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 cell apoptosis kit was purchased from Beyotime Institute of Biotechnology (Haimen, China). 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). CCAT2, CCAT2 small interfering RNA (siRNA), negative control (NC) siRNA, and fluorescein isothiocyanate (FITC)-labeld CCAT2, si‑CCAT2, and NC-siRNA were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). All of the other chemicals were analytical or reagent grade.
Cell culture and animals models
Cells, including the lung cancer cell lines (H520 and A549) and normal control cell lines (BEAS2B, CCD-8L, and LL24) were obtained from the American Type Culture Collection (ATCC). The cisplatin-resistant A549/DDP cell lines were obtained from the Bank of Cancer Cell Lines of the Chinese Academy of Medical Science (Beijing, China). All cells were cultured in the RPMI 1640 containing 10% FBS and supplemented with and 100 U/mL penicillin/streptomycin at 37℃ in a humidified incubator with 5% CO2. 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) and raised under standard condition with free access to food and water.
Transfection of cells with CCAT2, si‑CCAT2, and NC-siRNA (or FITC-labeld CCAT2, si‑CCAT2, and NC-siRNA) was performed using the Lipofectamine 2000 (Invitrogen, USA) in accordance with the manufacturer's guidelines. In brief, 1× 104 A549 cells were seeded in six-well plates. After grown for 70% confluency, the plasmids containing different RNAs plus lipofectamine 2000 was added and co-incubated with cells for 6 h. Then the cell uptake efficacy and transfection efficiency was respectively determined by fluorescent analysis under a fluorescent microscopy (Leica DMI4000 B, Germany) and quantitative analysis of the CCAT2 expression by RT-qPCR assay.
Colony formation assay
5 × 103 of the transfected A549 cells were seeded into six-well plates and cultured for 10 days with the culture medium were replaced with fresh medium every two days. Then the cells in each well were stained with 0.1% crystal violet for 20 min followed by washing three times with phosphate buffer saline (PBS, pH 7.4). Finally, the colony numbers of cells were qualitatively analyzed under a invert microscopy (LSM710, Leica, Germany).
Migration and invasion assay
The lateral migration ability of A549 cells after transfection was determined by the wound-healing assay. In brief, 5 × 104 of the transfected cells were seeded in the six-well plates and allowed to culture for 24h. 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 24 h using the invert microscope.
The vertical invasion ability of A549 cells after transfection was determined by the trans-well assay. For investigation, 5 × 104 of the transfected cells in 100 µL serum-free medium were seeded in the top chamber of the 24-well trans-well while the lower chamber was filled with 600 µL medium containing 10% FBS. After incubation for 24 h, the cells invaded into the lower surface of the insert chamber were stained with 0.1% crystal followed by qualitative analysis using the invert microscope and quantitative analysis by the microplate reader (Thermo Multiskan MK3, USA).
Cell growth assay
5 × 103 of the transfected A549 cells were seeded in 96-well plates and cultured for an overnight. Then 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. To detect the cell growth rate, 10 μL of CCK-8 reagent was added and incubated with cells for 4 h followed detection at 450 nm using the microplate reader.
Additionally, to investigate whether elevation of CCAT2 expression alleviated the cytotoxicity of DDP, the transfected A549 cells were seeded as above. Then 0.5 µg/mL of DDP were added into each well and co-incubated with cells for 12, 24, 48 h, respectively. After addition of 10 μL of CCK-8 reagent and incubation for 4 h, the absorbance of the cells were detected as above.
Cell apoptosis assay
To further investigate the effect of CCAT2 on the alleviation of cytotoxicity of DDP to A549 cells, cell apoptosis assay was performed. 5×104 transfected A549 cells were seeded in 6-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 protein 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 various primary antibodies (1:100) including anti-P-gp, -MRP, -IGFBP2, -p-AKT/AKT, and -Bcl2 for overnight. Subsequently, peroxidase-conjugated secondary antibodies (1:1000) 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; si-CCAT2, forward, GATCC GTGCAACTCTGCAATTTAACACTTCCTGTCAGATGTTAAATTGCAGAGTTGCACTTTTTG, and reverse, AATTCAAAAAGTGCAACTCTGCAATTTAACA TCTGACAGGAAGTGTTAAATTGCAGAGTTGCACG; NC siRNA, forward, GATCCGCCACTTTGAAGAACCCAATCCTTCCTGTCAGGATTGGGTTCTTCAAAGTGGCTTTTTG, 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.
In vivo tumor growth and progression assay
The A549 cancer-bearing mice models were established using the subcutaneous transplantation approach. In brief, 2 × 106 of the transfected A549 cells in 100 µL RPMI-1640 (FBS free) were subcutaneously injected into the right flanks of mice. Then the mice were grouped (n=10) according to the injected cells: Control group (non-transfected A549 cells), NC siRNA group (NC siRNA transfected A549 cells), CCAT2 group (CCAT2 transfected A549 cells), and si-CCAT2 group (si-CCAT2 transfected A549 cells). All mice from each group were raised under the standard condition with free access to food and water. For tumor growth observation, the tumor volume changes of mice in each group were carefully calculated every three days using the formula: Volume = 0.5 × length × (width)2. For survival investigation, the deaths in each group were carefully recorded for finally Kaplan-Meier curve analysis. Besides above, the effect of CCAT2 regulating the anti-tumor effect of DDP was further investigated in vivo. Tumor-bearing mice were established and grouped (n=10) as above. Then the mice in each group was treated with DDP (5mg/kg) via tail intravenous injection. The treatment was done every two days in a week (a total of three times). Then the tumor volume change and survival time of each mice were carefully calculated and recorded. At the end of in vivo experiments, all tumor tissues were obtained for further analysis after the alive mice were euthanized.
Immunohistochemistry (IHC) study and TUNEL assay
The obtained tumor tissues were prepared for 5 µm slides followed by incubated with primary antibodies (anti-miR-204-3p, 1:100) for an overnight. Then the primary antibodies were withdrawn and replaced with the streptavidin peroxidase-conjugated second-antibodies (1:1000). After incubation for 1h, the results were obtained using the invert microscope. The Tdt-mediated dUTP nick-end labeling (TUNEL) assay was performed according to the protocol of NeuroTACS II In Situ Apoptosis Detection Kit. The tumor slide samples were counterstained with hematoxylin while positive signals were developed by diaminobenzidine (DAB). Finally, the results were achieved by the 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 experiments performed here were repeated at least three times and the statistical significance was defined as P<0.05.