Bioinformatics analysis
The adenocarcinoma cancer related GEO (Gene Expression Omnibus) data GSE63805 from NCBI (https://www.ncbi.nlm.nih.gov/) was downloaded, GSE63805 array data containing 32 adenocarcinoma samples and 30 paracancerous samples, and through the NCBI tools GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) to analysis the sample expression values of miR-224-5p.
The websites TargetScan (www.targetscan.org), miRanda (www.microrna.org) and PITA (http://genie.weizmann.ac) were used to predict the potential targets of miR-224-5p. The online tool Venny 2.1 (http://tools/venny/index.html) to identify the overlapping the targets.The Starbase (starbase.sysu.edu.cn) website were used to evaluate the binding site and their expression correlation between miR-224-5p and the target IL6ST. The GEPIA database (http://gepia2.cancer-pku.cn)and Kaplan-Meier Plotter database (http://kmplot.com/analysis/) were used to explore the expression value and survival curve of IL6ST in human NSCLC.
Cell lines and cell culture
Three human lung cancer cell lines including A549 (adenocarcinoma), NCI-H1703 (squamous cell carcinoma) and NCI-H226 (squamous cell carcinoma) and BEAS-2B cells (Normal lung epithelial cells) were obtained from Keygen Cell Bank (Nanjing, China). mycoplasma contamination testing was conducted. A549 cells, NCI-H1703 cells and NCI-H226 cells were maintained in RPMI-1640 with 10% FBS (Gibco) and 1% penicillin streptomycin (Gibco) and cultured at 37°C with 5% CO2 in a humid atmosphere. BEAS-2B cells were maintained in DMEM with 10% FBS (Gibco) and 1% penicillin streptomycin (Gibco) and cultured at 37°C with 5% CO2 in a humid atmosphere.
Cell transfection
The negative control (NC), miR-224-5p mimic, miR-224-5p inhibitor were all purchased from Guangzhou RiboBio, the day before transfection, lung cancer cell lines A549 and H226 were seed into the 6-well plate and transfected when the cell density reached 70%. The miR-224-5p mimic, miR-224-5p inhibitor 5nM/well were treated according to transfection reagent Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientif) instructions for transfection. Briefly, 3μL of lipofectamine 2000 reagent was diluted in 100μL opti-MEM medium, the miR-224-5p mimic and inhibitors were diluted in 100μL of opti-MEM medium.Upon mixing above diluted reagents, the mixture was incubated at room temperature for 5 minutes and added into the cells, after 48 hours transfection, through the RT-qpcr to verification the ratio of transfection.
Cell viability assay
MTT assays were implemented to assess cell viability. A549 cells, NCI-H1703 cells and NCI-H226 cells were maintained in 96-well plates at 5 × 104 cells/mL for 48 h. Then, 20 μL MTT reagent was added to each well of the 96-well plate, and cell viability was measured after 4 hr of incubation at 37°C. The resulting formazan crystals were dissolved in 150 μL of DMSO solution. The OD value was measured at a wavelength of 570 nm using a Thermo Scientific Multiskan FC microplate reader (New York). These experimental results were repeated at least three times.
Wound healing assay
Cells were grown on 24-well plates to 100% confluence. The 100 μm wounds were scratched using sterile pipette tips. The scratches were observed by inverted light microscope at 0h, 12 h, 24 h and 48 h. Each group has three repeats. Image J software is used to measure the distance between scratch edges and the total width of each image to detect the percentage of the original distance. Distance (ratio of 0 h) =percentage of wound surface (12 h, 24 h, 48 h)/0 h data.
Transwell assay
Cells were inoculated into a cell coated with matrix gel (BD, USA) and inserted into a well in a 24-well plate. 1640 culture medium containing 10%FBS was added to the 24-well plate. Cells were incubated for 24 h. After 24 h, cells migrated to the lower surface were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min. Images of the cells were captured by a light microscope (Nikon, Japan).
3D culture assay
Mix matrigel with 1640 in a ratio of 3:1. Add the solution to a 96-well plate, 50μL per well. Cells were inoculated into 96-well plates at a density of 5 × 104 cells/well, and then incubated the cells for 24 h, cell images were captured using the light microscope (Nikon, Japan).
