rate of local invasion and early, distant metastases in the nasopharynx \[[@bb0015],[@bb0020]\]. In previous studies, nasopharynx carcinogenesis had been shown to be closely related to EBV infection \[[@bb0025],[@bb0030]\]. As a human herpesvirus 4 (HHV4) family member, EBV establishes a life-long and latent infection in \>90% of the world\'s population \[[@bb0035], [@bb0040], [@bb0045]\]. The clinical use of Epstein-Barr virus (EBV) as a surrogate biomarker for population screening, prognostication, and disease surveillance for NPC continues to increase \[[@bb0050],[@bb0055]\]. MicroRNA (miRNA) dysregulation plays a vital role during the tumorigenic process \[[@bb0060], [@bb0065], [@bb0070]\], from initiation and development to progression toward a metastatic phenotype \[[@bb0075]\]. BamHI A rightward transcripts (BARTs) are particularly abundant in EBV-associated carcinomas and encode a large number of miRNAs \[[@bb0080], [@bb0085], [@bb0090]\] involved in tumor progression and treatment \[[@bb0095]\]. For instance, the EBV-encoded miRNA BART1 induces tumor metastases by regulating the PTEN-dependent pathway \[[@bb0100]\]. EBV-miR-BART7 and BART13 were highly expressed in NPC and served as a poor indicator for NPC prognosis \[[@bb0105]\]. Previously, we had found that EBV-miR-BART22 expression is higher in NPC tissues compared with those of adjacent mucosal tissues \[[@bb0110],[@bb0115]\], which suggested that EBV-miR-BART22 was correlated with NPC pathogenesis. However, the function and molecular basis of EBV-miR-BART22 in NPC has not been reported.

Cancer stem cells (CSCs), a cancer cell subpopulation that can initiate tumorigenesis, exist in many different tumor types. Furthermore, CSCs are the key factors promoting malignant tumor phenotypes including tumor metastasis and chemoresistance \[[@bb0120], [@bb0125], [@bb0130], [@bb0135], [@bb0140]\]. However, the mechanisms by which CSCs occur in NPC have not been extensively investigated.

Bufotoxin is toxin found in the white sputum of the Chinese giant salamander, and its main components are resibufogenin (BR), cinobuafagin (CB), bufotalin (BT), gamabufotalin (GB), and bufalin \[[@bb0145]\]. Pharmacologically, bufotoxin has cardiotonic, anesthetic, detoxifying, pain relieving, reclaiming, and refreshing effects, and is widely used in clinical practice \[[@bb0150]\]. Recently, it was found that traditional Chinese medicine preparations containing cinobufotalin could inhibit the growth of several different tumor cells, in vitro \[[@bb0155],[@bb0160]\]. Clinical observations suggested that cinobufotalin would have inhibitory effects on liver, lung, and pancreatic cancers \[[@bb0165]\]. However, pre-clinical evaluations of chemically synthesized cinobufotalin have rarely been reported \[[@bb0170]\]. Furthermore, the difference between the cytotoxicity of cinobufotalin and DDP has never been compared in tumors.

In this study, we performed a more in-depth investigation into the role of EBV-miR-BART22 in NPC pathogenesis and evaluated the action and mechanism of chemically synthesized cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. We showed that cinobufotalin is a very promising antitumor agent that powerfully reversed EBV-miR-BART22-induced cisplatin resistance by inducing *MAP2K4* to antagonize *MYH9/GSK3β/β-catenin*-mediated tumor stemness and EMT signals in NPC.

2. Materials and methods {#s0025}
========================

2.1. Ethical statement {#s0030}
----------------------

All animal experiments were conducted in accordance with a protocol approved by the Animal Care and Use Committee of Southern Medical University. For the use of these clinical materials for research purpose, written informed consent was obtained from each patient. Approval from the Ethics Committee of the Nanfang Hospital was obtained.

2.2. Cell culture {#s0035}
-----------------

HONE1-EBV^+^ cells were a gift of Dr. S.W. Tsao, the University of Hong Kong. HONE1 and 5-8F cells were stored in the Cancer Research Institute of the Southern Medical University (Guangzhou, China). Both cell lines were cultured in RPMI-1640 (Corning) supplemented with 10% fetal calf serum (Corning) in a humidified chamber with 5% CO~2~ at 37 °C.

2.3. Clinical samples {#s0040}
---------------------

Sixty one (61) primary fresh NPC samples with TNM staging and thirty-six \[[@bb0180]\] non-cancerous fresh nasopharyngx(NP) samples as well as one hundred and sixty-three (163) paraffin-embedded NPC cases with clinical prognostic information(55 females and 108 males) and thirty-five \[[@bb0175]\] paraffin-embedded NP samples were collected from the Nanfang Hospital, Southern Medical University, China, at the time of diagnosis before any therapy.

2.4. Chemicals {#s0045}
--------------

Cinobufotalin Chemical Structure:

![](fx2.gif)

Cinobufotalin(CB) was purchased from MedChem Express (Monmouth Junction, NJ, USA) ([HY-N0880](http://www.medchemexpress.cn/Cinobufotalin.html){#ir0005}). Stock solutions were prepared using dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) as a solvent, and further dissolved to make the desired concentrations for experimental use. MG132 was purchased from Sigma-Aldrich (St. Louis, MO, USA), cycloheximide (CHX) was purchased from Abcam (Cambridge, MA, USA).

2.5. Lentivirus production and infection {#s0050}
----------------------------------------

Lentiviral particles carrying the hsa-miR-BART22 precursor were constructed by GeneChem (Shanghai, China). HONE1-EBV^+^ and 5-8F cells were infected with lentiviral or control vectors, respectively. Green fluorescent protein was used as a marker to monitor infection efficiency. Overexpression efficiency of miR-BART22 was validated by RT-qPCR.

2.6. RNA isolation, reverse transcription, and RT-qPCR {#s0055}
------------------------------------------------------

Total RNA was extracted with the TRIzol reagent (Invitrogen), and complementary DNA (cDNA) was synthesized with the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer\'s instructions. QRT-PCR was performed in triplicate with the SYBR Premix ExTaq (TaKaRa, Dalian, China). We also quantified expression of EBV-miR-BART22 using TaqMan microRNA assays (Applied Biosystems). Briefly, mature miRNAs were reversely transcribed, and qRT-PCR was performed using an All-in-One miRNA qRT-PCR Detection Kit following the manufacturer\'s protocol (GeneCopoeia). Each primer sequence of the genes used in this study is listed in Supplementary Table 3. The fold changes were calculated by using the 2^-DDCt^ method.

2.7. Transient transfection with mimics, inhibitor or plasmids {#s0060}
--------------------------------------------------------------

SiRNAs for c-Jun, *MYH9* and miR-BART22 mimics or its inhibitors were designed and synthesized by RiboBio, Inc. (Guangzhou, China). The sequences and controls are shown in Supplementary Table 4. *MAP2K4, MYH9* and *GSK3β* plasmids were purchased from Vigenebio Technologies (Shandong, China), and the *GSK3β*-K183R plasmids were purchased from Genechem (Shanghai, China). Twelve hours before transfection, HONE1-EBV^+^ and 5-8F cells were plated into 6-well plates (Nest, Biotech, China) at a density of 60% confluence. NPC cells were transfected with the mimics, inhibitor or plasmid using Lipofectamine TM 2000 (Invitrogen Biotechnology, China) according to the manufacturer\'s protocol. After 48 to 72 h, cells were collected for further experiments.

2.8. Cisplatin or cinobufotalin treatment in cells {#s0065}
--------------------------------------------------

*cis*-Diamminedichloroplatinum (cisplatin, DDP; Qilu Pharmo Co. Ltd., Jinan, China) was resuspended in phosphate-buffered saline (PBS) (0.5 mg/ml). Drug sensitivity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates in 100 μl RPMI-1640 medium supplemented with 10% FBS at 5 × 10^3^ cells/well. Once attached, cells were treated with 2.5, 5, 10, 20 or 40 μM Cisplatin or with 0.05, 0.1, 0.25, 0.5 0.75 or 1 μM Cinobufotalin. For Cinobufotalin combined with DDP, cells were treated by Cinobufotalin and DDP with the same contration at 0.05, 0.1, 0.25, 0.5 0.75 or 1 μM and incubated at 37 °C in 5% CO~2~ for 48 h. MTT cytotoxicity assays were then performed three times.

2.9. Nuclear and cytoplasmic extraction assay {#s0070}
---------------------------------------------

Cell nuclear and cytoplasmic extraction assay was conducted using NE-PER@ Nuclear and Cytoplasmic Extraction kit (Thermo Scientific Pierce, UK) according to the manufacturer\'s instructions. Briefly, cells were harvested and washed with PBS by pipetting, and were then incubated with ice-cold CER I for 10 min at 4 °C. After incubation, CER II extraction regent was added to the reaction mixture for another 1 min and the lysate was centrifuged at 16000 *g* for 5 min. The supernatant (cytoplasmic extract) was carefully transferred into a fresh microcentrifuge tube and stored in ice. The pellet was resuspended in NER extraction regent and incubated for 40 min on ice. The suspension was then centrifuged at 16000 *g* for 10 min, and the supernatant (nuclear extract) was transferred to a fresh microcentrifuge tube and stored on ice. The proteins were quantitated by BCA protein assay kit, and further analyzed by western blot analysis.

