1. Cell culture
All NSCLC cell lines were obtained from the Central Laboratory of Jiangsu Cancer Institute (Nanjing, Jiangsu). These cells were grown in DMEM/F12 or RPMI 1640 medium supplemented with 10% fetal bovine serum , 100 U/mL penicillin and streptomycin. All cell lines were cultured at 37 ℃, 5% CO2 in humid air, and conducted a mycoplasma contamination test every 3 months. The cells used in the experiment were all within 10 generations after thawing.
2. Tissue samples
The 100 NSCLC tissue samples included in the study were provided by Jiangsu Cancer Hospital and confirmed by experienced pathologists. According to the Declaration of Helsinki, this study had been approved by the Clinical Ethics Review Committee of Jiangsu Cancer Hospital, and a written informed consent form of each patient involved in the study was obtained. The histological grade and staging of NSCLC were reclassified according to the 8th edition of the American Joint Committee on Cancer (AJCC) cancer staging system.
3. Generation of plasmids and stably transfected cell lines
NCBI database was used to analyze the full length of ZNF488 gene sequence. Primer bank website was used to design ZNF488 primers: Forward: 5’-GAAAACAGATGGCGACTTAGCG -3’; Reverse: 5’-CTGCCGGTCCTTCATCCTC-3’. ZNF488 cDNA was amplified by PCR and insert it into pPMCV vector. The vector or pPMCV-ZNF488 plasmid was transfected together with the retroviral packaging vector PIK into 293FT cells. After transfection, the supernatant was collected and used to infect NCI-H520 and PC-9 cells, and the stable transfected cell lines were screened with puromycin.
4. RNA extraction, reverse transcription and qRT-PCR
Trizol reagent (Invitrogen) was used to isolate total RNA from tissues and cells. Reverse transcription was performed using M-MLV reverse transcription mold (Promega, Madison, WI). Quantitative RT-PCR was performed using the Bio-RAD CFX96 real-time system (Bio-Rad, Hercules, CA) and Platinum SYBR Green Qpcr SuperMix-UDG reagent (Invitrogen). Then extract RNA according to the instructions provided by the reagent supplier.
5. Western blot analysis
The total protein was separated on a 6%-12% SDS-polyacrylamide electrophoresis gel and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Block the membrane with 5% skimmed milk and incubate with the primary antibody against ZNF488 (Abcam), then incubate with the anti-mouse or anti-rabbit IgG secondary antibody. The band is detected by enhanced chemiluminescence, and GAPDH or α-tubulin is served as a loading control.
6. Wound healing assay
When the cells in the 6-well plate grow to 80-90% confluence, replace the complete medium with serum-free medium, and incubate for 24 hours. An 10ul pipette tip was used to create a wound on the surface of the adherent cells,and images were taken at 0 and 24 hours useing a fluorescent inverted microscope.
7. Transwell invasion assay
20,000 cells resuspended in serum-free medium were added to the upper chamber of the transwell chamber, while 500 µl of 20% serum-containing medium was added to the lower chamber.After culturing for 24-36 hours, the unmigrated cells in the upper chamber were remove. The cells that migrated through the holes to the lower chamber were fixed with methanol, and then stained with 1% crystal violet and counted.
100 NSCLC tissue samples were stained with specific antibodies and bio-conjugated secondary antibodies, and then incubated with avidin-biotin-peroxide complex. Visualize the target protein using 3-amino-9-ethylcarbazole chromogen. Two experienced doctors in the pathology department scored the staining intensity of the IHC staining results: no staining was scored 0 point; weak staining was scored 1 point ; medium staining was scored 2 points; strong staining was scored 3 points. Score based on the percentage of counted positive staining cells in the overall cells: <10%, 0 point; 10%-25%, 1 point; 26%-60%, 2 points; more than 60%, 3 points. Multiply the above two parameters, the obtained immune score was 0-9. A score of <4 was defined as low expression, and a score of ≥ 4 was defined as high expression.
9. Xenograft experiment
BABL/C nude mice were purchased from Guangdong Laboratory Animal Co. Ltd (Guangzhou,China). The animal experiments were conducted under the guidance of the Animal Research Ethics Committee of Nanjing Medical University. Six nude mice in each group were inoculated tumor cells suspension through the tail vein at a density of 1×106. The general condition of the nude mice was observed and recorded every three days. After 8 weeks of injection the lungs were taken out, and the metastases were observed and photographed. The removed lung tissues were fixed with formaldehyde. Tissue sections were made with paraffin, and stained with hematoxylin and eosin (HE).
10. Treatment for the patients
According to the clinical practice guidelines of oncology of National Comprehensive Cancer Network (NCCN) , the treatment plan was developed. The 100 NSCLC patients included in the study were all stage I-IIIa. All the patients underwent radical resection of lung cancer, and the surgical plan was thoracoscopic one-way lobectomy plus mediastinal lymph node dissection. The excised tumor tissue specimens were subjected to histopathological examination to determine the tumor type, differentiation and pathological stage. Then according to the pathological stage, patients in stage Ib-IIIa received adjuvant chemotherapy for 4 cycles while patients in stage Ia did not. The adjuvant chemotherapy regimen is NP regimen (vinorelbine 25mg/m2 for the 1st day and the 8th day plus cisplatin or nedaplatin 80mg/m2 for the 1st day, every 21 days a cycle) or GP regimen (gemcitabine 1000mg/m2, for the 1st day and the 8th day plus cisplatin or nedaplatin 80mg/m2 for the 1st day, every 21 days a cycle).
Follow-up was conducted by telephone or outpatient service from the first day of treatment to death or the deadline for follow-up. After treatment, the patients were followed up every 3 months for the first 2 years, every six months from the 3rd to the 5th year, and every year after 5 years. Follow-up content included physical examination, hematology test, imaging examination, etc.
12. Statistical analysis
Data were presented as mean±standard deviation (SD), and SPSS 25.0 software was used for statistical analysis. The Kaplan-Meier method was used to calculate the cumulative survival time of clinical samples. Cox regression model was used to perform multivariate analysis on the parameters that were different in single factor analysis. Use chi-square test or t-test to compare the differences between the two groups. When P-value <0.05, the difference was considered to be statistically significant. Use Graphpad 5.0 software to draw the results.