Repositories
Patients’ transcriptome data and clinical data were all downloaded in the Cancer Genome Atlas repository (https://portal.gdc.cancer.gov/). Data included a total of 535 tumor samples and 59 normal samples. All patients’ information was displayed in the table S1. 232 autophagy-related genes were selected from the Human Autophagy Database (http://www.autophagy.lu/). Names of these genes were listed in table S2.
Construction and bioinformatic validation of signature
We obtained the lncRNA expression data from TCGA repository and analyzed the correlation between lncRNAs and autophagy-related genes, |cor|>0.4 was regarded as the inclusion criteria. Then we screened the lncRNAs by difference analysis and univariate regression analysis and obtained the prognostic differential autophagy-related lncRNAs. Finally, LASSO was used to construct a risk signature and each patient’s riskscore was calculated. The calculation formula of risk score is as follows: . In order to verify the accuracy of the model, we divided all patients into high-risk and low-risk groups according to median riskscore, and plotted risk curves and survival curves to distinguish the prognosis under different risk modes as well. Then univariate and multivariate independent prognostic analyses were applied to compare the prognostic efficacy between the signature and clinical characteristics (age, gender, TNM). ROC curves were also plotted to show the sensitivity and specificity of the prognostic risk model.
Bioinformatics mechanism exploration
Gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) were performed to analyze the differences in pathway enrichment under different risk patterns. The eligibility criteria for GSEA are: |NES|>1,NOM p value<0.05,FDR<0.25. GSEA runs for 1000 times.
Cell culture and transfection
The human lung adenocarcinoma cell lines A549 and H1299, as well human normal bronchial epithelial cell line BEAS-2B were purchased from the American Type Culture Collection (ATCC). Cell lines A549 and H1299 were cultured in RPMI-1640 medium (Yuanpei Biotechnology Co., Ltd, Shanghai) with 10% fetal bovine serum (Sorfa life science, South America) and 1% penicillin – streptomycin (New cell & Molecular Biotech Co., Ltd, Suzhou). BEAS-2B cell was cultured in Opti-DMEM (Yuanpei Biotechnology Co., Ltd, Shanghai) with 10% fetal bovine serum and 1% penicillin – streptomycin. All cells were incubated at 37 ° C, 5% carbon dioxide.
Si-LINC01559#1, si-LINC01559#2 and si-NC were designed and synthesized in Ribobio technology and co-transfected into cells with Lipotransfectamine 2000 transfection reagent (Sangon Bioengineering Co., Ltd, Shanghai). Sequences of si-LINC01559#1 and si-LINC01559#2 were shown in table S3. The final concentration of siRNA transfection was 30nM.
RNA extraction and quantitative real-time PCR
Total RNA of cells was extracted by TRIzol reagent (Thermo Fisher, United States). The complementary DNA was reverse transcribed buy PrimeScriptTM RT reagent Kit (Takara, Kyoto, Japan). Quantitative real-time PCR (qPCR) was applied to detect expression quantity by using TB green Premix Ex TaqTM II kit (Takara, Kyoto, Japan) on LightCycler480 system (Roche, Swiss). Actin was used as internal reference. Sequences of forward and reverse primer of actin and LINC01559 were shown in table 1. The relative expression was calculated by the method of 2-ΔΔCT. All experiments were repeated for 3 times.
Cell proliferation assays
2000 cells were inoculated in each well of 96-well plates for 5 replicate wells. Cell viability was measured by using cell counting kit-8 (CCK-8) reagent (Dojindo Laboratories, Kumamoto, Japan), and OD450 was measured at 0, 24, 48 and 72 hours respectively. The cell proliferation ability was detected by EdU (Beyotime technology, Shanghai) assay according to manufacturer’s instructions. All experiments were repeated for 3 times.
Wound healing assays
2×105 cells were seeded into the 6-well plate and proliferated to over 90% fusion. Swept the wound by using aseptic 200µL pipette tip in a straight line and washed with PBS for 3 times. Cells were cultured in serum-free medium for 24 hours, and images at 0 and 24 hours were recorded. Experiments were repeated for 3 times.
Transwell assays
Transwell assays were performed as described before[26, 27]. Briefly, transfected cells were inoculated in transwell chambers with 200ml serum-free medium suspension which containing 2×104 cells. The bottom chamber was added with 600ml complete culture medium without penicillin – streptomycin. The migratory cells at the bottom of the chamber were observed with inverted phase contrast microscope at 24 h and counted.
Flow cytometry assays
H1299 cells were collected after transfected with siRNA for 48 hours. Resuspended the cells with 500μl buffers and add 5μl of Annexin-V FITC and propidium iodide (PI) respectively. Incubated in dark for 15 minutes and then determined on flow cytometry. All experiments were repeated for 3 times.
Western blot
The transfected cells were lysed with RIPA lysate for 30min to extract the total protein. Then total protein was divided by electrophoresis in SDS-PAGE system for 120V, 2 hours. Polyvinylidene fluoride (PVDF) membrane as the carrier and protein transferred for 400mA, 1 hour. The following antibodies were used: LC3B (Proteintech: 14600-1-AP, 1:1000), p62 (Abmart: T59081, 1:1000), tubulin (Abmart: M20005, 1:1000), Caspase 3 (Abmart: T40044, 1:1000), bcl-2 (Abmart: T40056, 1:1000), CDK1 (Abways: CY5176, 1:1000), cyclin B1 (Abways: CY5378, 1:1000), cyclin D1 (Abways: CY5404, 1:1000).
Immunofluorescence staining
The cells were evenly spread in the 6-well plate for cell slide. After treatment with paraformaldehyde and tritonX-100, cell was blocked with TBST (with 5% bovine serum albumin) for 1h. Then cells were incubated overnight with LC3B primary antibody (Proteintech: 14600-1-AP, dilution ratio 1:200). Cells were incubated in FITC-labeled secondary antibodies for 1h and finally observed under fluorescence microscope.
Statistics
All bioinformatics analyses were performed in R version 4.0.2, and all experimental data analysis was carried out in GraghPad Prism 9. The significance of the differences between the groups was assessed by the Student’s t test. Correlation coefficient between lncRNAs and autophagy-related genes expression were analyzed by spearman correlation analysis. Kaplan-meier method was performed to plotting survival curves. Univariate and multivariate regression analysis were used to evaluating association between parameters and clinical characteristics. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered significant; ns intended no significance.