Tumor sample collection
NSCLC tissues and matching normal lung tissues were obtained at Shandong Provincial Chest Hospital, after obtaining patient informed consent according to the protocol approved by the Shandong Provincial Chest Hospital institutional review board. All patients were diagnosed with NSCLC at Shandong Provincial Chest Hospital and underwent surgical resection. All tumor tissues were diagnosed by histopathology.
Total RNA from NSCLC tissues was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA). The cDNA was synthetized by PrimeScript Reverse Transcription Reagent Kit, and then qPCR was performed using SYBR Premix Ex Taq™. β-actin acted as the endogenous control. The specific primers of CKMT1 and β-actin were as follows, F: 5′-CTTCACCTCACTTTACCTTC-3′, R: 5′-TCTTTTACTTCTCTGCGTCT-3′ and F: 5′-CGTGACATTAAGGAGAAGCTG-3′, R: 5′-CTAGAAGCATTTGCGGTGGAC-3′. The relative levels of CKMT1 mRNA were calculated with 2−△△Ct method.
All samples were routinely fixed in formalin and then embedded in paraffin. The paraffin samples were continuously sliced into 5 µm sections. All tissues were stained using the streptavidin-peroxidase immunohistochemistry. Briefly, sections were deparaffinized in xylol and rehydrated in gradient ethanol. Subsequently, the sections were immersed in a sodium citrate solution (pH 6.0) and microwaved. To eliminate nonspecific staining, slides were incubated with 5% goat serum for 1 h. Then, the slides were incubated with rabbit polyclonal CKMT1 antibody (anti-CKMT1, 1:200, 15346-1-AP, Proteintech) for 2 h and incubated with a labeled polymer-HRP for 1 h. DAB chromogen solution was used for color reaction, and hematoxylin was used for counterstaining.
The scores for IHC were quantified independently by two trained pathologists at three 200X fields. The score for each sample was multiplied by the staining intensity score and the percentage score of positive cells. Staining intensity for CKMT1 was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Percentage of positive cells was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). A score > 3 points was considered to CKMT1 high expression.
Cell culture and transfection
The human normal pulmonary epithelial cell line (Beas-2B) and four NSCLC cell lines (H1650, H1299, A549 and H524) were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cell lines were cultured in RPMI-1640 medium (Sigma Chemical Co, St Louis, MO) supplemented with 10% fetal bovine serum (FBS, Sigma), 10 U/mL penicillin and 10 µg/mL streptomycin in a humidified atmosphere of 5% CO2/21% O2 at 37°C. Cells were exposed to a hypoxic chamber (MACS V A500 microaerophilic workstation, Don Whitley Scientific, Bingley, UK) with atmosphere containing 5% CO2, 1% O2 and residual N2 at 37°C to hypoxic conditions.
Specific siRNA sequences targeting CKMT1 (si-CKMT1) were synthetized by Genepharm Co. (Shanghai, China), and were transfected into cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). The CKMT1-specific siRNA sequences were designed as follows: 5′-ACGGTACCATGGCTGGTCCCTTCTCCCGT-3′.
Total protein was lysed using RIPA Buffer (Beyotime, Beijing, China) for 15 minutes on ice. The equal amounts of proteins (20–40 µg/lane) were separated by SDS-PAGE and transferred to PVDF membranes (EMD Millipore, Billerica, MA). The membranes were blocked with 5% nonfat milk for 1 h and incubated with the following primary antibodies: anti-CKMT1 antibody (1:1000, 15346-1-AP), anti-HIF-1α antibody (1:1000, 20960-1-AP), anti-E-cadherin antibody (1:2000, 20874-1-AP), anti-N-cadherin antibody (1:2000.16617-1-AP), anti-Snail1 antibody (1:2000, 13099-1-AP), and anti-Tubulin antibody (1:5000, 11224-1-AP), at 4°C overnight. All antibodies were purchased from Proteintech. Tubulin was used as an endogenous control. Then, membranes were incubated with appropriately HRP-conjugated secondary antibody for 1 h and visualized using the ECL system (GE Healthcare, Little Chalfont, UK).
Luciferase activity assay
The CKMT1 wild type (WT) or mutant type (MUT) promoter sequence was cloned into pGL3.0 vector, and the pGL3.0 recombinant plasmid was transfected into H1650 and H1299 cells using Lipofectamine 2000. After cultured under normoxic or hypoxic conditions for 48 h, cells were harvested and luciferase activity was detected using a luciferase assay system (Promega, Madison, Wisconsin).
Cells transfected or not transfected with si-CKMT1 or si-NC were seeded in 96-well plates. After incubating for a specified time, 10 µL of CCK8 solution was added to each well and incubated for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader.
The colony formation ability was determined with colony formation assay. Cells transfected or not transfected with si-CKMT1 or si-NC were seeded in 6-well plates, and cultured under normoxic or hypoxic conditions for 24 h. After another 2 weeks of cultivation, visible colonies were formed. The colonies were fixed with methanol and stained with 0.1% crystal violet. Colonies were analyzed by Elispot system (CTL) and pictured.
Transwell chamber (pore size of 8µm) coated with Matrigel was used to evaluate the cell invasion. Cells transfected or not transfected with si-CKMT1 or si-NC were suspended in FBS-free RPMI-1640 medium and were seeded to the upper chamber at a density of 2×103 cells/well, and the lower chamber was filled with RPMI-1640 medium containing 10% FBS. Invasion cells on the lower surface of the chambers were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet. Finally, the cells were counted in five randomly selected fields under an inverted microscope (200x magnification, Nikon TE2000).
Cells transfected or not transfected with si-CKMT1 or si-NC were cultured in FBS-free RPMI-1640 medium for 24 h, and were continue cultured under normoxic or hypoxic conditions for 24 h. Then, cells were harvested and washed twice with Annexin V binding buffer. Subsequently, cells were double stained with 5 µL Annexin V and 5µL PI, and analyzed by a flow cytometer (C6 Accuri, BD Biosciences).
Statistical analysis was performed using GraphPad Prism Software. Data are presented as the mean ± SD. The differences between the groups were calculated using a Student's t-test. P < 0.05 was considered statistically significant.