2.1 Derivation of GEO data and identification of differentially expressed genes (DEGs)
Herein, two microarray expression profile datasets GSE10096 and GSE43458 were downloaded from the GEO data resource. The annotation data of all the probe sets was on the basis of the GPL571 [HG-UL33A] Affymetrix Human Genome U133A 2.0 Array. In all, 30 non-malignant lung tissues, 80 lung adenocarcinomas and 9 bone metastases lesion samples were used. Then SangerBox (www.soft.sangerbox.com) was applied according to the instructions to uncover DEGs among lung tissues, adenocarcinomas and NSCLC bone metastatic lesions. The top 30 DEGs are presented in a heatmap.
2.2. Human specimens
Overall, 70 non-malignant lung tissues, 117 NSCLC tissues, as well as 37 lung cancer bone metastatic tissues were acquired from patients who underwent surgical resection in our hospital from 2007 to 2019. The tissues obtained during surgery were frozen in liquid nitrogen immediately within 2 hours following surgical excision. None of the participants was inoculated with radiotherapy, or chemotherapy prior to surgery. The approval of this study was granted by the Ethics Committee of Changzheng Hospital of the Second Military Medical University (Shanghai, China). Besides, the participants provided informed consent.
2.2. Cells and culture
The A549 and PC9 NSCLC cells were commercially provided by SIBCB of the Chinese Academy of Sciences (Shanghai, China). Cells were inoculated in medium (A549 with in DMEM and PC9 in RPMI-1640, Thermo Fisher Scientific,USA) enriched with 10% FBS (Cat No, GIBCO, USA). RAW 246.7 cell were inoculated in MEM α (Cat No, Thermo Fisher Scientific, USA) enriched with 10% FBS. The culture condition in the cell incubator was 37°C and 5% CO2.
2.3 IGF2 overexpression plasmid and siRNA
KpnI along with EcoRV (Cat Nos, TransGen Biotech, China) were employed to digest the pcDNA3.1 + plasmid. PCR was used to amplify IGF2 coding sequence, followed by insertion into pcDNA3.1 + through the Quick-Fusion Cloning Kit (Cat No, biotool, USA). The oligonucleotide primers for the PCR included; F: ATG GGA ATC CCA ATG GGG AA; R: TCA CTT CCG ATT GCT GGC CA. DNA sequencing was employed to validate the overexpression (OE) plasmid of IGF2. Two distinct siRNA oligos against IGF2 were supplied by IDT. siRNA-1: CGG CCT GGG AAG TAG GAC TAA; siRNA-2: GTA GGA CTA AGG ACC CGA ACT. RT-PCR along with Western blotting were employed to verify IGF2 expression post transfection.
2.4 Cell transfection
Cells in the log growth phase were used once they reached 5070% confluence. The DNA Transfection Reagent (Bimake) was used according to the operation manual
2.5 Immunohistochemistry (IHC)
Tissues were fixed in 4% PFA at 4˚C for 24 hours, and sectioned into 4-mm slices, followed by embedding in paraffin. Afterwards, 2% hydrogen peroxide dispersed in methanol was employed to block the endogenous peroxidase activity. Thereafter, the slides were inoculated in citrate buffer (pH 6.0) and then heated in a microwave oven for 12 minutes to retrieve the antigen. Subsequently, the slides were inoculated with rabbit anti-human IGF2 antibody (cat. no. ab9574; Abcam, Cambridge, MA, USA; 1:1,000). Thereafter, the samples were inoculated with the goat anti-rabbit secondary antibody (Cat No, Dako, Glostrup, Denmark; 1:2000). Then, DAB staining (Cat No, Dako) was done as described by the manufacturer. Finally, light counterstaining of the samples with hematoxylin was done, followed by mounting. Two experienced pathologists assessed the results and images were acquired.
The intensity of staining was scored as; strongly positive, 3; moderately positive, 2; weakly positive; and negative, 0. Four categories were used in scoring percentage positivity: >75%, 4; >50–75%, 3; >25–50%, 2; 5–25%, 1, and < 5%, 0. The staining score was determined through the multiplication of intensity of staining score with the positivity percentage, and was graded as high expression (score ≥ 3) or low expression (score ≤ 2).
