MiR‐2110 induced by chemically synthesized cinobufagin functions as a tumor‐metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR‐2110 was cloned and identified in Epstein–Barr virus (EBV)‐positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR‐2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR‐2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT‐PCR, and in situ hybridization were used to observe the expression of miR‐2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR‐2110 on NPC metastasis. Western blot, Co‐IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR‐2110 affecting NPC metastasis. Finally, miR‐2110 induced by CB participates in CB‐stimulated inhibition of NPC metastasis was explored. The data showed that increased miR‐2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR‐2110 markedly restored NPC cell migration and invasion. Mechanistically, miR‐2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4‐induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT‐stimulated epithelial–mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR‐2110 expression by suppressing PI3K/AKT/c‐Jun‐mediated transcription inhibition. Suppression of miR‐2110 significantly restored cell migration and invasion in CB‐treated NPC cells. Finally, a clinical sample assay indicated that reduced miR‐2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR‐2110 is a metastatic suppressor involving in CB‐induced suppression of NPC metastasis.

Natural Science Foundation of Guangdong Province, Grant/Award Number: 2019A1515110022; Shenzhen Key Medical Discipline Construction Fund, Grant/Award Number: SZXK039; Shenzhen Science and Technology Plan Projects, Grant/Award Number: JCYJ20220530154200002 NEDD4, which downregulated NEDD4-induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT-stimulated epithelial-mesenchymal transition signaling and ultimately suppressing NPC metastasis.Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR-2110 expression by suppressing PI3K/AKT/c-Jun-mediated transcription inhibition.Suppression of miR-2110 significantly restored cell migration and invasion in CB-treated NPC cells.
Finally, a clinical sample assay indicated that reduced miR-2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis.In summary, miR-2110 is a metastatic suppressor involving in CB-induced suppression of NPC metastasis.

| INTRODUCTION
2][3] The occurrence of NPC mainly involves genetic susceptibility, Epstein-Barr virus (EBV) infection, and other factors, such as smoking and air pollution.[6][7][8] MicroRNA (miRNA) is a type of endogenous small noncoding RNA that modulates gene expression change via recognizing cognate sequences and interfering with transcriptional, translational, or epigenetic processes. 6,95][16][17][18][19] In NPC, many human and EBV-encoded miRNAs have been reported to be involved in the carcinogenesis of tumors and chemoradiotherapy resistance. 20,213][24] These data demonstrated the significance of miRNAs in NPC pathogenesis.
MiR-2110 is located in chromosome 10q25.3,and it was initially cloned from EBV-positive NPC.6][27] However, it remains unknown whether miR-2110 is involved in the pathogenesis of other tumors, including NPC, and its related roles and mechanisms have not been reported.
We have previously explored the role and molecular basis of chemically synthesized cinobufagin (CB) in tumors.9][30] These data suggest that chemically synthesized CB is a new potential antitumor drug for the treatment of cancer.However, it remains unknown whether CB regulates other targets to inhibit tumor pathogenesis.
Here, we investigated the involvement of miR-2110 in the pathogenesis of NPC and its upstream and downstream regulatory mechanisms.We also examined whether miR-2110 is involved in CBinduced inhibition of NPC metastasis.

| Bioinformatics analysis
GEO database was used, and the constraints were set to Nasopharyngeal carcinoma & Homo Sapiens & Tissue Sample & Expression profiling by high throughput sequencing.Then, the edgr2 package is used in R language to analyze the data set, and the threshold was set to jlogFCj > 1, p < .05.
All the cells were maintained in a humidified and 5% CO 2 incubator at 37 C.

| Transient transfection with siRNAs, plasmid, and miR-2110 mimics/inhibitor
SiRNAs for phosphatase and tensin homolog (PTEN) or has-miR-2110 mimics and inhibitor were designed and composed by Guangzhou RiboBio (RiboBio Inc.) (Table S1).Plasmids of FGFR1 and c-Jun were obtained from Vigene Biosciences (Shandong, China).5-8F and HONE-1 cells were plated onto a six-well plate (Nest Biotech, China) and were kept at 30%-50% confluence for 12 h.Then, the siRNAs, mimics, inhibitors, and plasmids were transfected into NPC cells via using Lipofectamine TM 3000 (Invitrogen Biotechnology, China) in line with the manufacturer's protocol.Cells were collected for subsequent experiments after 72 h.

