Cell lines and reagent
Human normal lung epithelial cell line (BEAS-2B) and human lung adenocarcinoma cell lines (HCC827, H1650, H522, H1299, A549) were available from American Type Culture Collection (ATCC; Manassas, VA). All cell samples were cultured in Dulbecco's modified Eagle’s medium (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS; Gibco) and 1% mixture of Pen/Strep. Cell culture was performed in humidified incubator containing 5% CO2 at 37 °C. 0.5 µM of Colivelin, STAT3 activator, was available from Santa Cruz Biotechnology (Dallas, TX).
Isolation of total RNA and quantitative real-time PCR (RT-qPCR)
The isolation of total RNA was conducted with the application of Trizol reagent (Thermo Fisher Scientific, Waltham, MA). cDNA was then synthesized using PrimeScript™ RT reagent kit as instructed by supplier (Takara, Otsu, Janpan). SYBR Premix Ex Taq II (Takara) was employed on StepOne™ Real-Time PCR System (Applied Biosystems, Carlsbad, CA) for quantitative analysis, with GAPDH gene or U6 as internal reference. 2-ΔΔCt method was applied for calculating relative expression.
Plasmid transfection
To overexpress GATA6-AS1, the full-length cDNA sequences of GATA6-AS1 was cloned into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) after PCR amplification. To suppress GATA6-AS1 and SOCS1, the designed short hairpin RNAs (shRNAs) and control-shRNAs were purchased from GenePharma (Shanghai, China). Besides, miR-331-3p mimics and NC mimics, miR-331-3p inhibitor and NC inhibitor were acquired from RiboBio (Guangzhou, China). Transfection kit Lipofectamine2000 was used for 48 h of plasmid transfection as instructed (Invitrogen).
Cell counting kit-8 (CCK-8)
After transfection, cells of A549, H1299 and HCC827 were reaped at logarithmic growth phase and seeded to 96-well plates with 2 × 104 cells/well. Cell viability was monitored every 24 h using CCK-8 Kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). 10 μl of CCK-8 was added to each well for 1 h at 37°C with 5% CO2. Absorbance at 450 nm was examined by microplate reader.
EdU staining
Cell proliferation was also examined by EdU staining Kit as required by supplier (Ribobio). After transfection, each well of 96-well plate were added with 100 μL of EdU medium diluent, then cells were fixed by 4% paraformaldehyde and permeabilized by 0.5% Troxin X-100. DAPI dye was used for nuclear counterstain, and cell stained cells were evaluated by fluorescent microscope (Leica, Wetzlar, Germany).
TUNEL staining
Cells on coverslips were fixed for 10 min, then permeabilized in PBS. Cell apoptosis was detected in accordance with the protocol of In Situ Cell Apoptosis Detection Kit (Roche, Basel, Switzerland). After DAPI staining, cell samples were analyzed by manual counting under fluorescent microscope.
Flow cytometry assay
Transfected cancer cells (2 × 105) were reaped, then rinsed in precooled PBS and subjected to Annexin V-FITC/PI Apoptosis kit as instructed by provider (BD Biosciences, San Jose, CA). Following 15 min of incubation in darkroom, apoptotic cells were treated with FACS Calibur flow cytometer (BD Biosciences).
RNA pull down
The miR-331-3p fragment covering the GATA6-AS1 or SOCS1 binding sites including wild-type and mutant, were biotinylated into Bio-miR-331-3p-WT/Mut probes. The cell protein lysates collected from RIPA lysis buffer were mixed with miR-331-3p probes and control probes in magnetic beads. The RNA enrichment in pull-down mixture was assayed via qRT-PCR.
RNA immunoprecipitation (RIP)
Thermo Fisher RIP kit was commercially from Thermo Fisher Scientific for performing RIP assay in A549 and H1299 cells in light of the user manual. The collected cell lysates from RIP lysis buffer were employed for immunoprecipitation with magnetic beads and human Argonaute2 (Ago2) antibody (Millipore, Billerica, MA). Normal mouse immunoglobulin G (IgG; Millipore) served as negative control.
Luciferase reporter assay
The GATA6-AS1 or SOCS1-3’-UTR fragment covering the miR-331-3p binding sequences (wild-type and mutant) were used to insert into pmirGLO vector, and the constructs were named as GATA6-AS1-WT/Mut and SOCS1-3’-UTR-WT/Mut. Following co-transfection with indicated transfection plasmids, HEK-293T cells (ATCC) were subjected to Luciferase Reporter Assay System (Promega, Madison, WI) 48 h later.
Western blot
Cell protein samples collected from RIPA lysis buffer were subjected to electrophoresis on 12% SDS-PAGE, and then separated proteins were shifted to PVDF membranes. The primary antibody against SOCS1 (ab62584; Abcam, Cambridge, MA) and GAPDH (ab8245; Abcam) were used to probe membranes after specific dilution. At last, membranes were analyzed using ECL detection system as instructed (Bio-Rad lab, Hercules, CA).
Statistical analysis
All assays in this study were bio-repeated for more than two times, the measurement data were exhibited as the means ± Standard Deviation (S.D.) and analyzed by use of PRISM 6 (GraphPad, San Diego, CA). P-values below 0.05 were seen as the significant levels for statistical analyses in form of one-way analysis of variance (ANOVA) or Student’s t-test.