The non-small cell lung cancer patients were enrolled in the Second Hospital of Hebei Medical University in this study with written informed consents. This study was approved by the Ethics Committee of the Second Hospital of Hebei Medical University. All tumors were pathologically confirmed by at least three experienced experts.
The human non-small cell lung cancer cell lines A549, SPCA1, H1299, H358, PC9 and immortalized human bronchial epithelial cell 16HBE were obtained from the American Type Culture Collection (ATCC, VA, USA). The cell identities and mycoplasma-free were authenticated by DNA profiling. All cancer cells were maintained in RPMI modified medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. HBEC cell was kept in Airway Epithelial Cell Basal Medium supplemented with the Bronchial Epithelial Cell Growth Kit (ATCC, VA, USA). Cell culture was performed in the humidified CO2 (5%) incubator. Exponentially growing cells were used for the experimental analysis. Lipofectamine 2000 (Invitrogen, MA, USA) was used for cell transfection purpose. Transfection efficacy was evaluated with GFP. For transient knockdown, siRNAs were synthesized by the Ribobio (Guangzhou, China).
Trizol reagent (Invitrogen, Waltham, MA USA) was employed to extract total RNA from lung cancer cells. The RNA samples were quality-checked by agarose electrophoresis and quantified with Nanodrop 2000 (Invitrogen, Waltham, MA USA). The cDNA was prepared from each 1 μg RNA using the First Strand cDNA Synthesis Kit (Sigma, St. Louis, MO) following the manufacturer’s guide. For miR quantitative purpose, reverse transcription was performed with miScript II RT Kit (Qiagen, Valencia, CA, USA). Real-time PCR was performed with the SYBR Green Real-Time PCR Master Mixes (ThermoFisher, Waltham, MA USA). The endogenous GAPDH was employed as internal control. For real-time PCR with miR, the miScript SYBR Green PCR Kit (Qiagen) was used. U6 was employed as internal control for miR quantitation. The primers were provided as below:
PELI3 Forward primer: CTGGAAGGAAACCCTGAAGT
PELI3 Reverse primer: AGCGGCGTGGAGATGTG,
GAPDH Forward primer: ACAACTTTGGTATCGTGGAAGG;
GAPDH Reverse primer: GCCATCACGCCACAGTTTC.
miR-365a-5p reverse transcription primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACATC;
miR-365a-5p Forward primer: CTGAGGGACTTTTGGGGGCAG,
miR-365a-5p Reverse primer: GTGCAGGGTCCGAGGT;
U6 reverse transcription primer: GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAA.
U6 Forward primer: TGCGGGTGCTCGCTTCGGCAGC,
U6 Reverse primer: GTGCAGGGTCCGAGGT.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
Cell viability was evaluated by the MTT method. The commercially available MTT Cell Proliferation Assay Kit (Cayman, Ann Arbor, MI, USA) was adopted for this assay. Briefly, transient PELI3-knockdown A549 and H1299 cells (1000 cells/well) were seeded into 96-well plate in triplicate and cultured for 24 h. 10 μL MTT solution was added into each well and incubated the plate in the CO2 chamber for 3 h to allow the formazan formation. Dissolution was conducted with 100 μL of dissolving solution and incubated for another 4 h. The absorption was measured at 570 nm with 490 nm as reference.
The indicated cells were lysed in ice-cold RIPA buffer (Beyotime, Nantong, China) for 30 min. The cell debris was discarded after refrigerated high-speed centrifugation (12,000 rpm by 15 min). The protein concentration was measured by the BCA method (Bicinchoninic Acid Kit, Sigma). The protein samples were first separated by the SDS-PAGE followed by the PVDF membrane transfer. The membranes were subjected to non-fat milk blocking, primary antibody incubation and secondary hybridization consecutively. The protein blots were visualized by the enhanced chemiluminescence kit (ECL Substrate Kit, Abcam, Cambridge, UK).
The fresh or flash-frozen in liquid nitrogen tissue samples were dehydrated first and followed by the fixation and paraffin embedding. Fixed and embedded in paraffin. The tissue blocks were cut into 5 μΜ sections and followed by the deparaffinization and rehydration. Antigen retrieval was performed with sodium citrate (pH 6.0) and boiled in microwave for 20 min. The endogenous peroxidase was blocked with 5% normal goat serum. The primary antibody (1:100) was incubated at 4 °C overnight. After rinsing with tris-buffered saline and Tween 20 buffer and H2O2 incubation, horseradish peroxidase-conjugated secondary antibody was applied for 1 h at room temperature. The protein was visualized with DAB (Boster, Wuhan, China).
Cell proliferation in response to PELI3 deficiency was determined by the colony formation assay. 1000 exponentially growing cells were seeded into 6-well plate in triplicate. Consecutive cell culture was performed in CO2 hood for 2 weeks. The formed colonies were fixed with methanol and stained with 0.25% crystal violet.
Cell invasive capacity was determined with Basal Membrane Extract (BD BioSciences, CA, USA)-precoated Transwell chambers (Corning, NY, USA). The single-cell suspension (20,000 cells/100 μL) was prepared in serum-free medium and laid on the upper insert. The complete culture medium (650 μL) was filled into the bottom compartment. After 12 h, the carbonate filter was cautiously removed and subjected to fixation and staining with crystal violet.
The migrative capacity of indicated cells was determined by the wound healing assay. The log phase single layer cells were cultured in 6-well plate overnight. The straight scratch of uniform width was created with sterile tips. The closure of scratch was continuously monitored under light microscope.
Luciferase reporter assay
The PELI3 3′UTR region was sub-cloned into pGL4 dual luciferase reporter vectors with the following primers:
Either wild-type or mutated luciferase reporter plasmids co-transfected into A549 and H1299 cells with miR-365a-5p. After 24 h, the relative luciferase activity was measured with Luciferase Assay System (Promega, WI, USA) in accordance with the manufacturer’s instruction.
The direct binding between miR-365a-5p and PELI3 transcript was interrogated by the pulldown assay. Biotin-labelled miR-365a-5p was purchase from Ribobio (Guangzhou, China). The cell lysate was prepared from both A549 and H1299 cell lines, which was subsequently incubated with biotin-miR-365a-5p at 4 °C for 2 h. The mixture was then pulled down by the Streptavidin-couple Dynabeads (ThermoFisher). After elution, the enriched PELI3 transcripts were quantified by the real-time PCR.
Xenograft tumor model
To establish xenograft tumor model, PELI3 was stably silenced in A549 by shRNAs, which was subjected to puromycin (2 μg/mL) selection and knockdown efficiency was confirmed by real-time PCR. BALB/c nude mice (4-week) were purchased from the Vital River (Beijing, China) and housed in the specific pathogen-free environment with free access to drinking water and food. 1 × 106 cells resuspended in serum-free medium was mixed with equal volume of Matrigel (BD BioSciences) and subcutaneously inoculated into right flank of nude mice. The tumor growth was monitored regularly until the size approaching 1500 mm3 and subjected to sacrifice. The xenograft tumor was resected and representative macroscopic images were captured. For lung metastasis evaluation, 5 × 105 cells were tail vein injected and lung nodules were determined by H&E staining.
All data were acquired from at least three independent experiments. GraphPad PRISM 6.0 software was used for data analysis and processing. Statistical comparison was performed with ANOVA followed the Student t-test. The p value was calculated and p < 0.05 was considered as significantly different.