| Lentivirus
Lentivirus particles for miR-2110 and control vector were purchased from GeneChem (Shanghai, China).5-8F and HONE-1 were infected with lentiviral vectors or control vectors in line with manufacturer's protocol.The preliminary infection efficiency was judged by green fluorescent protein expression and quantitative real-time polymerase chain reaction (qRT-PCR) assays.

| RNA isolation, RT-PCR, qPCR, and primers
Total RNA was obtained from NPC cells by using TRIzol reagent (Takara, Shiga, Japan).Then, RNA was transformed into complementary DNA (cDNA).cDNA was used as a template to perform amplification with specific primers by using SYBR Premix ExTaq (TaKaRa, Dalian, China).Specific primers are shown in Table S2.

| Wound healing assay
5-8F and HONE-1 cells were transfected with different plasmids or si-RNAs for 24 h.Then, the cells with different treatments were scratched by a 1000 μL pipette tip across the center of the well.RPMI-1640 without serum was used to culture cells.The cell migration ability was assessed by measuring the distance traveled into the wound at 0 and 48 h after scratching.

| Immunofluorescence and confocal microscopy
NPC cells were plated onto coverslips in 48-well plates and cultured overnight to allow cell adherence.Then, 4% paraformaldehyde was added into NPC cells, and the cells were undergone permeabilization in 0.2% Triton X-100 for about 30 min.Subsequently, antibodies with different concentrations guided by instructions were added to NPC cells and incubated overnight.On the second day, the coverslips were incubated with DAPI for 5 min and visualized with a fluorescent confocal microscope.

| Boyden assay
The 24 μg/mL of Matrigel (R&D Systems, USA) was added on the top of chamber for 30 min in advance.A 8 Â 10 4 cells with specific treatment were put into 100 μL RPMI-1640 without serum.Then, the bottom chambers were filled with 500 μL RPMI-1640 mixed with 10% fetal bovine serum.The filter was fixed, stained, and photographed after 15 h.Cells were counted under a microscope in three random fields.

| Dual luciferase report assay
Dual luciferase report assay was used to examine whether miR-2110 directly targets FGFR1.The specific detection process is implemented according to our previous reports. 31Briefly, the 3 0 UTR fragment of FGFR1 including miR-2110-binding site was cloned into the psiCHECK-2 vector.Correspondingly, site-directed mutagenesis of FGFR1 3 0 UTR sites binding to miR-2110 was carried out using the GeneTailor system (Invitrogen), which is taken as the control of wild (wt) FGFR1 3 0 UTR fragment.The wild (wt) or mutant (mt) type of the FGFR1 3 0 UTR vectors and psiCHECK-2 was co-transfected into the cells with miR-2110 mimics and inhibitors.

| ChIP assay
The interaction between c-Jun and miR-2110 was tested by ChIP assay kit (Thermo Scientific, Waltham, MA, USA).In order to obtain DNA fragments, chromatin was crosslinked, isolated, and digested with Micrococcal Nuclease.The IgG or anti-c-Jun was added in the reaction system for immunoprecipitation.After eluting and purifying the recovered DNA fragments, qPCR and agarose gel electrophoresis assays were conducted to detect the combination of c-Jun with miR-2110 promoter.

| Co-IP assay
Co-immunoprecipitation (Co-IP) was performed with a Pierce coimmunoprecipitation kit (Thermo Scientific, USA) in line with the manufacturer's protocol.About 1000 μg of protein was mixed with 10 μg of special antibodies overnight at 4 C.The mixture was analyzed by western blot.

| Cycloheximide chase assay
NPC cells were transfected with FGFR1 or control plasmids for 48 h, and then, 20 μmol/L of MG132 (Sigma-Aldrich, MO, USA) was added into them for 6 h.At different time points post 50 μg/mL cycloheximide (CHX) treatment, the cells were prepared for western blot.

