Data acquisition and processing: The LUAD project of the TCGA (TCGA_LUAD) dataset and GSE68465 dataset was acquired from the GDC hub of the UCSC Xena website (http://xena.ucsc.edu/public) and the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds/), respectively. Data were processed as described in our previous study.
Online database analysis: A meta-analysis of NEDD4L transcriptional level in LUAD was performed by the Oncomine database (https://www.oncomine.org/resource/login.html). NEDD4L expression data of 9 groups of LUAD and normal tissues were retrieved and compared statistically. The default threshold was as follows: P < 1E-4, fold change > 2, and the top 10% of genes. The GEPIA database (http://gepia.cancer-pku.cn/index.html) was used to analyze the correlation between NEDD4L expression level and prognosis of LUAD patients.
Cell culture and treatment
Human LUAD cancer H1299 and PC9 cells were obtained from Cell Bank of Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 (H1299), DMEM (PC9) medium, containing 10% FBS, and maintained in an incubator with constant temperature and CO2. Cells were treated with gefitinib (ZD1839, Selleck).
Transfection and lentiviral transduction: Transfection was performed using Lipo8000™ (C0533, Beyotime). The siRNAs were acquired from GenePharma Company (Shanghai, China). The siRNA sequences are as follows: siEGFR#1, 5'-GUCCGCAAGUGUAAGAAGUTT-3'; siEGFR#2, 5'-GGAG
AUAAGUGAUGGAGAUTT-3'; negative control, 5'-UUCUCCGAACGUGUCACG
UTT-3'. The lentivirus was acquired from Genechem company (Shanghai, China).
The target sequence of shNEDD4L is as follow: 5'-GGAACUAAGCAGAAGGCUU
TT-3'. The method of lentivirus transfection of cells was described in our previous study.
Cells were plated in 96-well plates and incubated as described in our previous study. The viable cells were quantified by cell counting kit-8 (c0037, Beyotime) according to the manufacturer’s instructions.
Colony formation assay
The PC9 cells were seeded onto 6-well plates (200 cells/well) and cultured for 14 days. Cells were fixed using paraformaldehyde and stained using crystal violet as described in our previous study. Each group had three repetitions, and the t-test was used to analyze the difference in clone number between groups.
Cell cycle assay
Cells were first starved in 1% FBS medium for 12 hours, then spread onto 6-well plates and cultured in complete medium for 24 hours. Cells were treated using a cell cycle and apoptosis analysis kit (C1052, Beyotime) and detected using flow cytometry. The difference between groups was detected by t-test.
Gene set enrichment analysis: Patients from the TCGA_LUAD dataset were divided into high-expression and low-expression subgroups according to NEDD4L expression level. Gene set enrichment analysis (GSEA) was performed to investigate pathways enriched in the high-expression and low-expression subgroups. C2.cp.kegg.v7.1.symbols.gmt was chosen as the gene set database. Signaling pathways that meet the following criteria can be significantly enriched: nominal p-value < 0.05, q-value < 0.25, and normalized enrichment score > 1.
Cells were lysed on ice with pre-cooled RIPA for 10 minutes, and centrifuged at 4°C for 10 minutes (12000 rpm/min) to obtain the supernatant for subsequent detection. Western blotting was performed using antibodies against EGFR (#4267S, CST), phospho-EGFR (#3777, CST), mTOR (#2983, CST), phospho-mTOR (ab109268, Abcam), NEDD4L (ab46521, Abcam), and S6K (ab186753, Abcam), phospho-S6K (ab131436, Abcam), β-Actin (#3700, CST). Horseradish peroxidase-labeled Goat anti-rabbit IgG (H + L) (A0208, Beyotime) was used as secondary antibodies.
Patients and specimens
Twenty-four pairs of human LUAD tissues and para-carcinoma tissues were obtained from patients at Shanxi Provincial People's Hospital (Taiyuan, China). The use of clinical specimens was approved by the Ethics Committee of Zhoukou Normal University (ZKNU-2019043).
Immunohistochemistry and immunofluorescence assay: The carcinoma tissues and para-carcinoma tissues were fabricated into a tissue chip. Immunohistochemical (IHC) staining of FFPE sections was performed as described. The expression of target genes was assessed by the H-score system, and the formula for the H-score is as follows: Histoscore = Σ (I × Pi), where I = intensity of staining and Pi = percentage of stained tumor cells. Cells were seeded onto the special dish for laser confocal scanning, and the density was about 30%. Cells were treated with 10µM gefitinib for 24 hours. Immunofluorescence was performed as described in our previous study.
Xenograft lung adenocarcinoma model: For the xenograft lung cancer models, 6-week-old BALB/c nude mice were obtained from Weitong Lihua Experimental Animal Technology Co., Ltd (Beijing, China). H1299 cells (5 × 106 per mouse) were injected subcutaneously into the right side of mice, and the tumor volumes were measured 7 days later (Day 0). We measured the tumor volume every three days, and the formula is as follows: volume = (length*width2)/2. On day 18 of tumor development, mice were sacrificed and tumors were isolated and subjected to IHC analysis. All animal experiments were approved by the Ethics Committee of Zhoukou Normal University (ZKNU-2019043).
Correlation analysis was conducted by GraphPad Prism (version 8.0.1) with the Pearson method. The IHC score difference analysis was also conducted by GraphPad Prism software using paired t-test. The prognosis of LUAD patients from different subgroups was compared using the Kaplan–Meier method with a log-rank test. Target genes expression from different groups was compared by R software (version 4.0.2) using the Wilcox test. The “pheatmap”, "ggplot2", "ggpubr", "survival", "survminer", "plyr", "grid", and "gridExtra" packages in R (version 4.0.2) were used for visualization. A p-value of less than 0.05 was considered to be statistically significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001).