2.10. Western blot analysis {#s0075}
---------------------------

Cell lysates were separated by SDS-PAGE followed by blocking in 3% BSA (Bovine Serum Albumin), then incubated with primary antibodies against *PI3K* (1:1000), *p-PI3K* (Tyr^458^) (1:1000), *AKT* (1:1000), *p-AKT* (Ser^473^) (1:1000), *MAP2K4*(1:500), *c-Jun* (1:1000), *MYH9* (1:1000), β-catenin (1:1000), *GSK3β (*1:500), *p-GSK3β* (1:1000), *c-Myc* (1:1000), *E-cadherin (*1:1000)*, N-cadherin (*1:1000)*, Vimentin* (1:1000)*, Nanog* (1:1000)*, OCT-4* (1:1000)*, Sox2* (1:2000), *TRAF6* (1:1000)*, ubiquitin* (1:1000)*, β-actin* (1:1000); and *GAPDH* (1:1000). The immunoreactive bands were visualized with chemiluminescence (ECL) (Millipore, Bedford, MA, USA), and images were captured with Minichemi imaging system (Sage, Beijing,China). All blot figures include the location of molecular weight/size markers.

2.11. Antibodies {#s0080}
----------------

The following antibodies used in this study for western blotting (WB), Immunohistochemistry (IHC) and immunofluorescence (IF) were: *PI3K* (4292S, Cell Signaling), *p-PI3K* (Tyr458) (11508-2, Singalway Antibody), *AKT* (4691, Cell Signaling), *p-AKT* (Ser473) (4060S, Cell Signaling), *MAP2K4* (17340-1-AP, ProteinTech), *c-Jun* (9165S,Cell Signaling), *MYH9 (*60233-1-IG, ProteinTech), *GSK3β (*22104-1-AP, ProteinTech), *p-GSK3β* (5558S, Cell Signaling), *c-Myc* (AB32072, Abcam), *E-cadherin (*60335-1-IG, ProteinTech)*, N-cadherin (*66219-1-IG, ProteinTech)*, β-catenin (*51067-2-AP, ProteinTech)*, Vimentin (*10366-1-AP, ProteinTech)*, Nanog (*14295-1-AP, ProteinTech)*, OCT-4 (*60242-1-IG, ProteinTech)*, Sox2 (*11064-1-AP, ProteinTech)*, TRAF6 (*66498-1-IG, ProteinTech)*, ubiquitin (*10201-2-AP, ProteinTech)*, β-actin (*CW0264M), *GAPDH (*CW0100M) and *Histone* (\#5748, Cell Signaling), *CD133*(\#64326, Cell Signaling), *CD44*(\#3570, Cell Signaling).

2.12. Transwell and Boyden chamber analysis {#s0085}
-------------------------------------------

Transwell and boyden(BD Biosciences, NJ, USA) assay were performed to detect cell migration and invasion abilities. Cells were suspended in 100 μl RPMI-1640 without serum and seeded into the top chamber of the transwells coated with Matrigel (BD Biosciences, NJ, USA) or left uncoated, and the bottom chambers were filled with 500 μl RPMI-1640 supplemented with 10% FBS. After incubation, the filter was fixed, stained, and photographed. The number of stained cells that exhibited the ability to invade and migrate was counted. All experiments were performed three times.

2.13. In vivo metastasis assays in nude mice {#s0090}
--------------------------------------------

All mice (BALB/C, nu/nu) were 4-weeks-old, female, weighing 11-13 g, approved by the BEIJING HFK BIOSCIENCE CO., LTD. For in vivo metastasis assays, 50 μl of HONE1-EBV^+^ and 5-8F cells(5 × 10^6^) overexpressing miR-BART22 or their control cells were injected under the liver capsule of each mouse (*N* = 8 per group), and then carefully pushed its liver back into the abdominal cavity after cleaning and lightly pressing the pinhole with alcohol cotton balls for 1 min. The mice were maintained in a barrier facility on HEPA-filtered racks and fed an autoclaved laboratory rodent diet. All mice were euthanized after 6 weeks of study. Liver and colon tissues were subjected to fluorescent imaging using the LT-9MACIMSYSPLUS whole-body imaging system (Lighttools Research, Encinitas, CA, USA).

2.14. Establishing the subcutaneous xenograft mouse model {#s0095}
---------------------------------------------------------

The subcutaneous xenograft mouse model was also established to determine the tumor formation abilities. A serial number of 1 × 10^6^, 5 × 10^5^, 1 × 10^5^, and 5 × 10^4^ cells were injected into the mice (*N* = 6 per group), and tumor-initiating frequencies were calculated using extreme limiting dilution analysis (<http://bioinf.wehi.edu.au/software/elda/>).

2.15. DDP-treatment survival experiment on nude mice {#s0100}
----------------------------------------------------

To establish an NPC mouse model, 6 × 10^5^ miR-BART22-overexpressing HONE1 EBV^+^ or control cells, in 0.2 ml buffered saline, were intraperitoneally injected into the mice (*N* = 10 per group). Tumors were allowed to grow for 3 days, and then the animals were divided into four groups for treatment testing: the miR-BART22 + DDP group; the miR-BART22+ Normal saline (NS) group; the control cell + DDP (Mock) group and the miR-BART22+ NS group). Mice were intraperitoneally injected with NS or DDP(4 mg/kg) every 3 days respectively, and survival curves were analyzed using Kaplan-Meier analysis.

2.16. The cinobufotalin-treatment survival experiment on nude mice {#s0105}
------------------------------------------------------------------

5 × 10^6^ miR-BART22 over-expressing in HONE1-EBV^+^ and 5-8F cells were intraperitoneally injected into the mice. The mice were subsequently randomized into 4 groups (*N* = 10 per group). Ten days after injection, group 1 received the DDP (4 mg/kg), group 2 received cinobufotalin (4 mg/kg), group 3 received cinobufotalin with half dose (2 mg/kg), and group 4 received DDP combined with cinobufotalin (4 mg/kg, respectively) by intraperitoneal injection every 5 days. Finally, we observed the survival time of nude mice in different treatment group.

2.17. Immunohistochemistry (IHC) {#s0110}
--------------------------------

Paraffin sections (4 μm thickness) were prepared from in vivo experiments and used for immunohistochemistry to detect *E-cadherin* (1:200), *N-cadherin* (1:200), *OCT4* (1:250), *NANOG* (1:100)*, MAP2K4* (1:100) protein expression. The indirect streptavidin-peroxidase method was used based on the manufacturer\'s instructions. Stained tissue sections were examined separately by two pathologists.

2.18. In situ hybridization (ISH) {#s0115}
---------------------------------

In situ hybridization was conducted on paraffin-embedded specimens (4 μm thickness). Paraffin sections were deparaffinized in xylene and rehydrated in graded alcohols and distilled water. After treating with proteinase K at 37 °C for 30 min, sections were rinsed, fixed, and then prehybridized for 2 h. Hybridization was performed with miR-BART22 Digoxygenin (DIG)-labeled probes designed and synthesized by BersinBio (Guangzhou, China). Slides were hybridized with DIF-labeled LNA probes overnight at 37 °C and were then washed and incubated with anti-DIG-HRP Fab fragments for 1 h at room temperature. Signals were visualized with the 3,3′-Diaminobenzidine (DAB) substrate (Maixin Biotech. Co., Ltd., Fuzhou, China).

2.19. Tumorsphere formation {#s0120}
---------------------------

After digestion, single cell suspensions were seeded into 6-well ultra-low-attachment plates (Corning, Inc., NY, USA) at a density of 5 × 10^3^ cells/well and cultured in serum-free medium DMEM/F12 with FGF (20 ng/ml), EGF (20 ng/ml), and B27 (2%). After 2--3 weeks of incubation, tumor sizes were photographed using microscopy. Then single-cell suspensions were made to form new tumorspheres. The size and number of tumorspheres were analyzed after a continuous passage of three generations.

2.20. Side populations {#s0125}
----------------------

For side population (SP) analysis, 1 × 10^6^ cells (here we used HONE1 cells instead of HONE1-EBV^+^ with green fluorescence because of potential detection by the flow cytometer) were resuspended in DMEM containing 2% FBS, and treated with 5 μg/ml The Hoechst 33342 stain (Sigma-Aldrich, MO, USA) was applied for 90 min at 37 °C with gentle blending every 10 min. Samples were simultaneously incubated with 50 μmol/L of verapamil (Sigma-Aldrich, MO, USA), which served as negative controls. Cells were washed using ice-cold PBS and then subjected to flow cytometric analysis. Propidium Iodide was used to identify dead cells.

2.21. Luciferase reporter assay {#s0130}
-------------------------------

Luciferase reporter assay was used to examine the binding activation of miR-BART22 with PsiCHECK-2 vectors containing *MAP2K4* wt 3′-UTR or mut 3′-UTR. The vector was co-transfected with miR-BART22 mimics, inhibitor or control sequences into cells, and luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). To examine the effect of c-Jun on transcription activity of *MAP2K4, MYH9* and *ubiquitin*, fragments encoding c-Jun binding sites were cloned into pGL4.1-Basic luciferase reporter vector, and the c-Jun-binding site mutation vectors were constructed. These vectors and c-Jun plasmid were co-transfected into HONE1-EBV^+^ and 5-8F cells to detect the luciferase activity.

2.22. Ago2 immunoprecipitation (IP) {#s0135}
-----------------------------------

Cytoplasmic lysates were subjected to RNA immunoprecipitation (RIP) assays using an Ago2-specific antibody (Abcam, USA), and the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer\'s protocol.

2.23. Coimmunoprecipitation (Co-IP) {#s0140}
-----------------------------------

Co-IP was carried out using a Pierce Co-Immunoprecipitation kit (Thermo Scientific, USA) according to the manufacturer\'s instructions. Briefly, total proteins were extracted and quantified. A total of 1000 μg protein in 400 μL supernatant was incubated with 10 μg anti-Flag, anti-*MYH9* or anti-IgG antibodies for 12 h at 4 °C Beads were washed, eluted in sample buffer, and boiled for 10 min at 100 °C. Immune complexes were subjected to Coomassie brilliant blue staining, mass spectrometry, and western blot analysis. Anti-IgG was used as a negative control.

