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This study was approved by the Ethics Committee of Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology [Approval No.: 2026IEC(RYJ016)]. All patients provided informed consent, and the collection and use of clinical samples complied with the relevant ethical guidelines of the Declaration of Helsinki. The main experimental materials and instruments used in this study are listed in the Table of Materials.
Collection of clinical tissue samples and detection of CDCP1 expression
Surgical tissue specimens were collected from seven patients diagnosed with nasopharyngeal carcinoma at our hospital between January 2019 and December 2019. Simultaneously, nasal tissue samples from six patients with rhinitis were collected as controls. All tissue samples were immediately frozen in liquid nitrogen after collection for subsequent RNA extraction.
RNA extraction and real-time quantitative PCR detection
Total RNA was extracted from tissue samples according to the manufacturer’s instructions. For each sample, 50–100 mg of tissue was processed on ice in 1 mL of extraction reagent using a tissue homogenizer. Homogenization was carried out for 30 s and repeated 3x to ensure complete disruption of the tissue. After tissue disruption, each lysate received 200 µL of chloroform. The tubes were shaken vigorously by vortexing for 15 s, kept at room temperature for 3 min, and then spun at 12,000 × g. for 15 min at 4 °C to separate the phases. The clear aqueous layer was removed without disturbing the interphase and transferred into a new tube.
RNA was recovered from this aqueous fraction by adding isopropanol at a 1:1 volume ratio. Following a 10 min incubation at room temperature, the samples were centrifuged at 12,000 × g for 10 min at 4 °C to pellet the RNA. The supernatant was discarded, and the pellet was rinsed once with 1 mL of 75% ethanol. After a second centrifugation step at 7,500 × g. for 5 min at 4 °C, the ethanol wash was removed. The RNA pellet was then air-dried for 5–10 min at room temperature and resuspended in 20 µL of RNase-free water. RNA quantity and purity were determined spectrophotometrically.
For cDNA preparation, 1 µg of total RNA was used for reverse transcription. The reaction was carried out at 37 °C for 15 min and then heated to 85 °C for 5 s. Quantitative real-time PCR (qPCR) was subsequently conducted with TB Green Premix Ex Taq II on a Real-Time PCR System. Each 20 µL reaction contained 2 µL of cDNA template, forward and reverse primers at 0.4 µmol/L each, 10 µL of SYBR Green premix, and nuclease-free water. The amplification protocol began with denaturation at 95 °C for 30 s. This was followed by 40 cycles, with each cycle consisting of 95 °C for 5 s and 60 °C for 30 s for annealing/extension. Primer sequences are listed in Table 1.
Immunohistochemical detection of CDCP1 protein expression
Tissue specimens were first fixed in 4% paraformaldehyde for 24 h, followed by standard dehydration, paraffin embedding, and preparation of 4 µm-thick sections. The paraffin sections were then treated with xylene to remove paraffin and rehydrated sequentially through graded ethanol solutions. For antigen unmasking, the slides were placed in citrate buffer (pH 6.0) and heated in a pressure cooker for 3 min after the solution reached boiling. Endogenous peroxidase activity was quenched by exposing the sections to 3% hydrogen peroxide for 10 min.
After blocking of endogenous enzyme activity, the sections were incubated overnight at 4 °C with the anti-CDCP1 primary antibody diluted at 1:200. On the following day, the slides were treated with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature. Signal visualization was carried out using a DAB substrate kit, and the sections were subsequently counterstained with hematoxylin. Finally, the slides were dehydrated, mounted, and examined under an optical microscope.
Cell culture and grouping
CNE2 and HK1 human nasopharyngeal carcinoma cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37 °C in an incubator containing 5% CO₂. When cells reached 70%–80% confluence, they were transfected according to the following groups: Control group (Control): transfected with empty vector or negative control siRNA; CDCP1 overexpression group (CDCP1-OE): transfected with CDCP1 overexpression plasmid; CDCP1 silencing group (si-CDCP1): transfected with CDCP1 siRNA; Rescue experiment group (CDCP1-OE + U0126): treated with the ERK1/2-specific inhibitor U0126 (10 µmol/L) for 48 h on the basis of CDCP1. overexpression.