| Immunohistochemistry
Paraffin sections (4-μm thick) came from the lung tissues of experimental nude mice and the protein expression of E-cadherin (1:100), N-cadherin (1:100) or FGFR1 (1:100) were detected from these paraffin sections.These sections were placed into the citrate buffer and heat it to 100 C for antigen extraction.Then, 3% H 2 O 2 and goat serum were used to block the nonspecific antigens and endogenous peroxidase activity.Then, the slides were socked in antibody diluent of E-cadherin, N-cadherin, and FGFR1 overnight at 4 C.The sections were incubated with biotin-labeled secondary antibody and visualized by using a diaminobenzidine (DAB) substrate.Finally, the sections were counterstained with hematoxylin, mounted in neutral gum, and analyzed by brightfield microscope.The more percentage of positivestaining areas and cytoplasmic staining intensity of the cells are, the more expression of these proteins.

| In vivo metastasis assay
All mice were 3 weeks old, female, and weighed 7-9 g and were obtained from the Shanghai Laboratory Animal Center (Shanghai, China).They were divided into two groups (each group: N = 4) and one of them was injected 100 μL of 5-8F with miR-2110 lentivirus particles cells via tail vein and another group was injected overexpressing control cells for the same cells.Their lung tissues were removed after 7 weeks, and the metastatic tissues were analyzed under fluorescence microscopy.All animals were treated according to the principles and procedures outlined in the Southern Medical University Guide for the Care and Use of Animals.

| In vivo metastasis treatment with CB alone and CB coupled with miR-2110-inhibitor
The models of pulmonary metastasis were established by tail-vein injection with a control vector in 5-8F cells and were divided into three groups (each group: N = 4) including control group, CB combined with inhibitor of miR-2110 group and CB-treated group.Simply, three groups of cultured cells were collected to prepare suspension.One group simply added transfection reagent Entranster in vivo wrapped control miR control as control, and the other two groups added CB + Entranster in vivo or CB + Entranster in vivo wrapped miR-2110 inhibitor respectively, and then injected into the tail vein of nude mice respectively.After that, the same therapeutic reagent was injected into tail vein of the three groups of nude mice every 5 days.The injection amount of CB was 5 mg/kg for each mouse and miR-2110 inhibitor or its control is 12 μg for each mouse.The mice were killed 3 days after the eighth injection, and the lung fluorescence was observed by fluorescence microscope.

| In situ hybridization
The NPC tissue array including 123 NPC samples was purchased from biological company (Shanghai outdo biotech Co. Ltd.).The approval from the Ethics Committee was obtained from the company.In situ hybridization was conducted on paraffin-embedded specimens (4 μm thickness).The sections were added with proteinase K (2 mg/mL) at 37 C for 30 min with dewaxing for 1 h and processing with 3% H 2 O 2 .
Then, the sections were washed and prehybridized for 2 h at 37 C.
Hybridization was performed with miR-2110 digoxygenin (DIG)labeled probes designed and synthesized by Exiqon (Guangzhou, China).Slides and DIG-labeled LNA probes were mixed and hybridized for overnight at 37 C.The slides were incubated with alkaline phosphatase-conjugated sheep anti-DIG-Fab fragments for 1 h at room temperature.Signals were visualized with the 3,3 0 -DAB substrate (Maixin Biotech.Co., Ltd., Fuzhou, China).

| Transcription activity
The effect of c-Jun on the transcription activity of miR-2110 promoter was explored using luciferase reporter assays.Sequences of miR-2110 promoter containing c-Jun binding sites and their respectively control mutation sites were cloned to the pGL4.1-Basicluciferase reporter vector.Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) was utilized to examine luciferase activity after the plasmids were co-transfected with c-Jun plasmids into cells.

| Chemicals
Cinobufagin was a purified compound with the purity at 99.18% and purchased from MCE (HY-N0879) (https://www.medchemexpress.cn/Pseudobufarenogin.html).Powdered cinobufagin was dissolved using dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) for 25 mg/mL and further diluted to a suitable concentration for experiments.Then, we examined the expression of miR-2110 in NPC tissues via in situ hybridization (Figure 1C).Subsequently, we analyzed the relationship between the expression of miR-2110 and clinical characteristics in NPC patients of them.The data showed that there was no significant correlation between miR-2110 expression and age, sex, or M stage.However, we found a clear negative correlation between miR-2110 expression and clinical stage ( p = .025),T stage (p = .013),or N stage (p = .024)(Table 1).Besides, the results also showed that those who had higher expression of miR-2110 owned longer survival time than the patients with lower expression of miR-2110 (Figure 1D).