2.24. The chromatin immunoprecipitation (ChIP) assay {#s0145}
----------------------------------------------------

Chromatin immunoprecipitation assays were performed using a ChIP assay kit (Thermo Scientific, Waltham, MA, USA). According to the manufacturer\'s protocol, chromatin was crosslinked, isolated, and digested with Micrococcal Nuclease to obtain DNA fragments. The anti-c-Jun or IgG was added to the reaction systems for immunoprecipitation. After elution and purification, the recovered DNA fragments were subjected to qPCR and PCR. IgG served as a negative control.

2.25. Electrophoretic mobility shift assay (EMSA) analysis {#s0150}
----------------------------------------------------------

The electrophoretic mobility shift assay was conducted using an EMSA Kit (BersinBio, Guangzhou, China) according to the manufacturer\'s instructions. Nuclear extracts were obtained from cells, and concentrations were determined using the BCA assay kit. EMSA was performed in a reaction mixture containing nuclear extracts and biotin-labeled probes. Competition or super-shift assays were performed by adding 100-fold excesses of cold competitors (unlabeled wild-type or mutant probes), or polyclonal rabbit anti-c-Jun (Cell Signaling Technology) to the reaction mixture. After electrophoresis and incubation, signals were recorded and analyzed.

2.26. Immunofluorescence and confocal microscopy {#s0155}
------------------------------------------------

Cells were plated on coverslips in 48-well plates and cultured overnight to allow for cell adherence. After fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100, cells were incubated with antibodies. Cells were then counterstained with 0.2 mg/ml DAPI and visualized with a fluorescent confocal microscope (Carl Zeiss LSM800,Germany).*GSK3β (*1:50), *β-catenin (*1:100)*, MYH9 (*1:50), CD44(1:100), CD133(1:100).

2.27. The cycloheximide (CHX) chase assay {#s0160}
-----------------------------------------

Cells were transfected with scrambles or plasmids and were then incubated with 20 μmol/L MG132 for 0--12 h or left untreated. At different time points post 50 μg/ml CHX treatment, cells were harvested and prepared for Western blot analysis.

2.28. Statistical analysis {#s0165}
--------------------------

All the data were analyzed using SPSS 20.0 software (SPSS Inc. Chicago, IL, USA). The data are expressed as the means ± SD from at least three independent experiments. Statistical significance was determined using the Student\'s two-tailed *t*-test for two groups, the one-way ANOVA for multiple groups, and the two-way ANOVA for tumor growth and the MTT assay. Correlation between gene expression and clinicopathological characters were analyzed using the Chi-square test. Log-rank tests were performed on Kaplan-Meier survival curves to elucidate any significant relationships between gene expression and overall survival in patients. Univariate and multivariate survival analysis was performed using the Cox proportional hazards regression model. All statistical tests were two-sided and *P* \< .05 was considered statistically significant. \* P \< .05, \*\* *P* \< .01 and \*\*\* *P* \< .001.

3. Results {#s0170}
==========

3.1. EBV-miR-BART22 promotes tumor stemness and DDP chemoresistance {#s0175}
-------------------------------------------------------------------

Lentiviral vectors carrying miR-BART22 or control vectors were transfected into HONE1-EBV^+^ and 5-8F cell lines, respectively ([Fig. S1](#fig0050){ref-type="graphic"}a). MiR-BART22 levels were found to be increased in lentiviral-miR-BART22-transfected cells compared with control transfected cells using RT-qPCR (*P* \< .05) ([Fig. S1](#fig0050){ref-type="graphic"}b). Subsequently, the sphere-forming assay revealed that miR-BART22 stimulated sphere growth ([Fig. 1](#fig0005){ref-type="fig"}a). Next, the expression levels of CD133 and CD44 in miR-BART22-overexpressing cells were significantly higher than those in the control groups as measured using immunofluorescence ([Fig. 1](#fig0005){ref-type="fig"}b). Moreover, flow cytometry analysis showed that side population ratio was markedly up-regulated in overexpressed miR-BART22 NPC cells ([Fig. 1](#fig0005){ref-type="fig"}c). In the xenograft model, the estimated tumor-initiating frequency was 5 × 10^4^ when the HONE1-EBV^+^-miR-BART22 cells were inoculated, while the tumor-initiating rate was 1 × 10^5^ when the control cells were inoculated ([Fig. 1](#fig0005){ref-type="fig"}d).Fig. 1EBV-miR-BART22 promotes tumor stemness and DDP chemoresistance(a) The sizes (left panel) and number of spheres (right panel) generated by the miR-BART22-overexpressing and their control NPC cells are shown; three independent experiments were performed. Original magnification, ×100, scale bar, 200 μm; (b) Immunofluorescence staining clearly shows higher CD44 and CD133 expression in the miR-BART22-expressing cells compared to their control cells, ×400, scale bar, 25 μm; (c) Percentages of SP cells are shown in the panel; three independent experiments were performed, \*\**P* \< .01. HONE1 and 5-8F cells were transfected with BART22 mimics. Blank: HONE1 or 5-8F parental cells. (d) tumor formation in nude mice Injection of 10^6^, 5 × 10^5^, 10^4^ or 5 × 10^4^ cells with miR-BART22-overexpression or control cell, respectively in the left and right side (*n* = 6). (e) Effects on migration and invasion of miR-BART22 were measured by Transwell and Boyden Chamber assays in HONE1-EBV^+^-Lv-miR-BART22 and 5-8F-Lv-miR-BART22 cells. ×400, scale bar, 50 μm; \*\*P \< .01, \*\*\**P* \< .001. (f) In vivo intrahepatic and intestinal metastasis assays results after HONE1-EBV^+^-Lv-miR-BART22 and 5-8F-Lv-miR-BART22 injection. Graph described the numbers of mice which occur metastasis or not; χ^2^ test, \**P* \< .05. (g) Dose-response curves of HONE1-EBV^+^ and 5-8F treated with miR-BART22 or miR-mock 48 h after treatment with DDP. Parametric generalized linear model with random effects. (h) Animals were divided into four groups: control group (NC) + Normal saline (NS), NC + DDP, BART22 + NS and BART22 + DDP (each group: *N* = 10). Xenograft tumors are shown for each group after 25 days of DDP or NS treatment. (i) Survival analysis was plotted. Log-rank test.Fig. 1

For migration and invasion analysis, HONE1-EBV^+^-miR-BART22 and 5-8F-miR-BART22 cells or the respective controls were cultured in Transwell and Boyden chambers, respectively. As shown in [Fig. 1](#fig0005){ref-type="fig"}e, miR-BART22 increased the cell migration and invasion abilities compared with those of the control groups. In vivo experiments, mice injected with HONE1-EBV^+^-BART22 or 5-8F-BART22 cells were all displayed extensively intrahepatic dissemination, while four of eight(4/8) mice in the HONE1-EBV^+^ control group and five of eight(5/8) mice in the 5-8F control group developed intrahepatic dissemination. Besides, six of eight(6/8) mice in HONE1-EBV^+^-BART22 group and seven of eight mice(7/8) in 5-8F-BART22 group exhibited intestinal metastasis. Only one mouse with intestinal metastasis was detected in their corresponding control groups ([Fig. 1](#fig0005){ref-type="fig"}f). To study the influence of EBV-miR-BART22 on NPC chemotherapy efficacy, we first tested the viability of miR-BART22-overexpressing cells in different concentrations of DDP and inhibition rates were calculated 48 h after DDP treatment. Compared with the control cells, the IC50 of DPP increased from 4.1 to 11.6 μM in miR-BART22-overexpressing HONE1-EBV^+^ cells. Similarly, the IC50 of DDP in miR-BART22-overexpressing 5-8F cells increased from 6.6 to 14.3 μM ([Fig. 1](#fig0005){ref-type="fig"}g). Furthermore, we validated the chemotherapy resistance of miR-BART22-overexpressing cells in mice models. After miR-BART22-overexpressing or control cells were intraperitoneally injected into mice, mice were divided into four groups: \[[@bb0005]\] miR-BART22 + DDP, \[[@bb0010]\] miR-BART22 + Normal saline (NS), \[[@bb0015]\] normal control + DDP (NC + DPP), and \[[@bb0020]\] NS + NS cell groups ([Fig. 1](#fig0005){ref-type="fig"} h). The growth of tumors was recorded in each group of mice. Kaplan-Meier analysis showed the survival times of mice in the NC + DDP and NC + NS groups were much longer than those in miR-BART22 + NS and miR-BART22 + DDP groups. Compared with the other three groups, BRAT22 + NS group showed the shortest survival time, and the NC + DDP group showed the longest survival time ([Fig. 1](#fig0005){ref-type="fig"}i).

EBV-miR-BART22 inhibitors or controls were transfected into miR-BART22-overexpressing 5-8F and HONE1-EBV^+^ cells. RT-qPCR confirmed that the miR-BART22 inhibitor markedly inhibited miR-BART22 expression in both lines ([Fig. S1](#fig0050){ref-type="graphic"}c). The sphere-forming assay showed that the miR-BART22 inhibitor blocked the formation of tumor spheres ([Fig. S1](#fig0050){ref-type="graphic"}d). Using Transwell and Boyden Chambers, we observed that the miR-BART22 inhibitor reduced NPC cell migration and invasion ([Fig. S1](#fig0050){ref-type="graphic"}e). Subsequently, we examined the effect of the miR-BART22 inhibitor on DDP resistance. Result in [Fig. S1](#fig0050){ref-type="graphic"}f revealed that the IC50 of DDP for the HONE1-EBV^+^ BART22 + IN-NC cells was 12.0 μM, while that of the control cells with miR-BART22 inhibitor was 4.9 μM. The IC50 for DDP in HONE1-EBV^+^ cells was reduced from 5.4 to 3.2 μM after miR-BART22 Inhibitor transfection. Similarly, the IC50 of DDP was 14.7 μM in miR-BART22-overexpressing 5-8F cells, while the miR-BART22-inhibited control cells were 5.8 μM ([Fig. S1](#fig0050){ref-type="graphic"}f). Taken together, the EBV-miR-BART22 inhibitor reversed the stimulation of miR-BART22 on NPC progression and chemoresistance.

