NSCLC data from the GSE7670 (lung cancer), GSE10072 (LUAD), GSE19188 (NSCLC), GSE19804 (lung cancer), GSE27262 (NSCLC), GSE32863 (LUAD), GSE43458 (LUAD) and GSE75037 (LUAD) datasets were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/gds/?term=) (19-26). ‘limma’ and ‘impute’ (http://www.bioconductor.org/packages/release/data/annotation/html/org.Hs.eg.db.html) packages (R package) were used to explore the expression of SOCS2. The difference in SOCS2 expression was shown using a violin plot, which was drawn using R software (4.0.5). In addition, gene expression data from 1,145 samples were downloaded from TCGA database (https://cancergenome.nih.gov/); 1,037 were tumor samples (LUAD and LUSC) and 108 were paracancerous samples. The difference in expression of SOCS2 between paired normal adjacent tissue and tumor tissue was analysed by the ‘BiocManager’ (http://www.bioconductor.org/packages/release/data/annotation/html/org.Hs.eg.db.html) packages (R package). Gene expression data and gene mutation data of pan-cancer were downloaded through ucsc xena database (https://xenabrowser.net/). The expression levels of SOCS2 in pan-cancer were analyzed using the ‘ggpubr’ package (http://www.bioconductor.org/packages/release/data/annotation/html/org.Hs.eg.db.html). Tumor mutation burden (TMB) of SOCS2 in pan-cancer was explored and depicted in a radar chart via ‘fmsb’ package (http://www.bioconductor.org/packages/release/data/annotation/html/org.Hs.eg.db.html). A protein-protein interaction (PPI) network was generated to show the relationship between SOCS2 and other proteins using the STRING database (http://string-db.org/cgi/input.pl). The relationship between SOCS2 and the clinical prognosis of NSCLC was researched by performing a log-rank test in the KM-plotter database (https://kmplot.com/analysis/) using the following dataset ID: 232539_at (SOCS2). Clinical factors related to SOCS2 were evaluated through UALCAN database (http://ualcan.path.uab.edu/). The expression levels of SOCS2 in different NSCLC cell lines were analyzed using the Cancer Cell Line Encyclopedia website (https://sites.broadinstitute.org/ccle).

The LUAD A549 and wild-type LUSC H520 cell lines were purchased from the American Type Culture Collection. Both A549 and H520 cells were cultured in RPMI-1640 medium (Hyclone; Cytiva). 293T cells were cultured in high-glucose DMEM (both from HyClone; Cytiva). The two types of medium were supplemented with 10% fetal bovine serum (FBS; HyClone; Cytiva). The cells were incubated in an atmosphere containing 5% CO₂ at 37˚C.

For small interfering RNA (siRNA)-induced SOCS1 silencing, H520 and A549 cells were plated at a density of 3x10⁵/60-mm dish. After 24 h of culture, the cell culture medium was replaced with fresh medium. The cells were then transfected with SOCS2 siRNAs or control-siRNA (non-targeting control) purchased from Transheepbio (Shanghai Quanyang Biotechnology Co., Ltd.) using jetPRIME^® transfection reagent (Polyplus-transfection SA) at 37˚C. The final concentration of siRNA or control-siRNA was 0.05 µM. The duration of transfection was 24 h and the duration between transfection and subsequent experimentation was 72 h. The siRNA sequences were as follows: Control-siRNA, sense, 5'-UUCUCCGAACGUGUCACGUdTdT-3' and antisense, 5'-ACGUGACACGUUCGGAGAAdTdT-3'; SOCS2, sense, 5'-GCUGAAGCUAAUCUAAUUUdTdT-3' and antisense, 5'-AAAUUAGAUUAGCUUCAGCdTdT-3'.

For short hairpin RNA-SOCS2 (shSOCS2)-induced silencing of SOCS2, a second generation system used. The A549 and H520 cells were cultured for an additional 48 h in 10% FBS-RPMI 1640 medium. 293T cells were transfected with virus packaging plasmids (psPAX2; Addgene, Inc.; cat. no. 12260, 5 µg; pMD2.G, Addgene, Inc.; cat. no. 12259; 2 µg) and pLenti-EF1a-mcherry-P2A-Puro-CMV-MCS-3Flag (control; 5 µg) or SOCS2 knockout plasmids (5 µg) by transfection reagent (jetPRIME^® in vitro DNA and siRNA transfection reagent; Polyplus-transfection^® SA). The multiplicity of infection used to infect cells was 5. After the 293T cells were transfected for 48 h at 37˚C, the supernatant was collected and added to A549 and H520 cells for 24 h. The A549 and H520 cells were transfected with plasmid using jetPRIME transfection reagent according to the manufacturer's instructions. The duration of transduction was 3 days. A549 and H520 cells were selected using puromycin (4 ng/µl) for 1 week. The time interval between transduction and subsequent experimentation was 2-3 weeks. The concentration of puromycin used for maintenance was 4 ng/µl.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in compliance with the manufacturer's standard procedures. RNA samples were reverse transcribed into cDNA using a PrimeScript RT kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's recommendations. The LightCycler 480 Real-time PCR System (Roche Diagnotics) was used to detect the mRNA expression levels. The cDNA was analyzed in RT-qPCR using the SYBR Green PremixPro Taq HS qPCR Kit (Jiangsu Accuracy Biotechnology Co., Ltd.). The qPCR cycling conditions were as follows: Denaturation at 95˚C for 30 sec (one cycle); PCR at 95˚C for 5 sec, 55˚C for 30 sec and 72˚C for 30 sec (40 cycles); melting at 95˚C for 5 sec, 60˚C for 1 min and 95˚C for 10 min (one cycle); cooling at 50˚C for 30 sec (one cycle). The qPCR primers were designed using Primer3 (primer3.ut.ee/). Each sample was tested three times and 18S ribosomal RNA expression was used to normalize the results. The comparative threshold cycle value method, 2^-ΔΔCq (27), was used to evaluate the results. The primer sequences were as follows: 18S ribosomal RNA, forward 5'-AAACGGCTACCACATCCAAG-3' and reverse 5'-CCTCCAATGGATCCTCGTTA-3'; SOCS2, forward 5'-CTAAGGCTGACCAAGACCTGTTGA-3' and reverse 5'-GCCTGGACCTTTGACATAATGGA-3'.