Western blot analysis
Cells were incubated for 48 h, the culture medium was discarded, and RIPA (Beyotime) was used to lyse the cells to obtain proteins. Protein concentration was measured with a BCA kit (Beyotime) and analyzed via western blotting. The normalized proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes. The membranes were incubated with primary antibodies against CD 130 (Affinity, cat# AF6291, 1:1,000), Phospho-Jak2 (Tyr1007/1008) (C80C3) (Cell Singnal Technology, Rabbit mAb #3776,1:1000), JAK2 (Affinity, cat# AF6022, 1:1,000), Phospho-Stat3 (Tyr705) (M9C6) Cell Singnal Technology, Mouse mAb #4113,1:1000), STAT3 (Affinity, cat# AF6294, 1:1,000) and Tubulin beta (Affinity, cat# AF7011, 1:1,000) overnight at 4°C. The proteins were further incubated with HRP-labeled secondary antibodies (Affinity, 1:10,000) at room temperature for 2 h. Finally, the target proteins were visualized using chemiluminescence reagent (Affinity).
Reporter gene assays
The luciferase reporter gene vector of IL6ST was purchased from Guangzhou GeneCopoeia Inc. Before the transfection,the cells were seeded into a 6-well plate,when the cell density reached 70%, the IL6ST 3'-UTR sequences were cotransfected with miR-224-5p mimic, miR-224-5p inhibitor or their respective NCs into lung cancer cells using Lipofectamine® 2000 for 48 h at 37˚C, the luciferase activities within each group of cells were detected using the Dual Luciferase Reporter Gene Assay kit (Promega Corporation).
Immunofluorescence
Cells were added into a 24-well plate with cell climbing slices and incubation for 48 h. After 48 h, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 20 min. Infiltrate with 0.2% Triton X-100 (Sigma-Aldrich, USA) for 10 min (Tonkin et al., 2004). After 60 min of 5% BSA blockade, anti-p-STAT3 and anti-p-JAK2 primary antibodies were incubated with cells in a wet box at 4 °C overnight. Then, fluorescein-labeled (FITC) AffiniPure goat anti-mouse IgG (H + L) and tetramethylrhodamine-labeled (TRITC) AffiniPure goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA) were incubated with the cells at room temperature for 2 h. Cells were treated with a closure tablet containing DAPI. Cell images were obtained by TCS SP8 confocal microscopy (Leica, Germany).
Nude mouse xenograft model
Female mice (BABL/c nude, 5-6 weeks, 18-22g) were selected in animal experiments, and all mice were fed in specific pathogen-free (SPF) barriers. The H226 cells were divided into two groups: negative control (NC) (n=4), miR-224-5p mimics, (n=4). And the A549 cells were divided into two groups: negative control (NC) (n=4), miR-224-5p inhibitor (n=4). These four cells (1 × 106 /mouse) were used to build the cancer modes by subcutaneous injection on the left front leg of nude mice. The tumor volume (V = L×W2 /2) of the nude mice was organized and recorded daily until the end of the experiment. Mice were injected intraperitoneally with 100mg/kg pentobarbital to perform euthanasia. After euthanized, the xenograft tumors from each mouse were weighed and analyzed. All animal care and experimental procedures conformed to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Nankai University (Permit No. SYXK 2014-0003). Animal Ethics Registration Number: 2022-SYDWLL-000588, approval date: 2022.10.8. All animal experiments were performed in accordance with the ARRIVE guidelines.
H&E staining
Lung tissue were fixed in 10% formalin, dehydrated and embedded in paraffin. Tissue sections with thickness of 4 μm were stained hematoxylin and eosin (H&E). Images were randomly photographed with an upright transmission fluorescence microscope (Olympus) and analysed by Image-Pro Plus Version 6.0.
Statistical analysis
Data was presented using the Prism version 9.0 software as the means ± SD. Differences between experimental and control group were assessed by Student’s t test. Significant differences among multiple groups were detected by one-way ANOVA. P < 0.05 was considered as statistically significance (* p < 0.05, ** p < 0.01, *** p < 0.001).