3.2. EBV-miR-BART22 activates the *PI3K/AKT* and *GSK3β*/*β-catenin* signaling pathways {#s0180}
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We assessed the influence of EBV-miR-BART22 on the *PI3K/AKT* and *GSK3β/β-catenin* pathways. The data showed that overexpressed EBV-miR-BART22 induced phosphorylation of *PI3K/AKT* signaling. However, the expression change of total *PI3K/AKT* proteins was not observed. Further, total *GSK3β* and phosphorylated *GSK3β* proteins were found to be significantly downregulated, *β-catenin* protein expression was increased in EBV-miR-BART22-overexpressing NPC cells ([Fig. 2](#fig0010){ref-type="fig"}a). In addition, tumor stemness and EMT pathways were also activated in EBV-miR-BART22-overexpressing NPC cells, which included the upregulated expression levels of *Nanog, OCT-4, SOX2, N-cadherin, Vimentin, c-Myc,* and the downregulated expression level of *E-cadherin*. Interestingly, the oncogenic transcription factor c-Jun was also induced by EBV-miR-BART22 in NPC cells. Finally, immunohistochemical staining indicated higher expression of *N-cadherin, Nanog* and *OCT4*, and lower expression of *E-cadherin* in liver-transplanted EBV-miR-BART22-overexpressing NPC tumor tissues compared with liver-transplanted control tumor tissues ([Fig. 2](#fig0010){ref-type="fig"}b).Fig. 2EBV-miR-BART22 activates the *PI3K/AKT/GSK3β/β-catenin* signaling pathway (a) Protein levels of *PI3K, p-PI3K, AKT, p-AKT, c-Jun, β-catenin, GSK3β, p-GSK3β, c-Myc, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4* and *Sox2* were measured by western blot after miR-BART22 overexpression. Levels of related proteins were reduced or increased after BART22 inhibitor transfection or LY294002 treatment. GAPDH served as controls. (b) Immunohistochemistry analysis of E-cadherin, N-cadherin, Nanog and OCT4 expression. Original magnification, ×200, scale bar, 100 μm. (c) Western blotting analysis of the effect of miR-BART22 overexpression on *GSK3β* stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls.Fig. 2

In a subsequent investigation, simultaneous introduction of the miR-BART22 or PI3K inhibitor LY294002 in miR-BART22-overexpressing NPC cells reversed the expression pattern mediated by miR-BART22 upregulation.

Moreover, the cycloheximide (CHX) chasing assay showed that miR-BART22 reduced the half-life of the *GSK3β* protein. The effect of miR-BART22 on *GSK3β* stability could be obviously alleviated by the proteasome inhibitor MG132 ([Fig. 2](#fig0010){ref-type="fig"}c).

3.3. EBV-miR-BART22 directly targets *MAP2K4* {#s0185}
---------------------------------------------

Bioinformatics analysis predicts *MAP2K4* as a direct target of miR-BART22 ([Fig. S2](#fig0055){ref-type="graphic"}a). Western blot and RT-qPCR data demonstrated that miR-BART22 overexpression or miR-BART22 overexpression with the treatment of LY294002 resulted in downregulation or upregulation of *MAP2K4* mRNA and protein levels ([Fig. S2](#fig0055){ref-type="graphic"}b--c). Moreover, immunohistochemistry of xenografts originating from miR-BART22-overexpressing NPC cells indicated a significant reduction in *MAP2K4* protein expression, which was consistent with the in vitro findings ([Fig. S2](#fig0055){ref-type="graphic"}d). To verify if *MAP2K4* is a direct target of miR-BART22, wild-type (Wt) or mutant (mt) 3′UTR psi-CHECK vectors for *MAP2K4* were co-transfected with miR-BART22 mimics or its inhibitors into HONE1-EBV^+^ cells. Luciferase reporter assays confirmed that miR-BART22 directly bound to the 3′UTR of *MAP2K4* ([Fig. S2](#fig0055){ref-type="graphic"}e). To further validate this finding, we carried out RNA immunoprecipitation (IP). As shown in [Fig. S2](#fig0055){ref-type="graphic"}f, Ago2-bound miR-BART22 and *MAP2K4* mRNA levels were markedly increased in HONE1-EBV^+^ cells. Further, *MAP2K4* mRNA enrichment in the Ago2-IP was also increased by miR-BART22. These results indicated that *MAP2K4* is a direct target of miR-BART22.

3.4. *MAP2K4* reduces *MYH9* expression by downregulating *PI3K/AKT/c-Jun*-mediated stimulation {#s0190}
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Coimmunoprecipitation (Co-IP) combined with mass spectrometry were used in HONE1-EBV^+^ cells to screen for potential interacting proteins of MAP2K4. *MYH9* protein was screened as a potential interacting *MAP2K4* protein. Although exogenous Co-IP did not indicate the combination between *MAP2K4* and *MYH9* ([Fig. S3](#fig0060){ref-type="graphic"}a), we accidentally found that *MYH9* mRNA and protein levels were reduced in *MAP2K4*-overexpressing NPC cells compared with that of the control cells ([Fig. S3](#fig0060){ref-type="graphic"}b--c). Based on the ChIP-seq binding peaks in conjunction with UCSC, PROMO and JASPAR bioinformatics software, three c-Jun binding motifs at +22,898 to +22,906 (Site A), −7991 to −7979 (Site B), and −9578 to −9566 (Site C) were found within the transcription regulatory region of *MYH9* ([Fig. 3](#fig0015){ref-type="fig"}a). Knocking down c-Jun significantly reduced *MYH9* mRNA and protein levels by using qPCR and Western blot assays, respectively ([Fig. 3](#fig0015){ref-type="fig"}b--c). Further, the chromatin immunoprecipitation (ChIP) and the electrophoretic mobility shift assay (EMSA) verified that c-Jun bound to all the predicted sites inside the *MYH9* transcriptional regulation region in HONE1-EBV^+^ and 5-8F cells ([Fig. 3](#fig0015){ref-type="fig"}d--e). The luciferase reporter assays confirmed markedly upregulated luciferase activity in NPC cells after c-Jun cDNA transfection compared with that of the control cells ([Fig. 3](#fig0015){ref-type="fig"}f). *MAP2K4* was found to suppress *PI3K/AKT/c-Jun* signaling, which caused decreased the combination of c-Jun with *MYH9* promoter ([Fig. 3](#fig0015){ref-type="fig"}h) and thus suppressed *MYH9* mRNA and protein levels([Fig. 3](#fig0015){ref-type="fig"}g--i).

3.5. *MYH9* interacts with *GSK3β* and promotes *GSK3β* protein *ubiquitin* degradation {#s0195}
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The DOMINE database is used to predict that *MYH9* protein interacts with *GSK3β*. Subsequently, Co-IP analysis and immunofluorescence indicated that *MYH9* bound *GSK3β* and co-localized in the cytoplasm ([Fig. 4](#fig0020){ref-type="fig"}a). Further, we observed that *MYH9* downregulated *GSK3β* and phosphorylated *GSK3β* protein levels and upregulated *β-catenin* protein level. However, elevated *MYH9* did not change *GSK3β* mRNA expression ([Fig. S4](#fig0065){ref-type="graphic"}a--b). Subsequently, we employed the cycloheximide (CHX) chasing assay ([Fig. 4](#fig0020){ref-type="fig"}b) to examine the effects of *MYH9* on *GSK3β* protein stability. Co-IP assays indicated that *MYH9* overexpression promoted *GSK3β* and *ubiquitin* separation and facilitated the separation of *GSK3β* from the *ubiquitin* ligation complex composed of TRAF6 in HONE1-EBV^+^cells ([Fig. 4](#fig0020){ref-type="fig"}c). *GSK3β* K183 was identified as the ubiquitination site \[[@bb0175]\]. Plasmids with the *GSK3β* 183R mutant and wild-type *GSK3β* were transfected into MYH9-overexpressed HONE1-EBV^+^, and Co-IP assay showed that *GSK3β* K183R mutant induced the separation of *GSK3β* and *ubiquitin* and prevented the interaction of *GSK3β* and *ubiquitin* ligation complex composed of TRAF6 ([Fig. 4](#fig0020){ref-type="fig"}d). Finally, we detected the increased nuclear translocation of *β-catenin* in *MYH9*-overexpressing NPC cells ([Fig. 4](#fig0020){ref-type="fig"}e). By nuclear and cytoplasmic extraction assay, we confirmed that *MYH9* up-regulation induced the nuclear enrichment of *β-catenin*, whereas silencing *MYH9* impaired the nuclear enrichment of *β-catenin* in NPC cells ([Fig. 4](#fig0020){ref-type="fig"}f).

