The nasopharyngeal cancer CNE1 cell line (Catalogue number: WN-10371, Homo sapiens, epithelioid) derived from a 58-year-old female patient with nasopharyngeal carcinoma and 6-10B cell line (Catalogue number: WN-10197, Homo sapiens, epithelioid) were purchased from Wuhan Warner Bio Co., Ltd (Wuhan, China). Mycoplasma testing of the two cell lines measured by PCR were both negative. The cell lines used in the manuscript have been authenticated using Short Tandem Repeat (STR) analysis as described in 2012 in ANSI Standard (ASN-0002) by the ATCC Standards Development Organization (SDO). The cell lines were cultured in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 100 U/mL streptomycin, and 100 U/mL penicillin. All the cell lines were incubated in a humidified incubator with 5% CO${}_2$ at 37 °C and culture medium was replaced each 24 h. The third passages of nasopharyngeal cancer cell lines at the logarithmic phase of growth were used for further experiments.

The PinX1-overexpression plasmid (pcDNA3.0-PinX1) and the empty plasmid were constructed by Guangzhou Vipotion Biotechnology Co., Ltd (Guangzhou, China). Before transfection, CNE1 cells (2 $\times$ 10${}^5$ cells per well) were grown on 6-well plates (Corning Incorporated, Corning, NY, USA) to ca. 60%–70% confluency. 16 $\mu$L of Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA) and 400 $\mu$L of Opti-MEM (Invitrogen, USA) were mixed for 10 min at room temperature. Thereafter, the mixtures were incubated with 12 $\mu$L of plasmid (20 $\mu$M) in 400 $\mu$L of Opti-MEM (Invitrogen, USA) for 15–20 min at room temperature to form complexes of Lipofectamine™ 2000 and plasmid. Then, the complexes were added to the CNE1 cells in each well. The culture medium was discarded and replaced with fresh medium containing 10% FBS and antibiotics after culturing for 5 h. The cells were harvested after 2 d of transfection, and PinX1 expression was determined using reverse-transcription quantitative PCR (RT-qPCR) and western blotting. The cells were divided into the following groups: blank (cells without any transfection); Vector (cells transfected with the empty vector); Over-PinX1 (cells transfected with pcDNA3.0-PinX1); and Over-PinX1 + 3-MA (cells transfected with pcDNA3.0-PinX1 and treated with 3-methyladenine).

The proliferative capacity of the transfected and non-transfected CNE1 cells was measured via MTT Assay Kit (ab211091, Abcam, Cambridge, UK). Briefly, the cells were seeded onto 96-well plates at 1 $\times$ 10${}^5$ cells per well after culturing for 24, 48, and 72 hours. Microplate readers were used to measure the absorbance at 570 nm after adding MTT to the plates and incubating them for 2 h. To determine the mean values, the experiment was repeated three times. Cell viability curves were plotted with the culturing time as the abscissa and the optical density (OD) value as the ordinate.

Migration and invasion by transfected and non-transfected CNE1 cells were determined using Transwell assays. Migration analysis was performed with DMEM containing 10% FBS (Hyclone) in the lower chamber and 2 $\times$ 10${}^4$ cells in serum-free medium in the upper chamber. Invasion analysis followed a similar protocol, except that the chambers were covered with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA). Cells in the lower chambers were fixed and stained with 4% paraformaldehyde and 0.1% crystal violet, then counted under an OLYMPUS CX41 upright microscope. We randomly selected four fields of vision from each sample to determine the mean number of cells that had penetrated the Matrigel, providing an index of cell invasiveness.

