Identification of differentially expressed ADAM-encoding genes in TCGA data
In this study, TCGA (http://cancergenome.nih.gov/) RNA-seq data from a total of 551 LUAD patients were used, including 497 LUAD tissues and 54 healthy lung tissues. Twelve ADAMs were analysed, including ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30 and 33.The limma software package in R version (4.0.2) was used to identify ADAMs differentially expressed between tumour and healthy tissue. Genes with a p-value<0.05 and |log2(fold change)|>1 were considered to have significant differential expression. Subsequently, heatmaps were constructed to visualise the differential expression of ADAMs between the two groups.
cBioPortal database analysis
The cBioPortal Cancer Genomics Portal (http://cbioportal.org) is an open resource with data from 225 cancer studies. It was used to analyse the frequency of ADAM12 genetic alterations in LUAD (including mutations, deletions and copy number variation). All searches were performed according to the instructions provided on the cBioPortal website.
LinkedOmics database analysis
LinkedOmics (http://www.linkedomics.org/login.php) is an online tool used for the analysis of TCGA data .We used the LinkFinder module and Spearman correlation analysis to determine genes related to ADAM12 in LUAD. We then built a heatmap of co-expressed genes. After obtaining the strongly correlated co-expressed genes, we used R software to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these genes.
Immune infiltration database (TIMER and TISIDB)
We used the TIMER (https://cistrome.shinyapps.io/timer/) to analyse the correlation between ADAM12 expression and the infiltration of six major immune cell types, as well as to explore the relationship between the expression of ADAM12 and immune cell markers. We used the TISIDB database (http://cis.hku.hk/TISIDB/) to assess whether ADAM12 was related to LUAD immunotherapy outcome and immune cell subtypes, with the aim to elucidate the interaction between ADAM12 and tumour immunity.
Construction of a prognostic risk score model
We sought to develop a prognostic model of multiple ADAM12-related immune genes. We performed univariate Cox regression analysis, and used the Least Absolute Contraction and Selection Operator (LASSO) Cox regression algorithm to build a prognostic model based on the expression of selected genes. The risk score of the predictive model was calculated as follows:
risk score = coefficient 1 ×value1 + coefficient n × value n,
where the value is the relative expression of each selected gene's z-score conversion. This formula was applied to calculate the risk score for each patient in TCGA database.
Cell culture and cell transfections
Human bronchial epithelial cells BEAS-2B and the human LUAD cell line A549 were donated by Professor Huang Yi from Fujian Provincial Hospital. Human LUAD cell lines PC-9，H827 and H1299 were purchased from Central South University. All cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (10% FBS) under a humidified atmosphere of 37°C and 5% CO2. Human ADAM12 targeted small interfering RNAs (siRNAs) was designed and purchased from hippobiotec (Huzhou, China) and Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA) used for small RNA transfection. The siRNA sequence used in this study was as follows: ADAM12si#1:5'-CCUCAAGGCAACUAAGUAUdTdT-3';ADAM12si#2:5'-GCGAGAUGAGAGAUGCUAAdTdT-3';ADAM12si#3:5'-GCAGAUAACCAAGGUUUAAdTdT-3';sicontrol:5'-UUCUCCGAACGUGUCACGUdTdT-3'.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
TRIzol reagent ( Invitrogen, USA) was used to isolate total cellular RNA. Reverse transcription of ADAM12 was performed using the PrimeScript™ RT reagent Kit ( Takara, Japan). qRT-PCR was performed using the GoTaq® qPCR Master Mix kit ( Promega, USA). ADAM12 expression in NSCLC cell lines was determined via the 2-ΔΔCTmethod. GAPDH expression was used as an internal control. The primer sequences used in this study were as follows: ADAM12, 5'-ATTCAGCAGTCAGTCTCAGCAC-3' (forward) and 5'-CTTTTCAGCTTCTTCGCTGG 3'(reverse); GAPDH, 5'-GGTGTGAACCATGAGAAGTATGA-3' (forward) and 5'-GAGTCCTTCCACGATACCAAAG 3'(reverse).
Cells were lysed with RIPA buffer ( Beyotime) containing a protease inhibitor cocktail ( Thermo Scientific, Waltham, MA, USA) and PMSF. The protein concentration of samples was determined via the BCA method. 30-50 µg cell lysate was loaded and separated via SDS-PAGE. Protein was then transferred onto PVDF membranes ( Sigma, St. Louis, MO, USA), which were incubated with a specific primary antibody. They were then incubated with specific secondary antibodies. Autoradiography was employed for band density quantification ( Quantity One software; Bio-Rad). β-tubulin served as a loading control. The antibodies used were as follows: ADAM12 ( sc-293225, Santa Cruz, UA) and β-tubulin ( YM3139, Immunoway, China).
Cell colony formation assay
Cells were seeded in a 6-well plate ( 500 cells/well) and cultured with 10% FBS RPMI 1640 medium for 10 days. The excess liquid was removed, cells were fixed with formaldehyde, stained with crystal violet ( Sigma) and placed on an IX71 inverted microscope ( Olympus, Tokyo, Japan). Images were then obtained, and cells were counted. All experiments were repeated at least three times.
For the wound-healing experiment, cells were seeded into a 6-well plate ( 1 × 105 cells/well), and a single layer of cells was scratched with a 10 µL plastic pipette tip to form a uniform wound. The distance between the two edges of migrating cell sheets was photographed under a microscope and quantified. All experiments were repeated at least three times.
Transwell migration and invasion assays
For the migration assay, 2 × 104 cells in 200 µL serum-free medium were placed in the upper chamber of the Transwell system ( 8 μm pore size, BD Biosciences, San Jose, CA, USA). For the invasion assay, 5 × 104 cells in 200 µL serum-free medium were transferred to the upper chamber ( BD Biosciences) covered with Matrigel, and 600 µl RPMI 1640 medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation, the remaining cells in the upper chamber were removed, and those in the lower chamber were fixed with formaldehyde and stained with crystal violet ( Sigma). The cells were then imaged under an IX71 inverted microscope ( Olympus, Tokyo, Japan) and counted. All experiments were repeated at least three times.
TCGA data were analysed using R software ( version 4.0.2), and all other experimental results were analysed using GraphPad Prism 8 statistical software. The unpaired t-test was used for comparison between the two groups, ANOVA was used for analysing differences between multiple groups, and the Kaplan-Meier test was used to compare survival between groups. For experiments performed in triplicate, the data are expressed as the mean ±SD. p<0.05 indicated that the difference is statistically significant( *p<0.05, ** p<0.01, and *** p <0.001).