Interestingly, we unexpectedly observed that *MYH9* upregulated *ubiquitin* mRNA and protein expressions through stimulating *PI3K/AKT/c-Jun* pathway. Firstly we found that *ubiquitin* mRNA levels were induced in *MYH9*-overexpressing NPC cells compared with the control cells([Fig. S4](#fig0065){ref-type="graphic"}c). UCSC, PROMO, and JASPAR bioinformatic softwares predicted c-Jun as the potential transcription factor of *ubiquitin*([Fig. S4](#fig0065){ref-type="graphic"}d), c-Jun overexpression significantly enhanced *ubiquitin* mRNA expression and protein levels ([Fig. S4](#fig0065){ref-type="graphic"}e--f). The ChIP, EMSA and luciferase reporter assays showed the protein-DNA interactions between c-Jun and all the predicted sites of the *ubiquitin* promoter in HONE1-EBV^+^ and 5-8F cells ([Fig. 5](#fig0025){ref-type="fig"}a--c). Suppressing pPI3K using LY294002 in *MYH9*-overexpressing NPC cells significantly reduced *PI3K/AKT/c-Jun* ([Fig. 5](#fig0025){ref-type="fig"}d) and further decreased the binding of c-Jun with the *ubiquitin* promoter([Fig. 5](#fig0025){ref-type="fig"}e--f) and downregulating *ubiquitin* mRNA and protein levels([Fig. 5](#fig0025){ref-type="fig"}g,d). Finally, we observed that *ubiquitin* overexpression achieved by transfecting *ubiquitin* cDNA increased the level of *GSK3β* ubiquitination ([Fig. S4](#fig0065){ref-type="graphic"}g) and thus reduced *GSK3β* protein expression and upregulated TRAF6 expression in NPC cells with *MYH9* silenced ([Fig. S4](#fig0065){ref-type="graphic"}h). Collectively, *MYH9* interacts with *GSK3β* and promotes *GSK3β* protein ubiquitination degradation by activating *PI3K/AKT/c-Jun*-induced *ubiquitin* expression, which thus increases *β-catenin* expression and its nuclear translocation.

3.6. *MAP2K4* antagonizes the action of EBV-miR-BART22 {#s0200}
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*MAP2K4* plasmids or empty plasmids were respectively transfected into miR-BART22-overexpressing NPC cells and increased *MAP2K4* mRNA and protein expression levels were observed in miR-BART22-overexpressing NPC cells (Supplementary Fig. S5a--b). Overexpressed *MAP2K4* blocked tumor stemness, cell migration and invasion by the tumorsphere formation ([Fig. S5](#fig0070){ref-type="graphic"}c), Transwell and Boyden Chamber ([Fig. S5](#fig0070){ref-type="graphic"}d) assays. The DDP chemoresistance assay revealed significant downregulation of IC50 value in *MAP2K4*-overexpressing cells compared with that of the control cells ([Fig. S5](#fig0070){ref-type="graphic"}e).

Western blot analysis showed that cells with *MAP2K4* plasmids treatment inhibited *pPI3K, pAKT, c-Jun, MYH9,β-catenin, OCT4, NANOG, SOX2, Vimentin, N-cadherin*, and *c-Myc* expressions but increased the *E-cadherin, GSK3β*, and p-*GSK3β* expressions ([Fig. S5](#fig0070){ref-type="graphic"}f).

Further, we found the reduced *GSK3β* ubiquitination level in miR-BART22-overexpressing HONE1-EBV^+^ cells transfected with *MAP2K4* ([Fig. S5](#fig0070){ref-type="graphic"}g). Moreover, transiently transfecting *MAP2K4* into miR-BART22-overexpressing NPC cells reduced the binding of c-Jun with *MYH9* or the *ubiquitin* promoter using the ChIP assays ([Fig. S5](#fig0070){ref-type="graphic"}h--i). We also detected the decreased *ubiquitin* and *MYH9* mRNA levels after *MAP2K4* transfection in miR-BART22-overexpressed NPC cells ([Fig. S5](#fig0070){ref-type="graphic"}j--k).

3.7. Knocking down *MYH9* antagonizes the action of EBV-miR-BART22 {#s0205}
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SiRNAs-*MYH9* were transfected into miR-BART22-overexpressing NPC cells, and qPCR and Western blot analysis showed reduced *MYH9* mRNA and protein expressions ([Fig. S6](#fig0075){ref-type="graphic"}a--b). As expected, the percentage of SP cells was obviously reduced after interference with *MYH9* in the miR-BART22-overexpressing NPC cells ([Fig. S6](#fig0075){ref-type="graphic"}c). Assays using transwell and Boyden Chamber showed that *MYH9* knockdown reversed miR-BART22-induced cell migration and invasion ([Fig. S6](#fig0075){ref-type="graphic"}d). Western blot analysis also showed that siRNAs-*MYH9* treatment of NPC cells decreased *pPI3K, pAKT, c-Jun, β-catenin, OCT4, NANOG, SOX2, Vimentin, N-cadherin*, and *c-Myc* expression but increased the *E-cadherin, GSK3β* and *p-GSK3β* levels ([Fig. S6](#fig0075){ref-type="graphic"}e). In addition, *GSK3β* ubiquitination level was also reduced([Fig. S6](#fig0075){ref-type="graphic"}f). Further, Silencing *MYH9* also reduced the binding of c-Jun with the *ubiquitin* promoter using the ChIP assay ([Fig. S6](#fig0075){ref-type="graphic"}g--h) in miR-BART22-overexpressing NPC cells. Finally, reduced *ubiquitin* mRNA and protein levels were also found in miR-BART22-overexpressing NPC cells with *MYH9* knockdown ([Fig. S6](#fig0075){ref-type="graphic"}i--j).

3.8. Cinobufotalin reversed EBV-miR-BART22-induced DDP resistance {#s0210}
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To study the influence of cinobufotalin on EBV-miR-BART22 on NPC DDP chemotherapy efficacy, we firstly tested the viability of miR-BART22-overexpressing cells in using cinobufotalin combined with DDP. We observed that cinobufotalin markedly reduced the IC50 value in miR-BART-22-overexpressing NPC cells. Compared with the DDP group, the IC50 value of DDP in miR-BART22-overexpressing NPC cells was significantly reduced when combined with cinobufotalin. ([Fig. 6](#fig0030){ref-type="fig"}a--b). Next, we observed that the survival time of nude mice with DDP treatment was a little shorter than those using half-dose cinobufotalin and significantly lower than those with the treatment of the same dose cinobufotalin. More interestingly, nude mice with the combined usage of the same DDP and cinobufotalin dose showed the longest survival time compared to other three groups. The average survival times of nude mice injected with HONE1-EBV^+^-BART22 cells in the cinobufotalin, DDP, half-dose cinobufotalin, and DDP + cinobufotalin groups were 35.8, 28.6, 31.7, 43 days, respectively. The average survival times of the nude mice injected with 5-8F-BART22 cells in the cinobufotalin, DDP, half-dose cinobufotalin, and DDP + cinobufotalin groups were 36.4, 32, 33.2, and 44.3 days, respectively([Fig. 6](#fig0030){ref-type="fig"}c--d). In addition, the weights of the mice in each group were measured, and the changes in weight are shown in [Fig. S7](#fig0080){ref-type="graphic"}a--b. We found that the weight of the mice in the DDP and cinobufotalin half-dose group was markedly reduced compared with those in the DDP + cinobufotalin and cinobufotalin groups. The growth status of mice was observed at different times. Compared with the other three groups, cinobufotalin combined with the DDP group had better growth status and smaller tumor burden([Fig. S7](#fig0080){ref-type="graphic"}c).

3.9. Cinobufotalin inhibited EMT and tumor stemness signals by inducing *MAP2K4* in miR-BART22-overexpressing nasopharyngeal carcinoma {#s0215}
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Using transwell, Boyden, and sphere-forming assays, we showed that cinobufotalin markedly blocked cell invasion, migration, and the formation of tumorspheres induced by miR-BART22 in NPC cells([Fig. S7](#fig0080){ref-type="graphic"}d--f). Bioinformatics analysis predicted +902\~+908 of *MAP2K4* promoter as the binding site of c-Jun ([Fig. S7](#fig0080){ref-type="graphic"}g). In subsequent study, qPCR identified that c-Jun overexpression markedly decreased *MAP2K4* mRNA expression ([Fig. 6](#fig0030){ref-type="fig"}e). Next, the ChIP and luciferase reporter assays showed the protein-DNA interactions between c-Jun and the predicted site of the *MAP2K4* promoter in NPC cells([Fig. 6](#fig0030){ref-type="fig"}f--g). Using pPI3K inhibitor LY294002 in NPC cells, the expression level of *pPI3K/pAKT/c-Jun* was markedly reduced ([Fig. S7](#fig0080){ref-type="graphic"}h), which decreased the binding of c-Jun with *MAP2K4* promoter([Fig. S7](#fig0080){ref-type="graphic"}i) and thus increased the expression of *MAP2K4* mRNA and protein levels([Fig. S7](#fig0080){ref-type="graphic"}j,h). These data demonstrated that *pPI3K/pAKT/c-Jun* negatively modulated *MAP2K4* expression in NPC. In the subsequent investigation, we observed that *MAP2K4* mRNA and protein levels([Fig. 6](#fig0030){ref-type="fig"}h,j) were obviously induced by cinobufotalin via reducing *pPI3K/pAKT/c-Jun*-mediated transcription suppression of *MAP2K4* promoter([Fig. 6](#fig0030){ref-type="fig"}I). Furthermore, *MYH9*/*GSK3β/β-catenin* signal and its downstream tumor stemness and EMT signalings were observed to be significantly attenuated, which included the reduced expression levels of *N-cadherin, Vimentin, NANOG, OCT4, SOX2, β-catenin, c-Myc* protein levels and elevated *E-cadherin, GSK3β*, and *p-GSK3β* protein levels in miR-BART22-overexpressing NPC cells treated with cinobufotalin([Fig. 6](#fig0030){ref-type="fig"}j).

