Patients and Tissue specimens
A total of NSCLC samples (n=72) and paracancerous tissues (n=20) in the study were collected from NSCLC patients enrolled in Jinan Central Hospital from January 2011 to December 2014. The inclusion criteria of the samples in this study were as follows: 1) patients undergo radical surgery with no seriously surgical contraindications; 2) stage I–IIIa NSCLC patients; and 3) No radiotherapy or chemotherapy before surgery.
Follow-up
After operation, 62 patients had chemotherapy, 27 patients had radiotherapy, and 32 patients received therapy of EGFR-TKI. We recorded the location and tumor recurrence time .Prognostic analysis of NSCLC patients in this study included deaths.
Cell culture and infection
The human NSCLC cell line H1299 was obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured in DMEM (Hyclone, USA) supplementing with 10% FBS (Gibco, Thermo Fisher, USA), penicillin (100U/mL; Sigma Aldrich, Germany) and streptomycin (0.1mg/ml; Sigma Aldrich) at 37°C. The recombinant lentiviruses packaged with shRNA-KIF21B were obtained from Genechem (Shanghai Genechem Co., Ltd.). The target sequence of KIF21B was as follows:GGA GCT GAT GGA GTA TAA G;The sequence of NC control was as follows:TTC TCC GAA CGT GTC ACG T.
Immunohistochemical staining
The paraffin-embedded samples were sectioned at 4 μm and the expression of KIF21B was evaluated by immunohistochemistry (IHC) using the EliVisionTMplus kit (Maixin, Fuzhou, China) with anti-KIF21B antibody (ab121931; Abcam, UK) according to the instruction. The expression level of KIF21B was evaluated by staining intensity and proportion of positive stained cells. The staining intensity was scored strong staining (3), moderate staining (2), weak staining (1) and no staining (0). The proportion of positive stained cells was graded 0-10% (0), 11-25% (1), 26-50% (2), 51-75% (3), and >75% (4). The multiplication of the scores of the two groups was the final score of the expression level of KIF21B, and the score less than or equal to 6 was the low expression group, and the score more than 6 was the high expression group [11, 12].
Reverse Transkription Polymerase Chain Reaction analysis
We extracted total RNA from H1299 cells by an RNA extraction kit (CWBIO, Beijing, China). The reverse transcription was then conducted using a HiFiScript cDNA Synthesis Kit (CWBIO). KIF21B mRNA expression was measured by SYBR Premix Ex Taq II kit (Takara, Japan). The obtained data was analyzed using 2-ΔΔCt method. β-actin was used as the control. The primers used in this study were synthesized by GENEWIZ (Suzhou, China) and as follows: KIF21B forward, 5’-CGA GGA GAC GGA TGA GAA CG-3’; reverse, 5’-CCA CCA GGC TCT CTT CAC TG-3’; β-actin forward, 5’-CCC GAG CCG TGT TTC CT-3’, reverse, 5’-GTC CCA GTT GGT GAC GAT GC-3’.
Western blot
After being infected for 72h, H1299 cells were lysed in RIPA Buffer (CWBIO) and quantified using a BCA kit (CWBIO).We separated protein with 10% SDS-PAGE gel after that electrotransferred them to PVDF members (Millipore, MA, USA). Subsequently, we blocked the members in 5% nonfat dry milk for 1 h, and then incubated them with primary antibodies at 4°C overnight. After that, we incubated the membrane with secondary antibodies (dilution, 1:3000; Proteintech Group) for 1 h at room temperature. The protein signals were developed using an ECL kit (CWBIO) and analyzed using Image J software.
CCK8 assay
1×103 H1299 cells transfected with shRNAs were seeded in each 96-well plate at 37°C for 1h, 24 h, 48 h and 72 h, respectively. We incubated the cells were with 10 μl of CCK8 solution (Solarbio Science & Technology, Beijing, China) for another 1 h at 37 °C. Finally, a plate reader determined the OD value at 450 nm.
Colony formation assay
H1299 cells infected with shRNA-KIF21B lenti-virus (200 cells/well) were plated in a well plate and cultured for 1-2 weeks. We fixed the cells with 4% paraformaldehyde for 30 min, then stained them with 0.1% crystal violet for another 30 min. Finally, we counted the number of colonies.
Hoechst/PI assay
Cells infected with shRNAs (1×103 cells/well) were cultured in a well plate for 24 h. And then, we co-stained the cells with Hoechst (10 μg/mL; Beyotime, Shanghai, China) and PI (5 μg/mL; Beyotime) for 15 min. After washing with PBS for 3 times, cell fluorescence was observed and captured under a fluorescence microscope.
Wound-healing assay
Following infection of 24 h, the cells (5×105 cells/well) were cultured in a well plate for 12 h. A wound was generated using a pipette tip in each well. After 24 h of culture, the wound closure was measured.
Transwell assay
Transwell chambers (Millipore) coated with Matrigel was carried out to assess cell invasion, whereas cell migration assay did not add Matrigel. We plated about 1× 105 lentivirus-infected cells in the upper chamber, filled with medium containing FBS in the lower chamber. Subsequent to a period of 24 h, we used cotton swabs to wipe out the cells that were not through the membrane. We fixed the migrated or invaded cells in 4% paraformaldehyde for 30 minutes, and then stained them with 0.1% crystal violet for another 20 minutes. The number of migrated or invaded cells was quantified under a microscope (magnification, ×100) in five random microscope fields.
In vivo tumorigenic assay
According to the NIH guidelines, animal procedures were carried out. Approximately 5×106 H1299 cells which were infected with lentivirus-shRNA-KIF21B or lentivirus-shRNA- control were subcutaneously injected into the axillae of BALB/c nude mice. Five weeks after the infection, We euthanized the nude mice, and collected tumor samples to measure their weight.
Statistical analysis
The measurement data were presented as Mean± SD and analyzed using Student’s t-test. The enumeration data were analyzed using Fisher’s exact probability test. The Kaplan–Meier with log-rank test was performed for survival rate. Cox proportional hazard model was performed for multivariate analysis. We used SPSS (IBM SPSS Statistics 25, USA) to perform for statistical analysis. And P<0.05 was regarded as significant difference.