| Statistical analysis
A subsequent stratum analysis showed that high miR-2110 expression

| Increased miR-2110 suppresses NPC metastasis
To explore the effect of miR-2110 on NPC metastasis, we used lentiviral vectors carrying miR-2110 or a control for stable overexpression  Furthermore, western blot analysis was used to detect the key signals of EMT, which revealed that E-cadherin was upregulated and that N-cadherin was downregulated after overexpression of miR-2110 (Figure 2E).Subsequently, silencing miR-2110 via a specific inhibitor (Figure S1C) promoted the migration and invasion of NPC cells as detected by wound healing and Boyden chamber assays (Figure S1D,E).Then, western blot assay showed that E-cadherin is suppressed while N-cadherin is induced with silencing miR-2110 (Figure S1F).Taken together, these data revealed that miR-2110inhibits NPC metastasis.
Western blot analysis showed that miR-2110 overexpression or inhibition downregulated or upregulated, respectively, the protein level of FGFR1 (Figure 3C).In addition, immunohistochemical staining of pulmonary metastasis induced by miR-2110-overexpressing NPC cells demonstrated a significant decrease in FGFR1 protein expression, which agreed with the in vitro experiments (Figure 3D).Subsequently, to confirm whether FGFR1 is the direct target of miR-2110, the luciferase reporter assays were performed.The data showed that the luciferase reporter activities were downregulated with miR-2110 mimics, whereas the luciferase reporter activities were upregulated with miR-2110 inhibitors (Figure 3E).Meanwhile, this effect was not observed in the psiCHECK-2 vector group.These results demonstrated that FGFR1 is a direct target of miR-2110.Furthermore,

| The interaction between FGFR1 and PTEN
In the present study, GeneMANIA and STRING databases were utilized to predict the interaction of FGFR1 with NEDD4 or PTEN (Figure S3A,B).An immunofluorescence assay showed that FGFR1 colocates in the cytoplasm with PTEN in 5-8F and HONE-1 cells (Figure 4A).Previous studies have reported that NEDD4, an E3 ubiquitin ligase, downregulates the expression of PTEN in various cancers. 32Then, a Co-IP assay revealed that FGFR1 interacted with NEDD4, PTEN, and ubiquitin (Figure 4B), and FGFR1 overexpression enhanced the interaction between NEDD4 and PTEN (Figure 4C).In order to detect the relationship of NEDD4 on PTEN, the specific inhibitor of NEDD4 was imported into FGFR1-overexpression NPC cells, and the results of western blot showed that the downregulation of NEDD4 reversed the inhibition of FGFR1 on PTEN (Figure 4D).
Meanwhile, wound healing and Boyden assays also demonstrated that Si-NEDD4 offsets the tumor growth effect of FGFR1 (Figure S3C,D).
These findings indicated that FGFR1 induced the downregulation of PTEN via recruiting NEDD4.

| MiR-2110 induces degradation of PTEN by ubiquitination
FGFR1 overexpression decreased PTEN protein expression levels (Figure 5A).Yet, it did not alter PTEN mRNA expression (Figure 5B), do with proteasome.However, after treatment with CHX, PTEN had longer half-life in miR-2110-overexpressing NPC cells compared with control cells (Figure 5C).In addition, FGFR1 overexpression reversed the miR-2110-induced prolonged PTEN half-life (Figure 5D).These data revealed that miR-2110 induced the ubiquitination degradation of PTEN.

| CB induces miR-2110 via inactivating PI3K/ AKT/c-Jun-mediated transcription inhibition
We have previously demonstrated that CB promotes ENKUR and MAP2K4 through the PI3K/AKT/c-Jun axis in NPC.Here, we further showed that CB induced miR-2110 expression in NPC (Figure 6A).To explore how CB regulates miR-2110, we used JASPAR and ALEES-CAN websites to predict two c-Jun-binding sites in the miR-2110 promoter (Figure 6B).Subsequently, we demonstrated that c-Jun promotes the miR-2110 expression in NPC cells (Figure 6C).Further, we confirmed that c-Jun binds to the promoter region of miR-2110 via a ChIP and promoter report activation assays (Figure 6D-F).Furthermore, western blot analysis showed that the expression levels of p-PI3K, p-AKT, and c-Jun were downregulated after treatment with CB or LY294002 (Figure 6G).qRT-PCR also revealed that miR-2110 mRNA levels were increased after CB or LY294002 treatment (Figure 6A).These data demonstrated that CB inactivates the PI3K/ AKT/c-Jun pathway to induce miR-2110 expression.