3.10. Increased EBV-miR-BART22 and reduced *MAP2K4* expressions in NPC {#s0220}
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EBV-miR-BART22 expression was markedly upregulated in NPC tissues compared with NP tissues by qPCR analysis ([Fig. 7](#fig0035){ref-type="fig"}a,*p* \< .001). As show in [Fig. 7](#fig0035){ref-type="fig"}b, *MAP2K4* mRNA expression was significantly lower in NPC than in NP samples(p \< .001). Further, we found that miR-BART22 expression was negatively correlated with *MAP2K4* mRNA expression in NPC tissues ([Fig. 7](#fig0035){ref-type="fig"}c,γ = −0.2820,*p* = .0277). In line with mRNA data, the increased miR-BART22 expression was showed in NPC tissues compared to NP tissues ([Fig. 7](#fig0035){ref-type="fig"}d). Inversely, upregulated *MAP2K4* protein was observed in NP tissues compared to NPC tissues ([Fig. 7](#fig0035){ref-type="fig"}e). The clinical characteristics associated with miR-BART22 or *MAP2K4* are summarized in Table S1--2. Increased miR-BART22 expression was positively correlated with clinical stage(*p* = .006) (I-II vs. III-IV),tumor size (T classification; *p* = .032),lymph node metastasis (N classification; *p* = .008) (N0-N1 vs. N2--3). Reduced *MAP2K4* expression was negatively correlated with clinical stage(*p* = .015) (I-II vs. III-IV),tumor size (T classification; *p* = .035). Moreover, increased miR-BART22 expression had poorer overall survival rates by Kaplan-Meier survival analysis ([Fig. 7](#fig0035){ref-type="fig"}f) (*p* = .003). Inversely, increased *MAP2K4* expression had better overall survival rates by Kaplan-Meier survival analysis ([Fig. 7](#fig0035){ref-type="fig"}g) (p \< .001). NPC patients with low *MAP2K4* expression and high expression of miR-BART22 had the worst survival prognosis compared to other 3 groups in [Fig. 7](#fig0035){ref-type="fig"}h (p \< .001). We did not find a significant association between miR-BART22 expression and clinical stage in stratified analysis of survival analysis. However, we observed that increased *MAP2K4* expression showed the better survival prognosis compared to patients with reduced *MAP2K4* expression in III-V stage ([Fig. 7](#fig0035){ref-type="fig"}i--j) (p = .003).Fig. 3*MAP2K4* reduces *MYH9* by downregulating *PI3K/AKT/c-Jun*-mediated stimulation. (a)Bioinformatics analysis was used to predict the binding sites of c-Jun within promoter of *MYH9*. (b)(c) QPCR (*n* = 3 independent experiments, Student\'s *t*-test) and Western blot analysis of *MYH9* expression in c-Jun-silenced NPC cells and their control cells. (d) Chromatin immunoprecipitation analysis (comparison of all groups vs. IgG group) (n = 3 independent experiments, one-way ANOVA) of c-Jun binding to the *MYH9* promoter. (e) The protein-DNA interactions between c-Jun and *MYH9* promoter were determined using the electrophoretic mobility shift assay. (f) Luciferase reporter assays (comparison of all groups vs. control group) (n = 3 independent experiments, one-way ANOVA) were performed to confirm c-Jun binding to the *MAP2K4* promoter. (g) Protein levels of *PI3K, p-PI3K, AKT, p-AKT, c-Jun* and *MYH9* were measured by western blot in NPC cells treated with *MAP2K4* plasmids . (h) Chromatin immunoprecipitation analysis of c-Jun binding to *MYH9* promoter in *MAP2K4*-overexpressed HONE1-EBV^+^ and 5-8F cells. All data are presented as the means ± SD. Experiments were repeated three times. \*P \< .05. (i) QPCR analysis of *MYH9* expression in *MAP2K4*-transfected HONE1-EBV^+^ and 5-8F cells and their control cells (n = 3 independent experiments, Student\'s t-test). \*P \< .05, \*\*P \< .01.Fig. 3Fig. 4*MYH9* interacts with *GSK3β* to promote *GSK3β* ubiquitin protein degradation. (a) Immunofluorescence co-staining of *GSK3β* and *MYH9* to detect colocalization. The fluorescence intensities along the red arrow crossing the cytoplasm were calculated to show the colocalisation of *GSK3β* and *MYH9*, scale bar, 25 μm; Co-immunoprecipitation analysis of the effect of *MYH9* on the interaction with *GSK3β* in HONE1-EBV^+^cells. (b) Western blotting analysis of the effect of *MYH9* overexpression on *GSK3β* stability in NPC cells treated with cycloheximide and MG132 at different time points, GAPDH served as controls. (c) Co-immunoprecipitation analysis of the effect of *MYH9* on the interaction between *GSK3β, ubiquitin* and *TRAF6* in HONE1-EBV^+^cells. (d) Co-immunoprecipitation analysis of *ubiquitin, TRAF6* and wild *GSK3β* or mutant *GSK3β* in MYH9-overexpressed HONE1-EBV^+^ cells. (e) Immunofluorescence costaining of *MYH9* and *GSK3β* expression and localization in *MYH9*-overexpressed or *MYH9*-depleted HONE1-EBV^+^ and 5-8F cells. Scale bar, 25 μm. (f) Nucleic and cytoplasmic proteins were extracted for *β-catenin* detection by western blotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Fig. 4Fig. 5*MYH9* promotes *ubiquitin* transcription via *PI3K/AKT/c-Jun*.(a) Chromatin immunoprecipitation analysis (comparison of all groups vs. IgG group) (n = 3 independent experiments, one-way ANOVA) of c-Jun binding to the Ubc promoter. (b) The protein-DNA interactions between c-Jun and Ubc promoter were determined using the electrophoretic mobility shift assay. (c) Luciferase reporter assays (comparison of all groups vs. control group) (n = 3 independent experiments, one-way ANOVA) were performed to confirm c-Jun binding to the Ubc promoter.(d) Protein levels of *PI3K, p-PI3K, AKT, p-AKT, c-Jun* and *Ubc* were measured by western blot in NPC cells treated with *MYH9* plasmids or both treated with LY294002. (e-f) Chromatin immunoprecipitation analysis of c-Jun binding to Ubc promoter in HONE1-EBV^+^ and 5-8F cells treated with Ly294002. (g) QPCR analysis of Ubc mRNA levels in *MYH9* over-expressed NPC cells with Ly294002 transfected (n = 3 independent experiments, Student\'s t-test). All data are presented as the mean ± SD. Experiments were repeated three times. \*P \< .05.Fig. 5Fig. 6Cinobufotalin reversed EBV-miR-BART22-induced DDP resistanceby inducing the expression of *MAP2K4*.(a)(b) Dose-response curves of HONE1-EBV^+^ and 5-8F treated with miR-BART22 48 h after treatment with DDP, cinobufotalin or DDP combined with cinobufotalin. Parametric generalized linear model with random effects. (c)(d) Animals were divided into four groups: DDP, Cinobufotalin, Cinobufotalin with half dose, and DDP combined with Cinobufotalin (each group: N = 10). Survival analysis was plotted. Log-rank test (*P* \< .001). (e) QPCR analysis of *MAP2K4* mRNA levels in HONE-EBV^+^ and 5-8F cells with c-Jun over-expressed or control (n = 3 independent experiments, Student\'s t-test). (f) ChIP analysis for c-Jun binding to the transcriptional regulatory region of *MAP2K4* in HONE-EBV^+^ and 5-8F cells. (g) Luciferase reporter assays (n = 3 independent experiments, one-way ANOVA) were performed to confirm that c-Jun stimulated the activation of the *MAP2K4* promoter. (h) QPCR analysis of *MAP2K4* mRNA levels in HONE-EBV^+^ with cinobufotalin treated in 0.25, 0.5 μM or control (n = 3 independent experiments, Student\'s t-test). (i) ChIP analysis for c-Jun binding to the transcriptional regulatory region of *MAP2K4* in HONE-EBV^+^ and 5-8F cells with cinobufotalin treatment. All data are presented as the mean ± SD. Experiments were repeated three times. (j) Western blot analysis of *PI3K, p-PI3K, AKT, p-AKT, c-Jun, MAP2K4, MYH9, β-catenin, c-Myc, GSK3β, p-GSK3β, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4* and *Sox2* expression in BART22-overexpressed NPC cells with cinobufotalin treatment in different dose.Fig. 6Fig. 7Increased EBV-miR-BART22 and decreased *MAP2K4* expression in NPC.(a)(b) The expression of EBV-miR-BART22 and *MAP2K4* in NP and NPC samples was determined by qRT-PCR. Student\'s t-test, mean ± SD. (c) Correlations between miR-BART22 and *MAP2K4* expression levles were calculated. Two tailed Spearman\'s correlation analysis. Means ±SD.,*P* = .0277. (d) miR-BART22 expression in NP and NPC samples. (a)(b)Negative expression in NP tissues; (c)weak positive expression of miR-BART22 in NPC samples. (d)strong positive expression of miR-BART22 in NPC samples(original magnification ×400,scale bar, 50 μm). (e) *MAP2K4* expression in NP and NPC samples. (a)(b)strong staining of *MAP2K4* in NP samples. (c)Negative expression in NPC samples; (d)positive expression in NPC samples(original magnification ×400,scale bar, 50 μm). (f)(g) Kaplan-Meier survival analysis of overall survival of 163 NPC patients on the basis of miR-BART22 and *MAP2K4* expression.(log-rank test was used to calculate *P* values). (h) Kaplan-Meier survival analysis of overall survival in 163 NPC patients on the basis of low expression of miR-BART22 and high expression of *MAP2K4*. (log-rank test, P \< .001). (i) Stratified analysis was used to analyze the correlation of the expression of miR-BART22 with survival prognosis in clinical stage(I-V) stage. (j) Stratified analysis was used to analyze the correlation of the expression of *MAP2K4* with survival prognosis in clinical stage(I-V) stage.Fig. 7

4. Discussion {#s0225}
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In this study, we firstly demonstrated that EBV-miR-BART22 acts as an oncogene that promotes tumor stemness, metastasis and DDP chemotherapy resistance using in vitro and in vivo assays. It is well established that the activation of *PI3K/AKT* and *GSK3β/β-catenin* \[[@bb0180],[@bb0185]\] and their downstream tumor stemness and EMT signals is key factor in promoting tumor stemness \[[@bb0190]\], metastasis \[[@bb0195]\] and DDP chemoresistance \[[@bb0200]\], in tumors. In line with these reports, we observed that EBV-miR-BART22 activates the *PI3K/AKT* and *GSK3β/β-catenin* pathway and their downstream tumor stemness and EMT signals, which facilitated CSCs properties, metastasis, and chemoresistance of NPC cells.