Transfection was carried out according to the manufacturer’s instructions. Cells were plated 24 h before transfection in 6-well plates at 2 × 105 cells/well, with each well containing 2 mL of antibiotic-free medium. Transfection was initiated when the cultures reached approximately 70% confluence. The culture medium was then removed and replaced with fresh serum-free medium. For each well, either 2 µg of plasmid DNA or 100 pmol of siRNA was prepared separately from 5 µL of transfection reagent, with each component diluted in 250 µL of reduced serum-containing medium. Both mixtures were kept at room temperature for 5 min. The diluted nucleic acid and transfection reagent were then combined and allowed to stand for 20 min at room temperature to generate the transfection complexes. These complexes were added to the cells dropwise, gently distributed across the well, and incubated with the cells for 6 h. Once the incubation period was completed, the transfection mixture was removed, and the cells were supplied with complete medium containing 10% FBS.
Cell proliferation assay
At 48 h post transfection, CNE2 and HK1 cells from each group were collected and seeded in 96-well plates at a density of 3 × 103 cells/well with 100 µL of medium per well, with five replicate wells per group. After the cells had been cultured for 24, 48, or 72 h, 10 µL of MTT solution at 5 mg/mL was added to each well. The plates were then incubated again at 37 °C with 5% CO₂ for another 4 h. After this incubation, the culture supernatant was carefully discarded, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The plates were agitated for 10 min to ensure complete dissolution. Absorbance was then recorded with a microplate reader at 570 nm, using 630 nm as the reference wavelength.
Apoptosis activity assay
At 48 h post transfection, CNE2 cells from each group were collected, and Caspase-3 activity was detected using a Caspase-3 Activity Assay Kit according to the manufacturer's instructions. A total of 100 µL of lysis buffer was added to 1 × 106 cells, lysed on ice for 15 min, and then centrifuged at 12,000 × g. for 10 min at 4 °C. The resulting supernatant was transferred for subsequent analysis. For the Caspase-3 assay, each well received 50 µL of reaction buffer and 5 µL of Caspase-3 substrate (DEVD-pNA). The plate was then maintained at 37 °C for 2 h, and absorbance was measured at 405 nm with a microplate reader.
Western blot analysis
At 48 h after transfection, CNE2 and HK1 cells from each experimental group were harvested for protein extraction. Cells were disrupted in RIPA buffer containing protease inhibitors and kept on ice for 30 min. The resulting lysates were centrifuged at 12,000 × g for 15 min at 4 °C, after which the supernatant fractions were collected. Protein concentration was measured with a BCA protein assay kit.
For western blot analysis, 30 µg of total protein from each sample was mixed with 5x loading buffer and heated at 100 °C for 10 min to denature the proteins. The samples were separated on 10% SDS-PAGE gels at 120 V for 90 min, followed by transfer onto PVDF membranes at a constant current of 300 mA for 90 min. After transfer, the membranes were blocked with 5% non-fat milk for 1 h at room temperature.
The membranes were incubated overnight at 4 °C with primary antibodies against CDCP1 (1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), p-ERK1/2 (1:2,000), ERK1/2 (1:1,000), and GAPDH (1:5,000).
On the following day, the membranes were rinsed in TBST 3x, with each wash lasting 10 min. They were then exposed to horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After three additional TBST washes, the protein signals were visualized using an ECL chemiluminescence substrate. Band intensity was measured with ImageJ software, and GAPDH was used as the normalization control.
Statistical analysis
All experiments were conducted independently 3x. Results are presented as the mean ± standard deviation (SD). Differences among multiple groups were evaluated by one-way analysis of variance (ANOVA), and Tukey’s method was used for pairwise post hoc comparisons. A P-value of < 0.05 was considered statistically significant.