Using an Annexin V/propidium iodide (PI) apoptosis detection kit (Beyotime, Shanghai, China), flow cytometry was conducted to analyse the cell-cycle phases and apoptosis rates of the transfected and non-transfected CNE1 cells following the manufacturer’s instructions. Briefly, after 48 h of incubation in a 96-well plate, the CNE1 cells (blank, Vector, Over-PinX1, and Over-PinX1 + 3-MA) were collected and stained with Annexin V-fluorescein isothiocyanate (FITC) and PI, then incubated for 15–20 min in the dark at room temperature. Flow cytometry data were then acquired using a FACSCalibur HG flow cytometer (BD Biosciences) and analyzed by FlowJo 10 (Tree Star Software, San Carlos, CA, USA).

Total RNA of cultured cells was extracted using TRIzol reagent (Invitrogen), and cDNA was prepared using total RNA as a template based on the Bestar qPCR RT Kit (Applied Biosystems, Grand Island, NY, USA). RT-qPCR with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference was carried out on the Agilent Stratagene Mx3000 real-time qPCR Thermocycle Instrument (Agilent Stratagene, CA, USA). Thermal cycling conditions of PCR amplifications were degeneration at 95 °C for 2 min followed by 40 cycles of 30 s at 94 °C , 20 s at 58 °C, and 20 s at 72 °C, and extension at 72 °C for 10 min. Primer sequences used for qRT-PCR assays were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China), as follows: PinX1 (forward: 5${}^{\prime }$-CCA GAG GAG AAC GAA ACC ACG-3${}^{\prime }$; reverse: 5${}^{\prime }$-ACC TGC GTC TCA GAA ATG TCA-3${}^{\prime }$); hTERT (forward: 5${}^{\prime }$-CCG ATT GTG AAC ATG GAC TAC G-3${}^{\prime }$; reverse: 5${}^{\prime }$-CAC GCT GAA CAG TGC CTT C-3${}^{\prime }$); and GAPDH (forward: 5${}^{\prime }$-TGT TCG TCA TGG GTG TGA AC-3${}^{\prime }$; reverse: 5${}^{\prime }$-ATG GCA TGG ACT GTG GTC AT-3${}^{\prime }$). The relative expression levels of mRNA were calculated based on the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method, and were normalized to GAPDH levels.

Total protein of cultured cells was extracted using radioimmunoprecipitation assay (Kettner, #378) lysate (Beyotime, Nanjing, China). Protein samples (each well ca. 20 $\mu$g) was loaded onto a 10 % separation gel and isolated by SDS-PAGE. Proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA) and blocked with 5% non-fat milk for 1.5 h at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C, including anti-PinX1 (1:1000), anti-LC3B (1:2000), anti-p62 (1:20,000), anti-Beclin-1 (1:2000), anti-p-AKT (1:500), anti-p-mTOR (1:5000), anti-p65 (1:3000), anti-p-p65 (1:5000) and anti-GAPDH (1:10,000). The membranes were then washed and incubated with the HRP-labeled secondary antibody (1:20,000) for 1.5 h at room temperature. All of these antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Beyotime) and were quantified using Quantity One (Bio-Rad version 4.6.2) in Gel Doc XR imager system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative protein expression levels were calculated based on the gray value of folds and normalized to GAPDH. Experiments were repeated three times to obtain the mean values.

Female nude mice (4-week-old, a body weight of 17 g) provided by the Animal Laboratory of Southern Medical University were used to establish NPC mouse model. A total of 1 $\times$ 10${}^4$ logarithmically growing PinX1-overexpressing cells or their controls (Blank, Vector, or Over-PinX1) in 0.1 ml RPMI-1640 medium without FBS were subcutaneously injected into the right side of each nude mouse (n = 5 per group). Tumor size was measured once a week in the feeding environment using the equation V = (a${}^{\mathrm{2}}$*b)/2, where a is the length of short side and b is the length of the long side of the tumor graft, and tumor grafts were isolated at four weeks after injection. Differences in tumor-graft volume were compared among the groups of mice injected with the three cell groups. The in vivo experiments were approved by the Laboratory Animal Ethics Committee of Southern Medical University Zhujiang Hospital (Approval No: LAEC-2019-011) and were conducted in accordance with the National Laboratory Animal Care and Maintenance Guide.