To further explore the detailed molecular basis of EBV-miR-BART22 as a stimulator of tumor stemness and EMT signals, bioinformatics analysis was firstly used to predict *MAP2K4* as the target of EBV-miR-BART22. Subsequently, we identified EBV-miR-BART22 directly targets *MAP2K4* in NPC. Further, we used CoIP and mass spectrometry to screen *MYH9* as the potential interacting protein with *MAP2K4* in NPC cells. Unfortunately, we did not detect the combination of *MYH9* with *MAP2K4* based on exogenous co-IP. Interestingly, we observed the downregulated *MYH9* mRNA and protein expression levels in *MAP2K4*-overexpressing NPC cells, which suggested that *MAP2K4* suppressed the *MYH9* expression at the transcriptional level. In a previous study, c-Jun was reported to be an oncogenic transcription factor \[[@bb0205],[@bb0210]\] that could bind to the *MYH9* promoter and transcriptionally upregulate *MYH9* mRNA expression. In a subsequent study, our data further revealed that c-Jun was a downstream positive factor of *PI3K/AKT* signal \[[@bb0215]\], which hinted that *MAP2K4* downregulated *MYH9* expression by suppressing *PI3K/AKT/c-Jun* signals and further reduced the combination of c-Jun and *MYH9* promoter. In subsequent investigation, we confirmed this speculation.

*GSK3β* inactivation stimulates the *β-catenin*-induced stemness and EMT signaling pathways and thus promotes CSC growth, metastasis, and chemoresistance in tumors \[[@bb0220],[@bb0225]\]. Interestingly, we searched the DOMINE database and found that *GSK3β* was a potential protein binding to *MYH9*. Subsequently, *MYH9* was confirmed to bind to *GSK3β* and co-localized in cytoplasm of NPC. Furthermore, *MYH9* overexpression suppressed *GSK3β* protein expression, but not *GSK3β* mRNA level. We hypothesized that *MYH9* was involved in *GSK3β* protein ubiquitination degradation \[[@bb0230],[@bb0235]\]. To confirm this speculation, we transfected NPC cells with an *MYH9* plasmid and observed that *GSK3β* ubiquitination was markedly increased in NPC. In addition, we interestingly observed that *MYH9* stimulated *PI3K/AKT/c-Jun* transcription to upregulate *ubiquitin* expression, which increased E3 ligase TRAF6 level to promote *GSK3β* ubiquitination and protein degradation. Finally, we confirmed that TRAF6-mediated *GSK3β* ubiquitination was greatly reduced when the K183R site of *GSK3β* is mutated \[[@bb0240]\]. Taken together, our data demonstrated that *MYH9* not only interacted with *GSK3β*, but also induced *ubiquitin* and *TRAF6* expression and further promoted *GSK3β* ubiquitin protein degradation, which thus activated *β-catenin* expression and its nuclear translocation in NPC. In a subsequent study, we observed that *MAP2K4* transduction or *MYH9* knockdown in miR-BART22 over-expression NPC cells significantly downregulated *β-catenin*-stimulated tumor stemness and EMT signals, and thus suppressed NPC tumor stemness formation, metastasis and DDP chemotherapy resistance. Altogether, these results confirmed that miR-BART22 is an oncogene that targets *MAP2K4* and further stimulates *MYH9*-mediated *GSK3β* ubiquitin protein degradation, which thus activates *β-catenin* and its downstream tumor stemness and EMT signaling pathways to promote tumor stemness formation, metastasis and chemotherapy resistance. Interestingly, *MYH9* had been shown to mediate EBV infection into nasopharyngeal epithelial cells as a receptor in previous document \[[@bb0245]\]. We speculated that EBV-encoded miR-BART22 can be secreted from NPC cells, which possibly enters into nasopharyngeal epithelial cells, induces *MYH9* expression via targeting *MAP2K4*, and ultimately stimulates EBV infection to nasopharyngeal epithelial cells. However, this idea is still to be determined.

Cinobufotalin is one of the bufadienolides prepared from toad venom with anticancer activity \[[@bb0250], [@bb0255], [@bb0260]\]. Pre-clinical studies showed that chemically synthesized cinobufotalin could induce cell death in lung cancer cells \[[@bb0165]\]. However, comparison of tumor cytotoxicity between cinobufotalin and DDP and their combined application for tumor treatment have never been reported. In this study, we initially observed that cinobufotalin markedly reduced the IC50 value and significantly increased the DDP sensitivity to miR-BART22-overexpressing NPC cells. Further, using in vivo experiments for nude mice with injected miR-BART22-overexpressing NPC cells that simulated advanced NPC, we observed that cinobufotalin markedly prolonged the survival time of nude mice compared to those with the treatment of the same doses and administration time of DDP. More interestingly, we observed that the nude mice showed the longest survival times when cinobufotalin and DDP were used together compared with those that were treated with DDP or cinobufotalin alone. Mechanism analysis indicated that cinobufotalin induced *MAP2K4* expression by suppressing *PI3K/AKT/c-Jun* signaling and thus antagonized miR-BART22-induced *MYH9/GSK3β/β-catenin* and its downstream tumor stemness and EMT signaling, which has never been reported. Attributed to the fact that cinobufotalin induces chemosensitivity to DDP by stimulating *MAP2K4*, a downstream targeted gene of miR-BART22, to antagonize *MYH9/GSK3β/β-catenin* axis in miR-BART22-positive NPC cells, it may also reverse DDP chemoresistance via inducing *MAP2K4* in EBV-negative NPC cells, which is independent of EBV-miR-BART-22. These data suggested that cinobufotalin is a more promising anti-tumor agent than DDP in treating NPC. Furthermore, the coupled use of cinobufotalin and DDP may be a better choice for the treatment of miR-BART22-induced NPC chemoresistance.

In clinical samples, we observed that miR-BART22 was mainly expressed in NPC compared with nasopharyngeal epithelial (NP) tissues. In situ hybridization indicated that miR-BART22 was only expressed in NPC tissues. Increased miR-BART22 obviously promoted clinical progression and poor prognosis in NPC patients. Inversely, *MAP2K4* was predominantly expressed in NP tissues compared to NPC tissues. Reduced *MAP2K4* expression was negatively correlated with clinical progression and positively related to the overall survival time of NPC patients. Furthermore, miR-BART22 expression was negatively correlated with *MAP2K4* protein expression in NPC tissues. NPC patients with high miR-BART22 and low *MAP2K4* protein expression showed the worst survival prognosis compared to the other three group patients. These data demonstrated the significance of abnormal miR-BART22/*MAP2K4* signal in NPC pathogenesis.

Taken together, our study firstly revealed that EBV-miR-BART22 directly targets *MAP2K4* to stimulate *MYH9* expression, which thus induces *GSK3β* protein ubiquitin degradation and further promotes tumor stemness, metastasis and DDP chemotherapy resistance through activation of the *β-catenin*-stimulated stemness and EMT signaling pathways. Furthermore, we found that cinobufotalin could powerfully reverse EBV-miR-BART22-induced DDP resistance by inducing *MAP2K4* to antagonize *MYH9/GSK3β/β-catenin*-stimulated tumor stemness and EMT signals in nasopharyngeal carcinoma. Finally, the combination of increased miR-BART22 and reduced *MAP2K4* expression levels may be the significant marker to predict poor prognosis for NPC patients.

Funding sources {#s0230}
===============

This study was supported by National Nature Science Fund of China (81772872, 81572643, 81872198), Science and Technology Project of Guangdong Province (No.2016A020215233, 20140212, 2014A020212342), People\'s Livelihood Science and Technology grant of Guangzhou Municipal Science and Technology project (No. 201803010023), Scientific research project of Guangdong Provincial Bureau of Traditional Chinese Medicine (No.20193010), Guangzhou Science and Technology plan(No.201804010023, 201707010425), Nature Science Fund of Guangdong Province (2016A030313526, 2017A030313701), The Supporting plan for Special Talents in Guangdong Province (No. 2016TQ03R466), Seeding program of Shenzhen Hospital of Southern Medical University, Guangdong Medical Science and Technology Research Fund Project (No.A2016610). The content is solely the responsibility of the authors and the funders did not have any role in study design, data collection, data analysis, interpretation, or writing of the report.

Authors\' contributions {#s0235}
=======================

Conceptualization, Y.Y.L and W.Y·F; Methodology, Y.Y.L and W.Y·F; Investigation, Q.P.J, X.L, C.L, Z.B.T, M.Y,Z, J.Z, Z.C,X.L; Writing-Original Draft, Y.Y.L; Writing-Review & Editing, W.Y·F; Funding Acquisition, W.Y·F, Z.L, Q.P.J, J.Z, L.B.L, Y.Y.X; Resources, W.Y·F; Supervision, W.Y.F. All authors have read and approved the final manuscript.

