DDP was obtained from Melonepharma (Dalian, China). The cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Gaithersburg, MD). The RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco (CA, USA) while the 100 U/mL penicillin/streptomycin was purchased from Sigma-Aldrich (St. Louis, MO, USA). The primers were obtained from Sangon Biotech (Shanghai, China) while the primary and/or second anti-bodies were purchased from BD Bioscience (San Diego, CA, USA). All of the other chemicals were analytical or reagent grade.
Cell Lines And Animals
The lung cancer cell lines H520 and A549 and the normal lung tissues cell lines, including BEAS2B, CCD-8L, and LL24 cells, were purchased from the American Type Culture Collection (ATCC). All the cells were cultured in RPMI 1640 containing 10% FBS and supplemented with and 100 U/mL penicillin/streptomycin at 37 ℃ in a humidified incubator with 5% CO2. The cisplatin-resistant A549/DDP cell line was obtained from the Bank of Cancer Cell Lines of the Chinese Academy of Medical Science (Beijing, China).
The male Balb/c nude mice, age of 4–5 weeks and weight of 20 ± 2 g, were achieved from BK Lab Anima Ltd. (Shanghai, China). For establishment of tumor-bearing model, 2 × 106 A549 cells were subcutaneously injected into the right flanks of mice. Then the mice were raised under standard condition with free access to food and water. The experimental protocol related animals in the present study was approved by the Animal Experimentation Ethics Committee of Cancer Hospital of China Medical University.
For transfection of cells, the plasmids was applied and performed using the Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's guidelines. After the A549 cells were allowed to grow for 70% confluency in six-well plates, the plasmid plus lipofectamine 2000 was added and co-incubated with cells for 6 h. Then the old medium was removed and immediately replaced with fresh ones and culture for further use.
Colony Formation Assay
The transfected cells were seeded into the six-well plates at the density of 5 × 103 cells/per well. After 10 days of incubation, the cells were stained with 0.1% crystal violet for 20 min. Then the cells were washed three twice with PBS followed by calculation of the numbers of colonies using the Image J software.
5 × 104 transfected cells were seeded in the six-well plates and allowed to culture for 24 h. Then the old medium was replaced with fresh mediums and the cells were incubated until the full monolayer was formed. Subsequently, a 150 µl sterile polystyrene micropipette tip was applied to make an artificial wound of scratched cells. The scratch were photographed respectively at the 0 h and 48 h using the invert microscope (Chongqing Optical & Electrical Instrument, Co., Ltd., Chongqing, China).
Cell Invasion Assay
For investigation of the vertical migration and invasion ability of cells, the 24-well trans-well (8-µm pore size, Corning, NY, USA) was applied. For migration assay, 5 × 104 cells in 100 µL serum-free medium were seeded in the top chamber while the lower chamber was filled with 600 µL medium containing 10% FBS. After incubation for 24 h, the cells migrated into the lower surface of the insert chamber were stained with 0.1% crystal followed by quantitative analysis using the microplate reader (Thermo Multiskan MK3, USA).
Cells were seeded in 96-well plates at the density of 5 × 103 cells per well. After 24 h of incubation, the old medium in each well of the plates was replaced with fresh culture medium and continue to incubate for 12, 24, 48 h, respectively, in the presence of DDP or not. Following incubation with 10 µL of CCK-8 reagent for 4 h, the absorbance of each well was detected at 450 nm using the microplate reader (Thermo Multiskan MK3, USA).
Flow Cytometric Analysis
To determine the apoptosis of cells, 5 × 104 transfected A549 cells were seeded in 96-well plates and allowed to grow for overnight. Then the old medium in each well was replaced with fresh serum-free medium containing DDP (0.5 µg/mL). After incubation for 24 h, the cells in each well were harvested by centrifugation at 1000 g for 5 min. Then the apoptosis of cells was examined using the double staining (propidium iodide and annexin V) approach and determined by the FACSscan Flow Cytometer (BD PharMingen, Heidelberg, Germany).
Western Blot Analysis
Total proteins in the tumor tissues and cells was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) and performed in accordance with the manufacturer's instructions. Then the concentration of RNA was determined using the BCA protein assay kit (Pierce, Thermo Scientific). The tissue or cell lysis solutions were separated by the SDS-PAGE followed by transplant to the polyvinylidene difluoride (PVDF) membranes ((Millipore Corp., Billerica, MA, USA). After that, the membranes were blocked by 5% non-fat milk in TBST buffer for 1 h followed by co-incubation with primary antibodies (1:1000) for overnight. Subsequently, peroxidase-conjugated secondary antibodies (Yeasen, Shanghai, China) were introduced and co-incubated with the samples for 1 h before visualization of the signal using enhanced chemiluminescence (ECL) method using chemiscope 5600 (CLINX, Shanghai, China). The protein expression levels were normalized to that of GAPDH (Sangon Biotech, Shanghai, China) and semi-quantitative analysis was performed by densitometric scanning.
Real-time Quantitative PCR (RT-qPCR)
The RNA in tissues and cells was obtained as above. After that, the PrimeScript RT Master Mix (Takara, Tokyo, Japan) was applied to conduct the cDNA followed by analysis of the RT-qPCR using the SYBR Green PCR master mix (Takara, Tokyo, Japan). The primer sequences were as follows: CCAT2, forward, GGCCTGTAGGAAGAGTCAAATAG, and reverse, AGGTCAGGAATCAGGAGACA; sh-CCAT2, forward, GATCC GTGCAACTCTGCAATTTAACACTTCCTGTCAGATGTTAAATTGCAGAGTTGCACTTTTTG, and reverse, AATTCAAAAAGTGCAACTCTGCAATTTAACA TCTGACAGGAAGTGTTAAATTGCAGAGTTGCACG; sh-NC, forward, GATCC GCCACTTTGAAGAACCCAATCCTTCCTGTCAGGATTGGGTTCTTCAAAGTGGCTTTTTG, and reverse, AATTCAAAAAGCCACTTTGAAGAACCCAATC TCTGACAGGAAGGATTGGGTTCTTCAAAGTGGCG; AKT forward, GGACAACCGCCATCCAGACT, and reverse, GCCAGGGACACCTCCATCTC; BCl2 forward, TTCTTTGAGTTCGGTGGGGTC, and reverse, TGCATATTTGTTTGGGGCAGG; IGFBP2F forward, GGGACTGCTTTCCAATAG, and reverse, TTACAGTCTTTGGTCTCGG; GAPDH, forward, CGGAGTCAACG GATTTGGTCGTA, and reverse, AGCCTTCTCCATGGTGGTGAAGAC. The protein expression levels were normalized to that of GAPDH.
Immunofluorescence Assay And Immunohistochemistry (IHC) Study
Tumor tissues were obtained at the end of in vivo tumor growth experiments and subjected to preparation of 5 µm slides. Then primary antibodies were introduced and co-incubated with the slides or cells for overnight followed by incubation with streptavidin peroxidase-conjugated or FITC-labeled secondary antibodies. The results were observed by the confocal microscopy analysis (LSM710, Leica, Germany) or invert microscope.
The data in the present study were expressed as the means ± SD. The statistical analyses were performed using the GraphPad Prism 7.0 version program. The Student’s t-test was applied to evaluate the difference between two means and the comparison between more than two groups was carried out by one-way ANOVA. The statistical significance was defined as P < 0.05.