CNE1 cells in each group were grown on 6-well plates and fixed at 4 °C overnight using 4% paraformaldehyde (PFA) after washing with phosphate buffered saline (PBS). The cells were then blocked with 10% goat serum for 15 min at room temperature, and incubated with LC3B antibodies (1:200) for 1 h at 4 °C followed by incubation with Alexa Fluor 488 conjugated secondary antibodies (1:1000) for 1 h at room temperature. The cells were washed three times with PBS (5 min per wash) before each incubation step. Finally, the cells were mounted with DAPI staining solution and incubated for 10 min at room temperature in the dark, and analyzed under a Bx51 inverted florescence microscope (Olympus Corporation, Shinjuku, Japan).

Changes in tumor tissue morphology were observed via Hematoxylin-Eosin (H&E) Staining. The tumor tissue in each group was dehydrated by exposure to decreasing concentrations of ethanol, embedded in paraffin wax, and cut into sections 5 mm thick. Paraffin-embedded sections of tumor tissue were deparaffinized and rehydrated in decreasing concentrations of ethanol, then stained with hematoxylin and eosin (both from Servicebio, Wuhan, China), following the manufacturer’s protocols.

Immunohistochemistry assays were carried out to detect protein expression level of PinX1 in tumor tissues. Paraffin sections prepared from the in vivo experiments and the indirect streptavidin–peroxidase method were used following the manufacturer’s instructions. Paraffin sections were rehydrated using Histo-Clear (National Diagnostics, Atlanta, GA, USA) followed by a 100% to 70% ethanol gradient. Endogenous peroxidase activity was quenched using H${}_2$O${}_2$. Then, the sections were placed in avidin and biotin blocking solutions (Vector Labs, Newark, CA, USA) followed by addition of 2.5% normal horse serum (Vector Labs), and incubated with rabbit polyclonal anti-PinX1 (dilution, 1:100) overnight. After dehydration of the tissue in a 70% to 100% ethanol gradient and Histo-Clear, followed by mounting in Vectashield mounting medium, detection was performed using ImmPRESS HRP Anti-Rabbit Ig and ImmPACT DAB Peroxidase (Vector Labs).

Statistical analyses were carried out using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Data are recorded as the mean $\pm$ SD from at least three independent experiments. Comparisons were conducted using Student’s t-tests for two groups, one-way ANOVA for multiple groups, and a parametric generalized linear model with random effects for tumor growth and MTT assay. All statistical tests were two-sided.

We first examined the expression of PinX1 in 6-10B and CNE1 cells to determine its role in NPC development. Based on RT-qPCR and western blot analysis, PinX1 was strongly expressed in 6-10B cells, but weakly expressed in CNE1 cells (Fig. 1a). Therefore, the PinX1-overexpression plasmid (pcDNA3.0-PinX1) was introduced into the CNE1 cell line, to further explore its biological role in NPC. PinX1 expression was more than twofold greater in CNE1 cells treated with pcDNA3.0-PinX1 than those of the blank and vector groups, based on RT-qPCR and western blot analysis (Student’s t-tests, p 0.001; Fig. 1b).

Fig. 1.

PinX1 inhibits the growth of NPC cells by targeting telomerase. (a) The expressions of PinX1 in the 6-10B and CNE1 cell lines as determined by RT-qPCR and western blot analysis. (b) The expression of PinX1 in CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as determined by RT-qPCR and western blot analysis. (c) The mRNA expression of hTERT in CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as determined by RT-qPCR. (d) The cell proliferation curves of CNE1 cells and cells transfected with empty vector and pcDNA3.0-PinX1 were measured via MTT Assay. *** p 0.001 vs. control.

