H1299 and A549 cell lines were obtained from the ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) at 37˚C in a 5% CO 2 humidified atmosphere.
1) Transfection and interference
Based on the previous study results, H1299 cells were E6 and E7 low-expression cell lines, while A549 cells were E6 and E7 high-expression cell lines . The plasmid of pEGFP-N1-HPV16 E6, pEGFP-N1-HPV16 E7, and pEGFP-N1 were kindly provided by Prof Xudong Tang, Institute of Biochemistry and Molecular Biology, Guangdong Medical College, China. HPV16 E6 SiRNA and HPV16 E7 SiRNA were purchased from RIBOBIO (Guangzhou, China), KIF7 siRNA was purchased from General Biosystems (Anhui, China). Disordered siRNA was used as a nonspecific siRNA control.
The cells were performed in 6-well plates using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) as per instructions from the manufacturer. Transfection with empty vector and mock transfection were used as controls. The protein analysis was assessed 48 hours after transfection by western blotting. The mRNA analysis was assessed 24 hours after transfection by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR).
The assays were performed as previously described . Information about primary antibodies is as follows: HPV16 E6 (1:100, Bioss Biotechnology Co., Ltd, Beijing, China), HPV16 E7 (1:100, Bioss Biotechnology Co., Ltd, Beijing, China), KIF7 (1:1000, Proteintech, Wuhan, China), LKB1 (1:800, Proteintech, Wuhan, China), p-LKB1 (Phospho-Ser428) (1:800, Sangon Biotech, Shanghai, China), GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA), and Lamin B (1:800, WanLeibio, Shenyang, China).
Quantitative real-time PCR
Total RNA was extracted from cells with Trizol solution (TaKaRa, Dalian, China) and RNase RNA isolation kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. A total of 1μg RNA was subjected to reverse transcription reaction to obtain cDNAs by using a Prime Script TM RT reagent Kit (TaKaRa, Dalian, China). qRT-PCR was performed using SYBR® Premix Ex Taq II (TaKaRa, Dalian, China) on 7900HT Fast Real-Time PCR System (Applied Biosystems). Non-template controls were carried out every time for each primer pair to detect nonspecific amplification. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as the internal control. All reactions were run in triplicate. The fold change of mRNA expression was calculated using the 2−ΔΔct method. The detailed information of the primers is given in Table 1.
Nuclear plasma separation technology
H1299 cells were interference with SiKIF7. Nuclear and Cytoplasmic Protein Extraction Kit (P0028, Beyotime, Shanghai, China) was used to perform nuclei isolation. All the experiments were performed on ice, Cytoplasmic protein extraction reagent A with 1mM PMSF was added to the cell suspension, and then 10-15 minutes of ice bath after 5 seconds of violent oscillation at the highest speed, cytoplasmic protein was added to cytoplasmic protein extraction reagent B. After 5 seconds of violent oscillation at the highest speed, the supernatant was centrifuged at 4℃ at 12000g for 5 minutes. The nuclear protein extraction reagent with 1mM PMSF was added to the remaining precipitate, which was intensely vortex at the highest speed for 15-30 seconds and then placed in an ice bath for 30 minutes, during which time the nuclear protein was intensely vortex for 15-30 seconds every 1-2 minutes. After centrifugation at 4℃ and 12000g for 10 minutes, the supernatant was absorbed into the precooled plastic tube, which was nuclear protein.
SPSS 22.0 software was utilized for statistical analyses in this study. Each assay was performed at least 3 times. The data were expressed as mean ± SD. Statistical significance was determined by Student’s t-test, and a p value<0.05 was considered significant.