A549 and H520 cells were lysed with RIPA lysis buffer (Bio-Rad Laboratories, Inc.). Equal amounts of protein (10 µg protein/lane) were separated by SDS-PAGE (resolving layer, 10%; stacking layer, 5%) and transferred onto PVDF membranes (Pall Life Sciences). The membranes were blocked in 5% bovine serum albumin (BovoStar; Bovogen Biologicals Pty Ltd.) for 1 h at room temperature and then incubated with primary antibodies against SOCS2 (cat. no. ab109245; dilution, 1:1,000; Abcam) and GAPDH (cat. no. ab9485; dilution, 1:1,000; Abcam) overnight at 4˚C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies [dilution, 1:10,000, cat. nos. ZB-2305 (goat anti-mouse) and ZB-2301 (goat anti-rabbit); OriGene Technologies, Inc.] for 1 h at room temperature after washing. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection reagent (WesternBright™ ECL; cat. no. 180805-33; Advansta, Inc.).

H520 and A549 were suspended with pancreatin (0.25% Trypsin-EDTA 1X; Gibco; Thermo Fisher Scientific, Inc.) and counted. The cells were then added into 60-mm dishes (1,000 cells/dish) containing 5 ml culture medium and incubated at 37˚C in an atmosphere containing 5% CO₂ and saturated humidity for 7-14 days. Cell culture was terminated when macroscopic cell colonies were visible on the dishes. After discarding the medium, PBS was used to wash the cells, which were then fixed with 4% paraformaldehyde for 30 min at 25˚C and stained with 0.1% crystal violet for 30 mins at 25˚C. Finally, excess dye was gently washed from the cells with running water and the cells were observed under an optical microscope (magnification, x400; Leica Microsystems GmbH). Colony images were obtained by scanning the culture dishes.

Pancreatin (0.25% Trypsin-EDTA 1X; Gibco; Thermo Fisher Scientific, Inc.) was used to lyse the cells. The floating cells were then added into a 6-well plate (5x10⁵ cells/well). After the 6-well plate was at 100% confluence, the cell layer was scratched using a 100-µl micropipette tip. The cells were then cultured in RPMI-1640 medium containing 1% FBS. Images of the wound widths were obtained under an optical light microscope at 0, 6, 24, 30 and 36 h (Leica Microsystems GmbH).

A sulforhodamine B (SRB) assay was used to assess cell proliferation. Cells were cultivated in 96-well plates (1,500 cells/well) in an incubator at 37˚C. After the cells adhered to the plate, the 96-well plate was removed from the incubator and cell proliferation was assessed every 12 h. After removing the medium, 60 µl 10% cold trichloroacetic acid (TCA) was added to each well for fixation at room temperature. After all of the samples were fixed, the cold TCA was removed and the plate was washed five times with tap water. After drying overnight at room temperature, 60 µl 0.057% (wt/vol) SRB solution (MilliporeSigma) was added to each well for 1 h at room temperature, after which, 1% acetic acid was used to wash each plate five times. The plates were again dried overnight at room temperature, 150 µl 10 mM Tris base solution was added to each well and the plates were shaken for 20 sec at 2,000 rpm. The cell absorbance was measured at 562 nm using a microplate reader (Thermo Fisher Scientific, Inc.). GraphPad Prism7 (Dotmatics) was used to analyze all results of the cell proliferation assay.

A549 and H520 cells were cultivated in 96-well plates (3,000 cells/well). Different concentrations (A549: 0, 0.05, 0.1, 0.15 and 0.2 mM; H520: 0, 0.05, 0.1 and 0.2 mM) of carboplatin (MedChemExpress) were added to the plates at 37˚C. The SRB was subsequently performed as aforementioned.