Afterward, we examined in vitro the effects of PinX1 expression on hTERT expression and NPC cell growth. hTERT expression in CNE1 cells treated with pcDNA3.0-PinX1 was significantly suppressed relative to those of the blank and vector groups, based on RT-qPCR (Student’s t-tests, p 0.001; Fig. 1c). MTT assay revealed that PinX1-overexpression significantly suppressed cell growth (p 0.001; Fig. 1d). These results suggest that PinX1 substantially inhibits the growth of NPC cells by targeting telomerase.

Using CNE1 cells (untreated, treated with the empty vector, or pcDNA3.0-PinX1-transfected cells) subcutaneously injected, we performed an in vivo tumor-formation experiment. The tumor growth curve was obtained by calculating the volume of tumors in each group at 7, 14, 21, and 28 days after inoculation: the tumor growth rate in the PinX1-overexpressing mice was significantly lower than that in the blank and vector groups (Fig. 2a). At 28 d after implantation, the PinX1-overexpressing mice had smaller tumor burdens (Fig. 2b) and displayed higher PinX1 expression in tumor tissues than the controls, and their transplanted tumor tissues showed fewer obviously pathological mitotic cell nuclei and cellular atypia than the control groups (Fig. 2c). These results suggest that PinX1 significantly inhibits tumorigenesis in vivo.

Fig. 2.

PinX1 inhibits NPC tumorigenesis in vivo. (a) Tumour growth curves were plotted in CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1. (b) The tumour volume was measured periodically for each of the xenograft mice models bearing tumours originating from CNE1 cells and those transfected with empty vector or pcDNA3.0-PinX1 (n = 5/group). (c) Immunohistochemistry detection of PinX1 in xenografts derived from CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as well as Representative H&E staining of primary cancer tissues are shown. Magnification $\times$400. Scale bar: 30 $\mu$m. * p 0.05 vs. control, ** p 0.01 vs. control. H&E, Hematoxylin-Eosin.

To investigate the impact of PinX1 overexpression on autophagy, the DAPI–FITC–LC3 reporter was used to monitor the autophagic flux. PinX1-overexpressing CNE1 cells contained more blue-green puncta than the control groups, suggesting that PinX1-overexpression activated autophagy (Fig. 3a). By measuring the level of the autophagy marker, Beclin-1, we established whether PinX1-overexpressing cells were actually undergoing autophagy. Beclin-1 protein levels were markedly elevated in PinX1-overexpressing cells, which also exhibited an elevated LC3-II/LC3-I protein ratio. In addition, p62 protein levels were reduced in PinX1-overexpressing cells, relative to the controls (Fig. 3b). As a result of these observations, PinX1 overexpression induces autophagy. We further examined PinX1 overexpression’s role in cell invasion and migration through pharmacological inhibition of autophagy using 3-MA, a widely used specific inhibitor of autophagy [23], and monitored its effects on autophagy. By monitoring autophagic flux using the DAPI–FITC–LC3 reporter, we found that 3-MA treatment of PinX1-overexpressing cells reduced the number of blue-green puncta (Fig. 3a). Western blot analysis revealed that 3-MA reduced the LC3-II/LC3-I ratio, and abolished the PinX1-overexpression-induced reduction of p62 expression (Fig. 3b). These results indicate that 3-MA is a potent inhibitor of PinX1-overexpression-induced autophagy in CNE1 cells. Using the Transwell assay, we then determined the number of migrating and invading cells. A significant reduction in migration and invasion was found in PinX1-overexpressing cells compared with controls, while treatment with 3-MA markedly increased migration and invasion (Fig. 3c).

Fig. 3.

Overexpression of PinX1 induces autophagy in NPC cells via the AKT/mTOR signaling pathway. (a) Comparison of autophagy flux and (b) the protein levels of LC3-II, LC3-I, p62 and Beclin-1 in CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as well as pcDNA3.0-PinX1 + 3-methyladenine. (c) Transwell assay for measuring cell migration and invasion following transfection. (d) The protein levels of p-AKT and p-mTOR and (e) Comparison of autophagy flux in CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as well as pcDNA3.0-PinX1 + Chloroquine. ** p 0.01 vs. control, *** p 0.001 vs. control.

