IHC Staining
The expression levels of KIF3A were evaluated by immunohistochemistry (IHC) staining. We performed the streptavidin-peroxidase-conjugated method according to the reagent instructions.
Cell Culture and Tissue specimens
The HONE1, SUNE1, NP69 and 5-8F NPC cell lines were obtained from the Cancer Research Institute of Southern Medical University (Guangzhou, China). The NPC cell lines were cultured in RPMI 1640 (Biological Industries) supplemented with 10% foetal calf serum (FBS; Biological Industries, USA), and the cell lines were cultured in a humidified chamber with 5% CO2 at 37°C. 106 NPC and 22 normal nasopharyngeal specimens were obtained from Nan Fang Hospital, China. Consent from the patients and approval from the Ethics Committee of Nan Fang Hospital were obtained before these clinical samples were used for study purposes.
qRT-PCR
A total RNA kit was used to isolate RNA from the NPC cell lines. cDNA was generated with a reverse transcription kit (TaKaRa Company) in the Bio–Rad T100 Thermal Cycler; subsequently, the cDNA was used as a template and amplified with specific primers in the Roche LightCycler. The fold changes in KIF3A gene expression were analysed by the 2-ΔΔCt method.
In Vivo Tumorigenesis in Nude Mice
All the in vivo studies were performed following a protocol approved by the Animal Care and Use Committee of Southern Medical University. Approximately 4 × 106 SUNE1 cells in the logarithmic phase of growth that had been transfected with negative control or KIF3A-overexpressing cDNA (N=7 per group) were subcutaneously injected into 4-week-old female nude mice (BALB/c, nu/nu). After 15 days, the nude mice were sacrificed, and the tumour tissues were removed and weighed.
Western blotting analysis
NPC cell lysates were generated, total protein was extracted, and a BCA protein assay kit (TIANGEN, Beijing) was used to measure the protein concentration. The antibodies included anti-KIF3A (1:1000), anti-β-catenin (1:1000), anti-N-cadherin (1:1000), anti-Vimentin (1:1000), anti-c-Myc (1:1000), anti-CCND1 (1:1000), anti-β-actin (1:5000), and anti-GAPDH (1:5000).
Transfection
The siRNAs targeting KIF3A and relevant negative control was synthesized by RiboBio (Guangzhou, China). KIF3A and β-catenin plasmids were purchased from WZ Biosciences Inc. (Shandong, China). Twelve hours before transfection, NPC cells were plated in six-well plates at 50%-60% confluence. Subsequently, according to the manufacturer's protocol, plasmids or siRNAs were transfected into cells using Lipofectamine TM 3000 (Invitrogen, Guangzhou, China). After 24-72 hours, the cells were harvested for further experiments.
Cell proliferation analysis and EdU incorporation assay
Cell proliferation was measured by CCK-8 cell Counting Kit (Vazyme, Nanjing, China). NPC cells were seeded in 96-well plates (1500 cells/well). After the cells attached, 10 µL CCK-8 reagent was added to the wells and incubated with the NPC cells at 37°C for 1.5 h. The optical density (OD) was assayed at 450 nm from Day 1 to Day 4. An EdU incorporation assay was conducted with Cell-Light EdU Apollo 567 (RiboBio, Guangzhou, China). NPC cells in the logarithmic phase of growth were seeded in 96-well plates, and after the cells attached, EDU A solution was incubated with the NPC cells for 2 h. Subsequently, 4% paraformaldehyde was used to fix the NPC cells, and then, 0.2% Triton X-100 was used to permeabilize the cells. Apollo solution was added and incubated for 30 min after permeabilization to stain the cell nuclei, and then, DAPI was used to stain the cell nuclei for 10 min. The cells were imaged under an inverted fluorescence microscope.
Colony formation and cell cycle progression
For colony formation, 100 NPC cells were seeded in six-well plates and cultured in a humidified chamber with 5% CO2 at 37°C. The culture medium was renewed every 72 h. After 2 weeks of incubation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Then, the NPC cells were stained with crystal violet. Cell cycle analysis was performed according to the instructions of the cell cycle and apoptosis kit (Leagene, Beijing, China).
Cell migration assay
A Transwell assay was performed to measure the migration capability of NPC cells. The cells were mixed with serum-free RPMI 1640 and were added to the upper chamber of Transwell plates. Then, RPMI 1640 supplemented with 10% foetal bovine serum was added to the lower chamber of the Transwell plates. After 10-24 h, the chamber was fixed with paraformaldehyde and stained with Giemsa for 5 min. Then, deionized water was used to wash the chamber, and the chamber was photographed with a microscope after air drying.