Matrigel (Clontech; Takara Bio USA) was used to coat the Transwell inserts (Corning, Corning, NY) for the Transwell invasion assay at room temperature for 1 h. The lower chamber of the Transwell insert (pore size, 8.0 µm; 24-well plates) was filled with 1,000 µl RPMI-1640 medium containing 20% FBS (HyClone; Cytiva), and ~4x10⁴ cells and RPMI-1640 medium without FBS were added to the upper chamber. After the cells were cultured for 24 h, the liquid in the upper chamber was discarded. The chamber was removed and a wet cotton swab was used to remove the cells from the upper chamber. Subsequently, the cells on the lower membrane were fixed for 20 min with 100% methanol, stained with 0.1% crystal violet for 20 min and washed using PBS at room temperature. Images of the Transwell assay were obtained under an optical light microscope (Leica Microsystems GmbH).

GSEA was used to explore gene functions. The NSCLC data of 1,145 patients from TCGA database were downloaded and analyzed using GSEA software (http://www.gsea-msigdb.org/gsea/downloads.jsp). GSEA software explores gene function based on gene expression levels in cells. The gene expression of SOCS2 matched the gene function, GSEA provided the relationship between the functions and SOCS2 gene. The present study used the gene set database, c5.all.v6.2.symbols.gmt.

GraphPad Prism 7 was used to analyze the data. Data are presented as the mean ± standard deviation of three independent experimental repeats. For experimental data analysis, comparisons between two sets of data were performed using unpaired Student's t-test, whereas one-way ANOVA, followed by Dunnett's multiple comparisons test, was used for the statistical analysis of more than two groups. For gene expression analysis of UALCAN data, comparisons between two sets of data were performed by unpaired t-test using a PERL script with the Comprehensive Perl Archive Network module ‘Statistics::TTest’ (http://search.cpan.org/~yunfang/Statistics-TTest-1.1.0/TTest.pm), whereas more than two groups were compared using the Kruskal-Wallis test followed by Dunn's multiple comparisons test (28). All P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.

A Kaplan-Meier survival analysis showed that the high expression of SOCS2 was a protective factor for patients with NSCLC, whereas low expression of SOCS2 mediated a poor prognosis (Fig. 1E; P<0.001). For both LUAD and LUSC, the clinical factors related to SOCS2 included stage, sample type, TP53 mutation status, nodal metastasis status, histological subtypes, smoking habit, age, sex and ethnicity (Figs. 2 and S1). These findings suggested that SOCS2 has an important effect on the prognosis of patients with NSCLC.

GSEA was performed to further understand the effect of SOCS2 on NSCLC. GSEA software provided scores based on the pathways associated with gene function and gene expression level. SOCS2-related functions included ‘EMT’, ‘mesenchymal cell differentiation’, ‘regulation of endothelial cell differentiation’, ‘cell proliferation’, ‘migration’ and ‘angiogenesis’ (Fig. 3). It was thus hypothesized that SOCS2 was related to the progression of NSCLC and mediated EMT.

SOCS2 expression levels were detected in different NSCLC cell lines using data provided by the CCLE (Fig. 4A), and it was revealed that the expression levels of SOCS2 were relatively high in H520 and A549 cells. RT-qPCR and western blot analysis were used to verify the silencing effects of SOCS2 siRNAs and shSOCS2 on the A549 and H520 cell lines. According to the experimental results, SOCS2 siRNA (Figs. 4B and S2) and shSOCS2 (for longer duration experiments; Figs. 5A and S3) were successful at silencing SOCS2 expression in A549 and H520 cells. The two bands of GAPDH may be due to excessive voltage. A549 and H520 cells transduced with stable shSOCS2 plasmids underwent a cell proliferation assay. Knockdown of SOCS2 promoted the proliferation of NSCLC cells (Fig. 4C). A549 knocked out with shSOCS2-2 did not have significant proliferation levels, This may be due to the poor state of the cells, which can be considered a normal experimental error. A549 and H520 cells transduced with stable shSOCS2 plasmids were used for the wound-healing assay, and images of cell migration were captured at 0, 6, 24, 30 and 36 h (Fig. 4D). The results showed that the migration ability of A549 and H520 cells was stronger when SOCS2 was knocked down compared with that in the control cell lines. The colony formation assay was performed in A549 and H520 cell lines transduced with stable shSOCS2 plasmids and showed that silencing SOCS2 could accelerate proliferation (Fig. 4E). In addition, A549 and H520 cells transfected with a SOCS2 siRNA underwent a Transwell assay. When SOCS2 was silenced, the invasion ability of cells was increased (Fig. 5B). These findings indicated that silencing SOCS2 could promote the EMT of LUAD (A549) and LUSC (H520) cells. In addition, the SOCS2 gene is likely a tumor suppressor gene and low SOCS2 expression may cause a poor prognosis.

A549 and H520 cells transfected with a SOCS2 siRNA underwent treatment with different concentrations of carboplatin (A549: 0, 0.05, 0.1, 0.15 and 0.2 mM; H520: 0, 0.05, 0.1, and 0.2 mM). The results revealed that increasing concentrations of carboplatin inhibited A549 and H520 cell proliferation. However, silencing SOCS2 reduced the sensitivity of NSCLC to carboplatin (Fig. 5C); the cell lines in which SOCS2 was silenced exhibited increased proliferation compared with the control groups.