2.6 Colony formation assay
Cells were digested, counted, planted in 6-well plates in triplicate, grown for 14 days, washed with PBS, fixed in 4 % PFA, followed by 0.1 % crystal violet staining (Sigma, St Louis). The number of effective colonies consisting of more than 50 cells was counted.
2.7 Cell cycle assay
Five hundred A549 or PC9 cells were inoculated in 6-well plates and inserted with pcDNA3.1+, or OE-IGF2 plasmids via transfection 48 hours. We harvested the cells, followed by fixing them with ice-cold 70% ethanol. Thereafter, the cells were rinsed in PBS, and rehydrated in PBS enriched with propidium iodide (0.5 mg/mL dispersed in PBS with 0.1% sodium azide) and RNase A (1 mg/mL) for 30 min in the dark. Finally the FACSCalibur flow cytometer (Becton-Dickinson, USA) was employed to analyze the samples. Lastly, the FlowJo software (Tree Star, USA) was employed to analyze the data.
2.8 Transwell migration and invasion assay
The 8-µm pore size transwell insert without matrigel (3422, Corning, USA) and with matrigel (354480, Corning, USA) were utilized for transwell migration and infiltration assay respectively. Digestion of the cells was done, followed by counting. Overall, 1 × 105 cells in 100 µL medium enriched with no FBS were inoculated in the upper inserts and 500 µL medium augmented with 10% FBS was inoculated in the lower inserts as chemoattractant and incubated for 24 hours. After that, we removed the cells in the upper inserts, while the bottom surface-cells were fixed with 4% PFA, followed staining with 0.1% crystal violet for 15 minutes. After that, 5 random fields were counted with a microscope at 400 ×. Lastly, acetic acid elution was performed and the OD values read at 570 nm.
2.9 Western blot
The RIPA buffer (KeyGen Biotech, Shanghai, China) enriched with PMSF (KeyGen Biotech), protease inhibitor, as well as phosphatase inhibitor cocktail, was employed to lyse the cells. The Western blotting was conducted as documented previously . 20–40 µg protein extract was fractionated on a 10% SDS-PAGE gel and transfer-embedded onto PFDV membranes (GE Healthcare Life Sciences, Germany). Next, 5% non-fat milk was employed to block the membranes. Afterwards, the membranes were inoculated overnight with the primary antibody at 4°C. Lastly, the SuperSignal® ECL Kit (Cat No, Pierce, USA). Primary antibodies we used are listed Supplementary Data.
2.10 Reverse transcription-PCR (RT-qPCR)
The TRIZOL (Cat No, Invitrogen, USA) was employed to isolate total RNA and utilized to generate fist-strand cDNAs with the Prime Script™ RT Master Mix (Cat No, Takara, Japan). The SYBR® Premix Ex Taq™ II (Cat No, Takara, Japan) was employed to quantify the mRNA on the 7900HT Fast qRT-PCR System (Life Technologies Corporation, USA). GAPDH was utilized as the normalization standard. The primers are listed Supplementary Data.
2.11 Osteoclast differentiation assay
Raw246.7 cells were inoculated with the conditional medium from A549 transfected with OE-IGF2, sh-IGF2 and their respective control plasmids for 7 days. Thereafter, cells were fixed with 4% PFA, followed by TRAP staining with the TRAP Kit (Cat No, Sigma-Aldrich) as described by the manufacturer. The number of TRAP-positive multinucleated cells (≥ 3 nuclei, mature osteoclasts) was counted.
ELISA was carried out using IGF2 ELISA kit (Cat No, Abnova, Taipei City, Taiwan) as described by the manufacturer. A microplate reader was employed to document the absorbance at 450 nm. The absorbance at 630nm was used as a reference.
2.13 Statistical analysis
Results are given as means ± SEM unless specified. Two-tailed unpaired t-test was employed to compute significant differences between the mean values of groups. The Kaplan Meier plotter website (http://kmplot.com/) was used for OS (overall survival) and PFS (progression-free survival) analysis in NSCLC patients. The IGF2 probe set was 202409_at (although there were three IGF2 probe sets, the results were similar.) The patients were split on the basis of the median, and the univariate cox regression was performed. P < 0.05 signified statistical significance.