Wound-Healing Assay
For wound-healing assays, NPC cells were grown to approximately 100% confluence in 6-well plates. A medium-sized pipette tip was used to scrap artificial wound tracks. The NPC cells were cultured with serum-free medium. To analyse the migration capability of the cells, the wound width was imaged under an inverted microscope at 0 and 36 h.
Co-immunoprecipitation (COIP)
Briefly, a COIP assay was conducted with protein A/G immunoprecipitation beads (Bimake, Shanghai, China). Total protein extracted from NPC cells was coincubated with anti-IgG, anti-β-catenin, and anti-KIF3A antibodies overnight at 4°C. The beads were incubated with the antigen-antibody complexes for 45 min at 37°C after the beads were washed twice with wash buffer. Finally, the mixed example was eluted in buffer and boiled for five minutes at 95°C. The immune complexes were subjected to Western blotting assay.
Immunofluorescent Staining
NPC cells were seeded in 35-mm glass bottom cell culture dishes (SORFA LIFE SCIENCE, Zhejiang) and cultured overnight. The NPC cells were fixed with 4% paraformaldehyde, and then, 0.2% Triton X-100 was used to permeabilize the cells. Subsequently, the permeabilized cells were incubated with the indicated antibodies overnight at 4°C. Finally, the NPC cells were incubated with fluorescein-labelled secondary antibodies at 37°C for 60 minutes, and DAPI was used to stain the cell nuclei. The cells were photographed under a confocal microscope.
Nuclear and cytoplasmic extraction assay
Nuclear and cytoplasmic extraction assays were performed with NE-PER Nuclear and Cytoplasmic Extraction reagents (Thermo Scientific, UK) according to reagent instructions. In short, NPC cell pellets were obtained by trypsin digestion and added to ice-cold CER I for a 10-minute incubation at 4°C. After the incubation, ice-cold CER II was added to the complex and incubated for 1 minute at 4°C. Then, the lysed mixture was centrifuged at 16000 ×g for 5 minutes. The supernatant (cytoplasmic extract) was transferred into a new centrifuge tube and stored at -80°C. The wash buffer was added to the pellet, and after washing, the sample was recentrifuged. Then, the pellet was resuspended by mixing with ice-cold NER and incubated for 40 minutes at 4°C. Finally, the mixture was centrifuged at 16000 ×g for 15 minutes and transferred to a new centrifuge tube for storage at -80°C.
Statistical Analysis
GraphPad Prism 9 software was used for statistical analysis. The data are expressed as the mean ± SD from at least three independent experiments, A Student’s t test was performed to compare two groups, the comparisons among multiple groups used one-way analysis of variance (ANOVA). A P value of less than 0.05 was considered to indicate statistically significant differences, which are labelled as follows: *P<0.05, **P<0.01, ***P<0.001.
Decreased KIF3A expression correlates with unfavourable outcome
To explore the basic expression of KIF3A in NPC, we used qRT–PCR and Western blotting to detect KIF3A mRNA and protein expression, respectively, in NPC cells. The results indicated that KIF3A expression was obviously downregulated in NPC cells compared with NP69 cells. Then, we conducted immunohistochemistry to detect KIF3A expression in 106 NPC tissues and 22 nasopharyngeal epithelial tissues. Consistent with the results described above, the expression of KIF3A was significantly reduced in NPC tissues compared with nasopharyngeal epithelial tissues (P=0.018) (Table1). Subsequently, we found that decreased KIF3A expression positively correlated with T stage (T1-T2 vs. T3-T4, P=0.035) and M stage (M0 vs. M1, P=0.039) but did not correlate with other clinicopathological characteristics (Table2). In addition, survival analysis indicated that NPC patients with low KIF3A expression had shorter survival times than patients with high KIF3A expression (P=0.033).
Decreased KIF3A expression correlates with unfavourable outcomes. (A-B) qRT–PCR and Western blotting were performed to determine the mRNA and protein expression of KIF3A in nasopharyngeal epithelium and NPC cells. (C) IHC staining was used to evaluate KIF3A expression in nasopharyngeal epithelium and NPC samples. a: Strong staining of KIF3A in NP tissues; b: low expression of KIF3A in NPC tissues; c: high expression of KIF3A in NPC tissues. (D) Kaplan–Meier survival analysis of the survival rate of NPC patients based on KIF3A expression. Log-rank test was used to calculate P values.