| DISCUSSION
4][35][36] NPC is an EBV cancer and it is different from other head and neck cancers due to distinctive geographical, etiological, and biological features. 37,38ny studies have shown that the pathogenesis of NPC is closely related to miRNAs. 39,40R-2110 was cloned from EBV-positive NPC samples, but its role and molecular basis have not been reported in NPC.We have previously reported that reduced levels of miR-2110 are negatively Distant metastasis of tumors is the main cause of death in patients with NPC. 41,42Interestingly, we found that the reduced expression of miR-2110 was negatively related to lymph node metastasis of NPC, suggesting that miR-2110 is a metastasis suppressor in NPC.Previous studies have demonstrated that miR-2110 is absorbed by the long noncoding RNAs, AFAP1-AS1, and ARAP1-AS1, to reduce the targeting of SP1 and HDAC2, respectively, thereby promoting breast invasion. 25,26However, these studies lack animal models to verify the functions of miR-2110.In the present study, we established stable overexpression of miR-2110 in NPC cells.In vitro and in vivo studies showed that miR-2110 significantly inhibits the migration, invasion, and metastasis of NPC cells.After suppressing the expression of miR-2110, the migration and invasion abilities of NPC cells were significantly restored.These data supported that miR-2110 functions as a metastasis suppressor in NPC.
To explore the molecular basis for miR-2110-mediated NPC metastasis inhibition, bioinformatics analysis was applied to predict the target of miR-2110 and found that FGFR1 is its potential target.
4][45][46][47] In the present study, we confirmed that miR-2110 directly downregulated FGFR1 protein expression by binding to its 3 0 UTR in NPC cells.Interestingly, a previous study has reported that FGFR1 downregulates PTEN protein expression in stem cell leukemia/lymphoma syndrome. 480][51] Most anticancer effects of PTEN depend on its lipid phosphatase activity.3][54] Yet, the specific molecular mechanism of FGFR1 inhibiting PTEN protein expression has not been reported.
6][57] In addition, NEDD4 has also been reported as a tumor promoter in NPC. 58Interestingly, both NEDD4 and PTEN were predicted as the interacting proteins of FGFR1 based on GeneMANIA.
Subsequently, we identified the combination of FGFR1 and NEDD4 or PTEN.In addition, FGFR1 overexpression downregulated the protein expression of PTEN in NPC cells.Yet, PTEN mRNA level did not show the significant change.Further, we found that overexpressed FGFR1 recruits NEDD4 to ubiquitinate and degrade PTEN protein.
To explore the role of FGFR1 in offsetting miR-2110 action, CB, a cardiotonic steroid or bufotalin, is isolated from toad venom and has an antitumor effect on many cancers. 59,60Our group recently identified a chemically synthesized CB that significantly inhibits the proliferation, metastasis, and tumor stemness of NPC, lung adenocarcinoma, and hepatocellular carcinoma. 29,30,61In the present study, we demonstrated that CB also significantly upregulated miR-2110 expression in NPC.Further, we showed that chemically synthesized CB activates miR-2110 expression via inactivating PI3K/AKT-induced c-Jun expression, which is an oncogenic transcriptional factor that binds to the miR-2110 promoter and negatively modulates its expression in NPC.
We have previously demonstrated that CB inhibits the migration All statistical analyses were performed via using SPSS version 24.0 (SPSS, Inc., Chicago, IL, USA).Data were shown as the means ± standard deviations.Student's t-test was used to analyze two groups, and more than two groups' data were analyzed by analysis of variance.Chi-square testing was used to determine the relationship between miR-2110 expression and influence factors.The survival analysis was shown by using the Kaplan-Meier analysis method.All statistical tests were two sided, and a p-value of <.05 was considered statistically significant.*p < .05,**p < 0.01, and ***p < .001. 3 | RESULTS 3.1 | Reduced miR-2110 is negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival We have predicted that miR-2110 might be downregulated in NPC via bioinformatics analysis (Figure 1A).In order to verify this F I G U R E 1 Clinicopathologic characteristics of miR-2110 expression in nasopharyngeal carcinoma (NPC).(A) Bioinformatics analysis for the expression of miR-2110 in NPC and NP tissue.(B) The mRNA expression of miR-2110 in five NPC cells and NP cells.(C) In situ hybridization technology for detecting miR-2110 level in NPC tissue array.(D) Kaplan-Meier survival analysis with a log-rank test was complied according to miR-2110 expression in NPC tissue array.(E and F) Stratum analysis for the overall survival of early stage or advanced stage NPC patients was conducted according to miR-2110 expression in NPC tissue array.prediction, we have detected the mRNA level of miR-2110 in five NPC cells and NP cell.The results showed that the expression of miR-2110 in NPC cells were significantly lower than NP cell (Figure 1B).

