NSCLC specimens were collected from 94 patients who were treated in the First Affiliated Hospital of Chengdu Medical College (Chengdu, China) from April in 2019 to September in 2020. The collection of clinical samples was approved by the ethics committee of Chengdu Medical College (approved ID: BR20re19), and each participant signed an informed consent form. All the patients were given no treatment before surgery. The collected specimens were stored in a liquid nitrogen tank.
Cell maintenance and transfection
The A549 and A549/R (cisplatin resistant) cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China), and cultured in DMEM (Dulbecco's Modified Eagle Medium) media (WISENT, Nanjing, Jiangsu, China) in a CO2 incubator at 37 °C. The DMEM media was supplemented with 10% FBS (fetal bovine serum), 100 mg/mL streptomycin and 100 U/mL penicillin (WISENT). The FBS was bought from Thermo Fisher Scientific (Waltham, Massachusetts, USA). All the other cell culture materials were bought from WISENT.
The cell infection was conducted according to its instruction book (Genechem, Shanghai, China). sh-ASAP1-IT1: 5’-GCU GCG ACA AUA GAC AUC GGA GUU U-3’, and sh-NC: 5’-CUC UCG GAA CAU GUC ACA U-3’. The mimic microRNAs and inhibitors were purchased from Ribobio (Guangzhou, China), miR-509-3p-related sequences were listed, Mock, 5’-UCU CCG AAC GUG UCA CGU U-3’; and anti-miR-509-3p, 5’-CCG UGG UUC AUA CUG GUA-3’; miR-509-3p mimic, 5’-UAC CAC AGG GUA GAA CCA CGG-3’. The pmirGLO-ASAP1-IT1-WT (wild type) or -Mut (Mutant), and pmirGLO-Yap1-3’UTR (untranslated region)-WT or -Mut (Mutant), and pcDNA-sh-NC, pcDNA-sh-ASAP1-IT1 were from Thermo Fisher Scientific.
Hematoxylin-eosin (H&E) staining
The NSCLC specimens were fixed in 4% paraformaldehyde, embedded in paraffin and subjected to sectioning. Then, H & E staining was performed as described previously . All the chemicals and reagents were bought from Servicebio (Wuhan, Hubei, China).
RNA extraction and qRT-PCR (quantitative real time polymerase chain reaction)
The total RNA was extracted using TRIzol reagent (Life Technologies, Rockville, MD, USA). The reverse transcription kit (Thermo Fisher Scientific) was applied to synthesize cDNA (complementary DNA) from the total RNA. Quantitative RT‐PCRs were used to evaluate the levels of miRNAs, lncRNAs, or mRNAs using the SYBR Premix Ex Taq II (TaKaRa, Dalian, China) . The qRT-PCR was carried out under the thermal cycling conditions: 95 °C × 5 min, and 40 cycles of 95 °C× 30 s, 60 °C × 30 s, and 72 °C × 1 min. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control form RNA and lncRNA, U6 was used as the internal control of miRNAs. The relative RNA levels were calculated following the 2−ΔΔCT method. The PCR primers were as follows: U6, 5’-TGC GGG TGC TCG CTT CGG CAG C-3’ (forward), 5’-GTG CAG GGT CCG AGG T-3’ (reverse); miR-509-3p, 5’-UAC CAC AGG GUA GAA-3’ (forward), 5’-CTCTACAGCTATATTGCCAGCCA-3’ (reverse); GAPDH, 5′-CAC CCA CTC CTC CAC CTT TG-3′ (forward), 5′-CCA CCA CCC TGT TGC TGT AG-3′ (reverse); Cyclin A1, 5’-ATAACGACGGGAAGAGCGG-3’ (forward), 5’-CAGGGTACATGATTGCGGGA-3’ (reverse); Cyclin B1, 5’-CAGGTTGTTGCAGGAGACCA-3’ (forward), 5’-CATGGCAGTGACACCAACCA-3’ (reverse); PCNA (proliferating cell nuclear antigen), 5′-CGCCCTGGTTCTGGAGGTAA-3′ (forward), 5′-GGCTGAGACTTGCGTAAGGG-3′ (reverse);Bcl-2, 5′-TTCTTTGAGTTCGGTGGGGT-3’ (forward), 5’-GAAATCAAACAGAGGCCGCAT-3’ (reverse); Bax, 5’-GGG TTG TCG CCC TTT TCT AC-3’ (forward), 5’-AGT CGC TTC AGT GAC TCG G-3’ (reverse);caspase-3, 5′-GGGGAGCTTGGAACGGTACG-3’ (forward), 5’-CCACTGACTTGCTCCCATGTAT-3’ (reverse); YAP1,5′-TGCTGTCCCAGATGAACGTC-3’ (forward), 5’-GGTTCATGGCAAAACGAGGG-3’ (reverse); ASAP1-IT1, 5’-AAACATCATCCCCAGAGTGG-3’ (forward), 5’-GCCTTGCTCACCTCTGAAAC-3’ (reverse);SOX2 (SRY-box transcription factor 2), 5’-CAT GAA GGA GCA CCC GGA TT-3’ (forward), 5’-ATG TGC GCG TAA CTG TCC AT-3’ (reverse); CD44, 5’-GCC ACC AGA GCT ATT CCC AA-3’ (forward), 5’-GGT CTT CGC CCA GCC TTT CT-3’ (reverse);CD133, 5’-GCC ATG CTC TCA GCT CTC C-3’ (forward), 5’-TCC TGA AAA GGA GTT CCC GC-3’ (reverse).All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.
Total protein samples were prepared from the tissues or cells using RIPA (radio immunoprecipitation assay) buffer (Beyotime) for 30 min. The lysates were centrifuged at 12, 000 g for 15 min to obtain the supernatants, and they were de-natured at 98 °C for 20 min. 50 μg proteins were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). All the prepared gels were transferred to the 0.22 μm PVDF (polyvinylidene difluoride) membranes (Thermo Fisher Scientific) for 2 h, and the proteins-carried PVDF were blocked with 1% bovine serum albumin. Then, the PVDF membranes were incubated with the primary antibody overnight in a 4 °C refrigerator. The membranes were incubated with the HRP (horseradish peroxidase)-conjugated secondary antibody diluted at 1: 50 000 (Bioworld Technology, Nanjing, China) for 1 hour. The anti-YAP1 and anti-Tubulin-α were bought from Thermo Fisher Scientific.
Sphere formation and colony formation of cancer stem cells
Cancer stem cells were sorted from A549 cells or transfected-A549 cells by sphero-cyst medium . The 3 × 104 cells were seeded in 6-well ultra-low cluster plates (Thermo Fisher Scientific). They were maintained in DMEM/F12 serum free medium (Thermo Fisher Scientific) containing epidermal growth factor (20 ng/mL), beta-fibroblast growth factor (20 ng/mL), insulin (4 μg/mL), and B27 (2%). All reagents were from Sigma. Finally, the number of spheres was counted under an inverted microscope (Leica, Oskar-Barnack-Straße, Germay) post 10 days incubation. The biomarkers of cancer stem cells SOX2, CD34, and CD133 were used to identify the cancer stem cells using qRT-PCR and Western blot.
The A549 cancer stem cells were isolated using magnetic bead kit, and 3 × 104 stem cells were seeded in six-well plates with ultra-low adhesion. They were cultured in sphere formation media for a week, and post another 20 days, the number of spheres was calculated under a microscope.
Cell apoptosis and cell cycle analysis
To determine cell apoptosis of A549-dereived stem cells and other cells, cells were collected at 80 g × 4 °C × 5 min for annexin-V/FITC (fluoresceine isothiocyanate)/PI (propidiumiodide) staining after 48 h infection with the indicated plasmids or miRNAs. Briefly, cells were incubated with annexin-V and PI for 10 min and 5 min, respectively, in a dark room using the annexin V-FITC/PI staining kit (Beyotime, Beijing, China). Finally, the cell cycle was evaluated using Beckman Coulter Navios EX flow cytometry (Beckman, Shanghai, China).