We next investigated the mechanism whereby PinX1 overexpression activates autophagy in NPC cells. Autophagy can be potently induced by inhibiting the AKT/mTOR signaling pathway. Therefore, we determined the state of AKT/mTOR signaling in CNE1 cells by measuring the changes in the levels of phosphorylated AKT and mTOR. Significantly lower expression levels were observed in PinX1-overexpressing CNE1 cells than those in the controls (Fig. 3d), indicating suppressed AKT/mTOR signaling in these cells. Adding chloroquine to PinX1-overexpressing cells did not cause significant differences in phosphorylated AKT and mTOR levels relative to the untreated PinX1-overexpressing cells, although it did inhibit autophagic flux, as revealed by the immunofluorescence assay (Fig. 3e), indicating that PinX1 might directly modulate AKT phosphorylation. Together, these results suggest a mechanism whereby PinX1 overexpression inhibits AKT/mTOR signaling in NPC cells, thereby activating autophagy.

To determine whether the inhibition of cell proliferation and apoptosis induced by PinX1 overexpression is caused by the induction of autophagy, the effect of 3-MA on these processes in PinX1-overexpressing cells using a MTT assay was investigated. Compared with the controls, the CNE1 cell proliferation was remarkably inhibited by PinX1 overexpression, and this effect was reversed by treating PinX1-overexpressing cells with 3-MA (Fig. 4a). We next investigated how inhibiting autophagy affected apoptosis in PinX1-overexpressing cells. Flow cytometry analysis revealed an obviously higher apoptosis rate in PinX1-overexpressing cells than in the control groups; further, treating PinX1-overexpressing cells with 3-MA markedly reduced the rate of apoptosis, relative to the untreated PinX1-overexpressing cells (Fig. 4b). These results indicate that inhibiting autophagy reverses the inhibition of cell proliferation and induction of apoptosis, that resulted from PinX1 overexpression. Furthermore, we monitored the effect of inhibiting autophagy on the cell cycle in PinX1-overexpressing cells. The percentage of cells in the G0/G1 phase was higher, and that of cells in G2/M phase was lower, in PinX1-overexpressing cells than in the control groups (Fig. 4c). Further, treating PinX1-overexpressing cells with 3-MA significantly reduced the percentage of cells in the G0/G1 phase and increased that of cells in the G2/M phase. These findings indicate that inhibiting autophagy reverses the deceleration of cell-cycle progression caused by PinX1 overexpression in CNE1 cells.

Fig. 4.

Autophagy inhibitor 3-MA reverses the effects of PinX1 overexpression on cell proliferation, apoptosis, and the cell cycle in NPC cells. (a) The cell proliferation curves, (b) the apoptosis rate and (c) the cell cycle of CNE1 cells and those transfected with empty vector and pcDNA3.0-PinX1 as well as pcDNA3.0-PinX1 + 3-methyladenine. ** p 0.01 vs. control, *** p 0.001 vs. control.

To further elucidate the signaling pathway involved in PinX1-overexpression-induced apoptosis, we assessed NF-$\kappa$B/p65 signaling in CNE1 cells, by measuring changes in p65 and p-p65 levels. Expression of p65 and p-p65 was significantly lower in PinX1-overexspressing CNE1 cells than in the controls (Fig. 5). In addition, 3-MA treatment of PinX1-overexpressing cells increased the expression of p65 and p-p65 relative to untreated PinX1-overexpressing cells. These results demonstrate that PinX1 overexpression promotes autophagy, thereby suppressing the NF-$\kappa$B/p65 signaling pathway, thus inducing apoptosis.

Fig. 5.

The protein levels of p65 and p-p65 in CNE1 cells and cells transfected with empty vector and pcDNA3.0-PinX1 as well as pcDNA3.0-PinX1 + 3-methyladenine as determined by Western blot.