in 5 -
8F and HONE-1 cells (FigureS1A), and the overexpression efficiencies were evaluated by qRT-PCR (FigureS1B).Wound healing and Boyden chamber assays showed that overexpression of miR-2110 significantly inhibited the migration and invasion of NPC cells (Figure2A,B).In vivo experiments were conducted to explore the effect of miR-2110 on NPC metastasis.Models of pulmonary metastasis were divided into two groups, and control vector or stable miR-2110 expression 5-8F cells were administered to mice via tail vein injection.Compared with the control group, miR-2110 overexpression resulted in fewer metastatic tumors and smaller tumor sizes (Figure2C).Immunohistochemistry analysis showed higher expression of E-cadherin and lower expression of N-cadherin in the miR-2110 group compared with the control group (Figure2D), which demonstrated that increased miR-2110 suppresses NPC metastasis in vivo.

FGFR1
plasmids were transfected into miR-2110 overexpressing NPC cells (Figure S2A), and wound healing and Boyden chamber assays were performed.The results showed that FGFR1 reversed the miR-2110-induced inhibition of NPC cell migration and invasion F I G U R E 4 (A) Immunofluorescence staining was applied to show the interaction between FGFR1 and PTEN in 5-8F and HONE-1 cells.(B) Co-IP assays were applied to show the interaction with FGFR1, PTEN, UBC, and NEDD4.(C) The effect of FGFR1 on the interaction with FGFR1, PTEN, UBC, and NEDD4.(D) The protein level of FGFR1, PTEN, and NEDD4 was showed with or without Si-NEDD4.(Figure S2B,C).These results indicated that FGFR1 is a direct target of miR-2110 and reverses miR-2110-induced suppression of NPC metastasis.
which suggested that FGFR1 suppresses PTEN expression possibly occurring at protein modification level.To verify this hypothesis, western blot analysis was performed and the half-life of PTEN remained unchanged after treatment with MG123 (Figure 5C,D), which demonstrated that the protein change of PTEN had nothing to F I G U R E 5 FGFR1 recruits NEDD4 to degrade PTEN protein by ubiquitination.(A and B) The protein and mRNA level of PTEN in nasopharyngeal carcinoma (NPC) cells with or without FGFR1 over-expression.(C and D) The effects of miR-2110 and FGFR1 on PTEN protein stability were analyzed in NPC cells incubated with cycloheximide at different time points with or without MG132.CHX, cycloheximide.

F I G U R E 6
Cinobufotalin (CB) induces miR-2110 suppression of PI3K/AKT/c-Jun-mediated transcription inhibition.(A) The expression of miR-2110 was analyzed in nasopharyngeal carcinoma (NPC) cells with CB and LY294002 treatments.(B) Two binding sites of c-Jun to miR-2110 from bioinformatics analysis websites.(C) The effect of c-Jun on miR-2110 expression level.(D and E) The binding of c-Jun to miR-2110 promoter via ChIP and gel electrophoresis assays.(F) Luciferase reporter assays were performed to confirm c-Jun binding to the miR-2110 promoter.(G) The protein levels of p-PI3K, p-AKT, and c-Jun were displayed in NPC cells with CB, LY294002 or untreated.