For analyzing cell cycle, the cells were cultured in 12-well plates (Thermo Fisher Scientific). After transfection with the indicated miRNAs or plasmids for 48 h, they were subjected to suspension and fixation in 75% ethanol at 4°C for 12 h. Then, they were washed with PBS (phosphate‐buffered saline) for 3 times. Afterwards, they were resuspended in 500 μL staining buffer containing propidium iodide (5 mg/ml)/RNase (10 mg/ml) at 37 °C for 30 min in a dark room. Finally, cell apoptosis rate was evaluated using the flow cytometry. Each treatment group consisted four wells, and each assay was repeated for 3 times independently.
Cell counting kit‐8 (CCK-8) assay
The A549-dereived stem cells or A549 cells were plated into a 96-well plate (Thermo Fisher Scientific) at the density of 4 × 103 cells per well for determination of cell viability. CCK‐8 reagent (Sangon, Shanghai, China) were added into medium when cultured 36 h. After incubation for 1 h at 37 °C, the formation of water‐soluble formazan was examined in a microplate reader (Bio-Rad) with the light length of 450 nm. All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.
Dual luciferase reporter assay
To determine the effects of miR-509-3p on luciferase activity of pmirGLO-ASAP1-IT1, 2 × 104 A549 cells per well were seeded in 24-well plates. They were co-transfected with pmirGLO-ASAP1-IT1-WT (wild type), pmirGLO-ASAP1-IT1-Mut (Mutant), as well as Mock (negative control), miR-509-3p mimic, and anti-miR-509-3p. To examine the effects of miR-509-3p on luciferase activity of pmirGLO-Yap1-3’UTR, the cells were co-transfected with pmirGLO-Yap1-3’ UTR-WT, pmirGLO-Yap1-3’ UTR-Mut (Mutant), as well as Mock, miR-509-3p mimic, and anti-miR-509-3p. After transfection for 48 h, they were collected and lysed. The supernatants were used for measurement of luciferase activity using the Dual-Luciferase Assay Kit on GloMax 20/20 Luminometer following the manufacturer’s instructions (Promega, Madison, USA). All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.
RNA immunoprecipitation (RIP) assay
RIP assay was conducted to examine the interaction between ASAP1-IT1 and miR-509-3p using the RIP RNA-binding protein immunoprecipitation kit Magna (Millipore, Massachusetts, USA). Briefly, the cell lysate was incubated with anti-Ago2 (argonaute-2), and anti-IgG was used as a negative control. The antibodies were purchased from Bioworld Technology (Nanjing, China). At last, the collected immunoprecipitated RNA samples were used to determine ASAP1-IT1 and miR-1301-3p content by qRT-PCR analysis. All experiments were performed in quadruplicate, and each assay was repeated independently for 3 times.
Mouse tumorigenesis assay
The A549-dereived stem cells were insfected with the mentioned shRNAs or plasmids as indicated. Post 48 h infection, the A549-dereived stem cells were collected at 80 g × 4 °C×5 min. Afterwards, 4 × 106 cells were inoculated into 5-week-old nude mice. The BALB/c nude mice were bought from the Model Animal Research Center of Nanjing University (Nanjing, China), and divided randomly for each group (3 mice for each group). The animal experiments were approved by the research ethics committee of Chengdu Medical College (approved animal protocol No.: CMC20LM22). The tumor volume was calculated every 3 days using the formula, tumor volume = (length × width2)/2. All the tumor-carried nude mice were sacrificed on the 24th day post inoculation.
Statistical analysis was conducted using SPSS software package (version 20.0, SPSS Inc., NY, USA) and GraphPad Prism 6 (GraphPad Software, CA, USA), and p value < 0.05 was statistically significant. The data were obtained from three independent experiments, and all data are expressed as mean ± standard deviation (S.D.). The statistical significance was examined using Two-tailed Student’s t-test for two-group comparisons and one-way analysis of variance (ANOVA) test with post-hoc analysis for multi-group comparisons.