Cell lines and patient
NPC cell line (SUNE1, 5-8F, 6-10B, CNE1, CNE2, and HNE1) and human normal nasopharyngeal cell line NP69 were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cell lines were cultured in RPMI1640 (Gibco, USA) medium containing 10%FBS (Gibco, USA) at 37 °C and 5%CO2.
Patients: Thirty patients with NPC and their adjacent tissues were treated in the first affiliated Hospital of Zhengzhou University. All patients were pathologically diagnosed as NPC after operation. Samples were collected immediately after operation and stored at -80 °C.
miR-18a-5pinhibitor, inhibitorNC, miR-18a-5pmimics, mimicsNC, agomiR-NC, and agomiR-18a-5p were purchased from Guangzhou RiboBio Co., Ltd. Small interference RNA (siRNA) synthesized by targeted SMAD2 was synthesized by Shanghai Shenggong Bioengineering Co., Ltd. On the basis of the reference manual, Lipofectamine® 3000 (Invitrogen, USA) was used to transfect inhibitor, mimics, siRNA, and their respective controls into NPC cells for gene or miRNA overexpression or silencing.
Quantitative real-time PCR assays（qRT-PCR）
Total RNA was extracted from NPC tissues and cells. CDNA was synthesized using reverse transcription system kit (TransGeneBiotech, Beijing, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was achieved using the ABI7500 instrument (7500 bot ABI Co.). SMAD2 was normalized with GAPDH as internal reference and miR-18a-5p with U6 as internal reference. The difference of relative expression of target gene mRNA between the control group and the experimental group was compared with the value of 2−ΔΔCt, and the experiment was repeated thrice. The primers used in the experiment are shown in Table 1.
At 48 h after transfection, total proteins were extracted with RIPA cleavage buffer (Beyotime Biotechnology, Shanghai, China). After adding 10 μL of the sample buffer to boil for 10 min at 95 °C, SDS-PAGE was achieved at 100 V. After electrophoresis, the protein was transferred to NC membrane at 100 mA for 120 min, sealed with 5% skimmed milk powder for 60 min, and incubated overnight at 4 °C. After the first antibody was incubated, the membrane was shaken and washed with 1 × TBST solution (Solarbio, Beijing, China) at room temperature, 5 min × 3 times, and hybridized with horseradish peroxidase-labeled goat anti-rabbit IgG, at room temperature for 120 min. At 20 min intervals, the photoluminescence reaction was performed using the ECL kit (Solarbio, Beijing, China), and protein imprinting was observed. Antibodies are shown in Table 2. The experiment was repeated thrice.
Cell proliferation and plate clone formation
Cell proliferation was tested by the MTT method, and the cells in the treatment group (5 × 103 per 100 μL) were inoculated into a 96-well plate. Each process included three repeats. After culturing for 0, 12, 24, 48, and 72 h, cell proliferation was evaluated with aseptic MTT solution (Beyotime) according to the instruction method. A spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) was used to measure the absorbance of the 490 nm wavelength.
Plate clone formation experiment: Treated cells were cultured in a Petri dish (1.5 × 103/plate). The cells were stirred gently and incubated with 5% CO2 at 37 °C for 14 days; the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Next, the cells were dyed with 0.5% crystal violet at room temperature for 15 min. Then, such cells were washed with water. Colonies of more than 50 cells were counted under a light microscope.
Treated cells (5 × 106) were inoculated in a 6-well plate. When the cell coverage reached 80%, the tip of the 200 μL pipette was used to gently scratch the monolayer through the center of the hole. Pores were washed twice with the medium to remove isolated cells. After adding fresh medium, the cells were cultured for 24 h, and cell migration was observed and photographed for 0 and 24 h under a microscope.
Transwell invasion and migration assay
A 24-well Transwell chamber (8 μm aperture, BD Biosciences) was used for Transwell invasion detection. Approximately 2 × 104 cells were added to the superior chamber, and the parietal chamber was coated with Matrigel matrix. The DMEM containing 10% FBS (Thermo fisher, USA) was filled into the lower chamber. After culturing at 37 °C for 48 h, the Transwell was washed twice with PBS, and 5% glutaraldehyde was fixed at 4 °C for 0.5 h. The cells on the upper surface were wiped with a cotton ball, observed and photographed under a microscope, and counted. No matrix was added to the apical chamber in the Transwell migration experiment, and the other processes were the same as those in the Transwell invasion experiment.
Dual-luciferase reporter assay
To identify the probability of binding between miR-18a-5p and SMAD2 3′UTR, a psiCHECK luciferase reporter vector (Sangon Co., Ltd., Shanghai, China) was constructed by inserting wild-type (wt) and mutant (mut) 3′UTR of SMAD2. Next, CNE1 and 6Mel 10B cells were inoculated in 48-well plates and cultured for 24 h. Afterward, miR-18a-5p/NC and psiCHECKwt/mut plasmids were co-transfected into the cells. Finally, luciferase activity was measured by luciferase assay reagent (Promega, Fitchburg, WI, USA).
Tumour xenograft model
Fifteen male nude mice (6 weeks) were purchased from Southern Medical University (Guangdong, China) and raised under aseptic conditions (12 h black and white photoperiod, 25 °C, 60% UV, 70% humidity). CNE1 cells were inoculated into the feet of male nude mice. When the tumor volume reached 60 mm3, antagomiR-18a-5p (10 nmol/50 mL), antagomiR-NC, and PBS were injected twice a week as negative control, and intratumoral therapy was performed eight times. Tumor volume measurement method: volume (cm3) = (length × width2)/2. After 4 weeks, the nude mice were euthanized, and the tumor weight was measured and photographed. The nursing and experimental procedures of the mice were approved by the Experimental Animal Ethics Committee.
The transplanted tumor tissue of mice was placed in 4% paraformaldehyde fixation solution and fixed in a refrigerator at 4 °C. After gradient dehydration of different concentrations of ethanol, xylene transparent treatment, paraffin embedding, and slicing, immunochemical staining was performed on the transplanted tumor tissue, which was finally sealed with xylene transparent and neutral gum, covered with glass slides, and dried naturally for follow-up observation. Antibodies are shown in Table 2.
Data were processed by SPSS22.0 statistical software, and the measurement data were expressed in the form of mean ± standard deviation. The comparison between the two groups was obtained using t-test. In the current paper, *, # indicated P < 0.05.