3. 7 |
Suppression of miR-2110 restores cell migration and invasion in CB-treated NPC cellsWe have previously shown that CB inhibits migration and invasion of NPC cells.Here, silencing of miR-2110 in CB-treated NPC cells recovered the migration and invasion abilities as detected by wound healing and Boyden chamber assays (Figure7A,B).Moreover, western blot analysis demonstrated that the expression levels of p-PI3K, p-AKT, FGFR1, and N-cadherin were upregulated while the expression levels of PTEN and E-cadherin were downregulated after silencing miR-2110 in CB-treated NPC cells (Figure7C).In the models of NPC lung metastasis with CB alone or CB + miR-2110 inhibitor treatment, we found that compared with the control treatment group, CB treatment showed the worst metastatic ability.Interestingly, using miR-2110 inhibitor in CB treatment, the metastatic ability of NPC cell can be partially restored (Figure7D).These results indicated that silencing miR-2110 restores the CB-induced inhibition of NPC migration, invasion, and metastasis.

FGFR1
cDNA was transfected into NPC cells with miR-2110 overexpression.The data showed that FGFR1 significantly restored the migration and invasion abilities of NPC cells via suppressing PTEN.In addition, the downstream PI3K/AKT-induced EMT signal inhibited by PTEN is reactivated by FGFR1.These data demonstrated that miR-2110 directly targets FGRF1 to reduce PTEN ubiquitination and degradation.Increased PTEN inactiviated PI3K/AKT-induced EMT signal and ultimately suppressed NPC migration, invasion, and metastasis.
and invasion of NPC.To determine whether miR-2110 participates in CB-mediated inhibition of NPC migration and invasion, CB-treated NPC cells were exposed to a miR-2110 inhibitor in the present study, which significantly increased the invasive and migratory abilities of CB-treated NPC cells.In addition, the expression levels of PTEN and E-cadherin were downregulated, and the expression levels of FGFR1, PI3K/AKT, and N-cadherin were upregulated.Finally, in vivo experiments indicated that inhibition of miR-2110 partially restores the metastatic ability of NPC cells in CB-treated NPC cells.These data demonstrated that miR-2110 modulated FGFR1/PTEN/PI3K/AKT to suppress EMT signal, which thus participated in CB-induced inhibition of NPC migration, invasion, and metastasis.In summary, the present study revealed that reduced miR-2110 is an unfavorable factor promoting NPC progression and poor prognosis.Further, miR-2110 is induced by CB via suppressing the PI3K/AKT/c-Jun pathway and directly targeting FGFR1 to attenuate its expression, thus decreasing the interaction between NEDD4 and PTEN, which suppresses the ubiquitination and degradation level of PTEN.As a result, PI3K/AKT and downstream EMT signaling are further suppressed, which reduces NPC metastasis.The present findings emphasized the critical role of miR-2110/FGFR1/NEDD4/PTEN in NPC metastasis, providing a potential therapeutic target for NPC patients with distant metastasis.AUTHOR CONTRIBUTIONS Z.L, C.C, and W.Y.F conceived the study, secured research funding, supervised all aspects of the work.S.Y.F, R.T.H, M.M.Z, L.Z.P, and X.D designed the research, prepared the figures and tables, and wrote the paper.S.Y.F, L.Z.P, X.N.L, J.Y.X, and L.R.P designed and performed experiments, interpreted data.X.N.L, Y.Y.L, Z.H.L, and J.Y.X was contributed with data analysis.B.X.Z participated in manuscript revision.Y.Y.X operate advanced instruments and image analysis.All authors read and approved the manuscript.
Correlation of miR-2110 protein expression with clinicalpathological parameters.Note: χ 2 test was applied to access the correlation between the clinicopathologic parameters and miR-2110 expression.
T A B L E 2 Univariate and multivariate Cox regression analysis in 123 nasopharyngeal carcinoma patients.positivelycorrelatedwith the survival time of advanced-stage, but not early-stage disease (Figure 1E,F).Univariate and multivariate Cox regression analyses in 123 patients with NPC showed that miR-2110 expression, N and M classification was independent prognostic factors (Table2).