Declaration of Competing Interest
=================================

Prof. Fang has a pending patent on Cinobufotalin (No. 201910399834.7). All authors declare that no support, financial or otherwise, has been received from any organization that may have an interest in the submitted work.

Appendix A. Supplementary data {#s3140}
==============================

Fig. S1Related to [Fig. 1](#fig0005){ref-type="fig"}. miR-BART22 inhibitor suppresses malignant phenotype of NPC cells.(a) HONE-EBV^+^ and 5-8F cells were transfected with Lv-miR-BART22 and LEV. Green fluorescent protein (GFP) expression was used to monitor the transfection efficiency. (b) miR-BART22 expression was measured by qRT-PCR following overexpression of Lv-miR-BART22. (c) HONE1-EBV^+^ and 5-8F-Lv-miR-BART22 cells were transfected with miR-BART22 inhibitors or controls. MiR-BART22 expression was measured by qRT-PCR. (d) The sizes (left panel) and number of spheres (right panel) generated by miR-BART22-overexpressed NPC cells with miR-BART22 inhibitor and control treatment are shown; Three independent experiments were performed. Original magnification, ×100, scale bar, 200 μm; (e) Transwell and Boyden Chamber assays, ×400, scale bar, 50 μm. (f) Dose-response curves of HONE1-EBV^+^ and 5-8F treated with miR-BART22 inhibitor or controls. All data are presented as the mean ± SD. Experiments were repeated three times.Fig. S1Fig. S2miR-BART22 directly targets *MAP2K4*.(a) miR-BART22 and its putative binding sequences in the 3′UTR of *MAP2K4*. A mutation was generated in the complementary site that binds to the seed region of miR-BART22. (b) *MAP2K4* expression was detected by qPCR in miR-BART22-overexpressing or miR-BART22-inhibited NPC cells, normalized to β-Actin. Student\'s *t*-test. Mean ± SD., \**P* \< .05, \*\**P* \< .01. (c) Western blot of *MAP2K4* in miR-BART22-over-expressing or miR-BART22-inhibited NPC cells, GAPDH was used as a loading control. (d) *MAP2K4* expression was evaluated by immunohistochemistry in xenografts derived from NPC cells. Original magnification, ×200, scale bar, 100 μm. (e) Luciferase reporter assay was used to determine *MAP2K4* as the direct target of miR-BART22.One-way ANOVA and Dunnett\'s multiple comparison test. \**P* \< .05. (f) Left panel: miR-BART22 and *MAP2K4* mRNA levels were increased in Ago2 IP. Right panel:*MAP2K4* mRNA was increased in miR-BART22 binding-Ago2 complex after miR-BART22 was over-expressed. Student\'s *t*-test, \*P \< .05; \*\*P \< .01.Fig. S2Fig. S3Related to [Fig. 3](#fig0015){ref-type="fig"}. *MAP2K4* reduced the expression of *MYH9*.(a)Co-immunoprecipitation analysis identified the combination of *MYH9* with *MAP2K4*. (b)(c) *MYH9* mRNA and protein levels were analyzed by QPCR and Western blot in *MAP2K4*-overexpressing NPC cells. Mean ± SD, \*P \< .05, \*\**P* \< .01.Fig. S3Fig. S4Related to [Fig. 4](#fig0020){ref-type="fig"}, [Fig. 5](#fig0025){ref-type="fig"}. *MYH9* promote *GSK3β ubiquitin* protein degradation.(a) Western blot analysis of *GSK3β, p-GSK3β* and *β-catenin* expression in *MYH9* overexpressed NPC cells. (b) QPCR (comparison of all groups vs. control group) (*n* = 3 independent experiments, one-way ANOVA) analysis of *GSK3β* expression in *MYH9* overexpressed NPC cells. (c) QPCR(n = 3 independent experiments, Student\'s *t*-test) analysis of Ubc expression in *MYH9*-overexpressed and their control NPC cells. (d) Bioinformatics analysis was used to predict the binding sites of c-Jun within promoter of Ubc. (e)(f) QPCR(n = 3 independent experiments, Student\'s *t*-test) and Western blot analysis of Ubc expression in c-Jun-overexpressed and their control NPC cells. (g) Co-immunoprecipitation and Western blotting analysis of the effect of *ubiquitin* on *GSK3β* expression in *ubiquitin*-overexpressed HONE1-EBV^+^ and the control cells. (h) Western blotting analysis the effects of *ubiquitin* on *Ubc, GSK3β*, and *TRAF6* expression in *ubiquitin*-overexpressed NPC cells with *MYH9*-silenced.Fig. S4Fig. S5*MAP2K4* suppresses malignant phenotypes caused by miR-BART22 in NPC cells.(a)(b) QPCR(n = 3 independent experiments, Student\'s t-test) and Western blot was used to examine the *MAP2K4* mRNA and protein expressions in miR-BART22-overexpressed NPC cells and their control cells. (c) Tumor sphere formation experiment generated by the miR-BART22 NPC cells transfected with *MAP2K4* and their empty control cells are shown; three independent experiments were performed. Original magnification, ×100, scale bar, 200 μm; (d) Transwell and Boyden Chamber assays, (e) Dose-response curves of HONE1-EBV^+^ and 5-8F treated with miR-BART22 inhibitor or controls. Experiments were repeated three times with similar results, mean ± SD, \**P* \< .05. (f) Western blot analysis of *PI3K, p-PI3K, AKT, p-AKT, c-Jun, c-Myc, GSK3β, p-GSK3β, β-catenin, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4, Sox2, Ubc* and *TRAF6* expression in BART22-overexpressed NPC cells with *MAP2K4* transfection. β-actin served as controls. (g) Co-immunoprecipitation analysis of *ubiquitin* in BART22 overexpressed HONE1-EBV^+^ cells with *MAP2K4* transfected. (h--i) Chromatin immunoprecipitation analysis of c-Jun binding to the transcriptional regulatory region of *MYH9* in BART22 overexpressed HONE1-EBV^+^ and 5-8F cells with *MAP2K4* transfected. All data are presented as the mean ± SD. Experiments were repeated three times. (j)(k) QPCR was used to examine the Ubc and *MYH9* mRNA levels in BART22 overexpressed NPC cells with *MAP2K4* transfected (n = 3 independent experiments, Student\'s t-test).Fig. S5Fig. S6Knocking down *MYH9* antagonizes the action of EBV-miR-BART22.(a)(b) QPCR(n = 3 independent experiments, Student\'s t-test) and Western blot analysis of *MYH9* depletion in miR-BART22-overexpressed NPC cells and their control cells. (c) Percentages of SP cells are shown in the panel; three independent experiments were performed, \*\**P* \< .01. (d) Transwell and Boyden Chamber assays. Experiments were repeated three times with similar results, mean ± SD, \**P* \< .05. (e) Western blot examined the expressions of *PI3K, p-PI3K, AKT, p-AKT, c-Jun, β-catenin, c-Myc, GSK3β, p-GSK3β, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4* and *Sox2* in BART22-overexpressed NPC cells with *MYH9* silenced. GAPDH served as controls. (f) Co-immunoprecipitation analysis of *GSK3β* ubiquitination levels in BART22-overexpressed HONE1-EBV^+^ cells with *MYH9* silenced. (g)(h) Chromatin immunoprecipitation analysis of c-Jun binding to the transcriptional regulatory region of Ubc in BART22-overexpressed NPC cells with *MYH9* silenced. (i) QPCR analysis of Ubc mRNA level in BART22-overexpressed NPC cells with *MYH9* silenced (Student\'s t-test). All data are presented as the mean ± SD. Experiments were repeated three times. (j) Western blot analysis of Ubc expression in BART22-overexpressed NPC cells transfected with *MYH9*, GAPDH served as controls.Fig. S6Fig. S7Related to [Fig. 6](#fig0030){ref-type="fig"}. Cinobufotalin reversed EBV-miR-BART22-induced DDP resistance by inducing the expression of *MAP2K4*.(a)(b) A fitted curve was used to determine the weight of each group. (c)Animals were divided into four groups: cinobufotalin, DDP, half-dose cinobufotalin, and DDP + cinobufotalin groups (each group: *N* = 10). Xenograft tumors are shown for each group at different days. (d--e) Migration and invasion were measured by Transwell and Boyden Chamber assays in HONE1-EBV^+^-BART22 and 5-8F-BART22 cells with Cinobufotalin treatment. (f) The sizes (left panel) and number of spheres (right panel) were examined in miR-BART22-overexpressing NPC cells with the treatment of cinobufotalin; three independent experiments were performed. Original magnification, ×100, scale bar, 200 μm; mean ± SD, \*P \< .05, \*\*P \< .01, \*\*\**P* \< .001. (g) Bioinformatics analysis to identify the binding sites of c-Jun within promoter of *MAP2K4*. (h) Western blot analysis of *PI3K, p-PI3K, AKT, p-AKT, c-Jun, MAP2K4* expression in NPC cells with Ly294002 treated, GAPDH served as controls. (i) ChIP analysis of c-Jun binding to the transcriptional regulatory region of *MAP2K4* in HONE1-EBV^+^ and 5-8F cells with Ly294002 treated. (j) qPCR analysis of *MAP2K4* mRNA level in NPC cells with Ly294002 treated. All data are presented as the mean ± SD. \*P \< .05, \*\*P \< .01, Experiments were repeated three times.Fig. S7Supplementary material: Supplement Table 1--2 Related to [Fig. 7](#fig0035){ref-type="fig"}. Supplement Table 3. qPCR Primer sequence. Supplement Table 4. Mimics or Inhibitors sequence.Image 1

Not applicable.

Supplementary data to this article can be found online at <https://doi.org/10.1016/j.ebiom.2019.08.040>.

[^1]: These authors contribute to the equal work.