KIF3A suppresses NPC proliferation in vitro and in vivo
Western blotting assays were used to determine the expression level of KIF3A after transfection of the KIF3A plasmid into HONE1 and SUNE1 cells. The results indicated that the KIF3A protein expression in KIF3A-overexpressing cells was obviously increased compared with that in negative control cells. To determine the biological function of KIF3A in NPC, a Cell Counting Kit-8 assay and colony formation assay were conducted, and the results indicated that the growth of KIF3A-overexpressing HONE1 and SUNE1 cells was markedly attenuated compared with that of negative control cells. Furthermore, EdU (5-ethynyl-2′-deoxyuridine) incorporation assays and cell cycle analysis showed that the cell cycle progression of KIF3A-overexpressing cells was significantly inhibited compared with that of NC cells. To further demonstrate the role of KIF3A in carcinogenesis in vivo, we performed a subcutaneous tumorigenesis study by inoculating LV-NC-GFP SUNE1 cells or LV-KIF3A-GFP SUNE1 cells into nude mice. After 15 days, the nude mice inoculated with LV-KIF3A-GFP SUNE1 cells exhibited lower tumour weights than the nude mice inoculated with LV-NC-GFP SUNE1 cells. The results described above demonstrate that KIF3A overexpression suppresses NPC proliferation in vivo and in vitro.
KIF3A suppresses NPC proliferation in vitro and in vivo. (A) Western blotting was used to measure KIF3A protein expression after the transfection of the negative control empty plasmid or KIF3A plasmid into HONE1 and SUNE1 cells. CCK-8 assay (B), colony-formation assay (C), EdU incorporation assay (D) and flow cytometry analysis (E) were performed to evaluate changes in the proliferation and cell cycle progression of HONE1 and SUNE1 cells after transfection with the KIF3A plasmids or LV-GFP-KIF3A. Student’s t test. Mean ± SD, **p<0.01, ***p<0.001. (F) The in vivo effect of KIF3A on proliferation was elucidated with a xenograft mouse model in which mice were inoculated with SUNE1-LV-NC and SUNE1-LV-KIF3A cells. Each group included 7 mice. Student’s t test, Mean ± SD, **p<0.01.
Knockdown of KIF3A expression reverses NPC proliferation
After transfection of specific small interfering RNA (siRNA) targeting KIF3A, Western blotting assays showed that simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected HONE1 and SUNE1 cells reversed the changes in KIF3A protein expression. In addition, the CCK-8 assay showed that simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected NPC cells reversed the growth inhibition caused by upregulated KIF3A expression. The EdU assay also showed that EdU staining was enhanced after the simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected HONE1 and SUNE1 cells. Together, these data show that knockdown of KIF3A expression in oe-KIF3A-transfected NPC cells restored NPC growth and cell cycle progression.
Knockdown of KIF3A expression reverses NPC proliferation. (A) Western blotting was used to evaluate silencing efficiency after the simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected HONE1 and SUNE1 cells. (B-C) CCK-8 and EdU incorporation assays after the simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected HONE1 and SUNE1 cells. Student’s t test. Mean ± SD, *p < 0.05, **p<0.01, ***p<0.001.
KIF3A suppresses NPC migration, and knockdown of KIF3A expression reverses this phenotype
To investigate the biological effect of KIF3A on NPC cell migration capability, a wound healing assay was conducted, and we observed that KIF3A-overexpressing HONE1 and SUNE1 cells had significantly suppressed wound healing abilities compared with NC HONE1 and SUNE1 cells. Subsequently, Transwell assays showed that overexpression of KIF3A significantly reduced the numbers of migrated HONE1 and SUNE1 cells compared with the NC. Furthermore, recovery experiments indicated that simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected NPC cells reversed the inhibitory effect on metastasis mediated by upregulated KIF3A expression.
KIF3A suppresses NPC migration, and knockdown of KIF3A expression reverses this phenotype. (A-B) Wound healing assays and Transwell assays were performed to evaluate the migration capability of NPC cells transfected with the KIF3A plasmid. Student’s t test. Mean ± SD, *p < 0.05, **p<0.01. (C-D) Wound healing and Transwell assays after simultaneous knockdown of KIF3A expression in oe-KIF3A-transfected HONE1 and SUNE1 cells, Student’s t test. Mean ± SD, *p < 0.05, **p<0.01.
KIF3A suppresses NPC proliferation and migration via the wnt/β-catenin signalling pathway
To explore the potential mechanism by which KIF3A functions, we used Western blotting to analyse the expression of β-catenin, cell cycle-associated proteins, and EMT markers. We observed that the expression of CCND1, c-Myc, N-cadherin, vimentin, and β-catenin was downregulated in KIF3A-overexpressing HONE1 cells and SUNE1 cells compared with the NC cells. In addition, recovery experiments indicated that interfering with KIF3A expression significantly restored the levels of CCND1, c-Myc, N-cadherin, vimentin, and β-catenin. Finally, we determined whether β-catenin was involved in KIF3A-regulated NPC proliferation and migration. Simultaneous upregulation of β-catenin expression in oe-KIF3A-transfected NPC cells reversed changes in CCND1, c-Myc, N-cadherin, and vimentin protein expression mediated by KIF3A overexpression.
KIF3A suppresses NPC proliferation and migration via the wnt/β-catenin signalling pathway. (A) Changes in the expression levels of KIF3A, N-cadherin, Vimentin, CCND1, and c-Myc were detected by Western blotting after transfection with the KIF3A plasmid in HONE1 and SUNE1 cells. β-actin was used as a loading control. (B) Changes in the expression levels of KIF3A, N-cadherin, Vimentin, CCND1, c-Myc were detected by Western blotting after transfecting the KIF3A plasmid or KIF3A plasmid + siRNA targeting KIF3A in HONE1 and SUNE1 cells. GAPDH was used as a loading control. (C) Changes in the expression levels of N-cadherin, Vimentin, CCND1, c-Myc were detected by Western blotting after transfection with the KIF3A plasmid or KIF3A + β-catenin plasmids in HONE1 and SUNE1 cells. GAPDH was used as a loading control.
β-catenin reverses the inhibitory effect exerted by KIF3A overexpression in NPC
To further explore the role of β-catenin in KIF3A-mediated NPC proliferation and metastasis, we transfected KIF3A or KIF3A + β-catenin plasmids into HONE1 and SUNE1 cells. Using CCK-8 assays, EdU incorporation assays and Transwell assays, we observed that simultaneous upregulation of β-catenin expression in oe-KIF3A-transfected HONE1 and SUNE1 cells reversed the inhibitory effect on proliferation, migration, and invasion caused by upregulated KIF3A expression.
β-catenin reverses the inhibitory effect exerted by KIF3A overexpression in NPC. (A-B) CCK-8 and EdU incorporation assays were conducted after transfection with the KIF3A plasmid or β-catenin+KIF3A plasmids in HONE1 and SUNE1 cells. (C) A wound healing assay was conducted to assess the migration capability of NPC cells treated with the KIF3A plasmid or β-catenin+KIF3A plasmids. Student’s t test. Mean ± SD, *p < 0.05, **p<0.01.
KIF3A interacts with β-catenin and suppresses β-catenin nuclear translocation
To explore the potential molecular mechanisms by which KIF3A represses the Wnt/β-catenin pathway, we used the BioGRID database to predict potential proteins that interact with KIF3A. Interestingly, we found that β-catenin may be a potential candidate interacting protein. Furthermore, a study reported that KIF3A interacts with β-catenin during spermatogenesis in Eriocheir sinensis, but this interaction has not been reported in H. sapiens. Therefore, we explored whether there was an interaction between KIF3A and β-catenin.
Endogenous coimmunoprecipitation (CO-IP) was performed and confirmed that KIF3A interacts with β-catenin. In addition, double colocalization by immunofluorescence demonstrated the colocalization of KIF3A and β-catenin in NPC cells. Nuclear and cytoplasmic extraction assays were performed to analyse the distribution of β-catenin in the nuclei and cytoplasm, and the results indicated that overexpression of KIF3A suppresses the levels of β-catenin in the cytoplasm and nuclei. These results demonstrate that KIF3A interacts with β-catenin in NPC and suppresses β-catenin nuclear translocation.
KIF3A interacts with β-catenin and suppresses β-catenin nuclear translocation. (A) A CO-IP assay was performed to detect the interactive relationship between KIF3A and β-catenin in NPC cells. (B) Colocalization of KIF3A and β-catenin by immunofluorescence in HONE1 and SUNE1 cells. (C) Western blotting was conducted to detect changes in β-catenin protein expression in the nucleus and cytoplasm. GAPDH was used as a loading control in the cytoplasm, and histone was used as a loading control in the nucleus.