Patients
Three lung squamous cell carcinoma (LUSC) patients were included in this study. The 6 tissue samples (3 LUSC samples and 3 paired adjacent normal samples) were utilized to carry out the RNA sequencing. Each patient submitted the signed informed consents, and this study were approved by the ethical committee of our hospital.
RNA isolation and sequencing
Total RNA was extracted from samples using the Trizol kit (Invitrogen, Carlsbad, CA, USA). Total RNA was further purifed with the Ribo-Zero Magnetic kit (EpiCentre, Madison, WI, USA). Illumina Hiseq Xten platform (Illumina, San Diego, CA, USA) was performed to conduct sequencing of mRNA. xpression levels of lncRNA and mRNA were compared using edgeR (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html). MRNA and lncRNAs with |log2FC|>1 and p value<0.05 was defined as significant DEmRNAs and DElncRNAs. Volcano plot was generated in R package (https://www.r-project.org/). Hierarchical clustering analysis of top 100 DEmRNAs and DElncRNAs was structured by heatmap.2 (http://127.0.0.1:28428/library/gplots/html/heatmap.2.html).
Functional annotation
Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were retrieved via GeneCoDis3 (http://genecodis.cnb.csic.es/analysis). Significantly enrichment was defined by p value <0.05.
PPI network construction
The top 50 up-regulated and down-regulated DEmRNAs were applied to establish the PPI network using BioGRID and Cytoscape 3.6.1 (http://www.cytoscape.org/). The nodes and edges to represent the proteins and interactions between two proteins, respectively.
DEmRNA-DElncRNA interaction analysis
To study the nearby DEmRNAs of DElncRNAs with cis-regulatory effects, DEmRNAs transcribed within a 100 kb window up- or down-stream of DElncRNAs between LUSC and normal controls were sought. Furthermore, the DEmRNAs co-expressed with DElncRNAs were screened as well. The pairwise Pearson correlation coefficients between DEmRNAs and DElncRNAs were analyzed. DElncRNA-DEmRNA pairs with p<0.001 and | r | ≥0.999 were served as significant mRNA-lncRNA co-expression pairs.
Validation in The Cancer Genome Atlas (TCGA)
The expression pattern of selected DEmRNAs and DElncRNAs were validated with TCGA dataset. We downloaded from the TCGA dataset, which consisted of 501 patients with LUSC and 49 normal controls.
Receiver operating characteristic (ROC) curve analyses
In order to evaluate the diagnostic value of DEmRNAs and DElncRNAs in LUSC, the “pROC” package was performed to generate ROC, and the area under the ROC curve (AUC) represents the diagnostic value. When AUC value was greater than 0.8, the DEG was is thought to be able to distinguish between case and normal controls with good specificity and sensitivity.
Survival analysis
To evaluate the prognostic of candidate genes, survival analysis was generated using clinical data from TCGA. Kaplan–Meier curve was plotted using the survival (https://cran.r-project.org/web/packages/survival/index.html) in R.
Cell culture and Cell transfection
Human lung squamous cell carcinoma cell lines NCI-H520 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in endothelial cell growth medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco) in 5% CO2 at 37 °C. Small interfering RNA targeting MIR205HG (si-MIR205HG) and scramble siRNA of MIR205HG (si-NC) were purchased by Guangzhou Ribobio (Guangzhou, China). For transfection, NCI-H520 cells were inoculated with 60 %-70 % fusion and allowed to adhere overnight. Subsequently, the corresponding plasmid was then transfected into the cells by Lipofectamine 2000 according to the manufacturer’s instructions. After 48h, cells were collected for subsequent experiments.
CCK8
Cell viability was measured using CCK8 kit (CCK-8 reagent). In brief, the treated NCI-H520 cells were inoculated on 96-well plates for 12 h, and 10 uLCCK-8 reagent was added to each well for 37°C for 4 h. The absorbance value of each well at 450 nm was determined using a microplate reader.
Transwell assay
Cell migration capacities were detected using by transwell assays. Cells resuspended in serum-free medium were placed into the upper chamber of a 24-Transwell plate with 8 µm pore filter (BD Biosciences, Franklin Lakes, NJ, USA).Then, 500 µl of growth medium containing 10% FBS was added into lower chamber. After incubation for 24 h, the cells that moved through the underside of the membrane filter were fixed with 4% paraformaldehyde and stained with 0.25% crystal violet. The number of migrated or invaded cells was counted and the images were photographed under a light microscope (Olympus, Tokyo, Japan).
Real-time quantitative RT-PCR
Total RNA was isolated from NCI-H520 cells using a RNA simple total RNA kit Tiangen, Beijing, China) and then reverse-transcribed with the Fast Quant RT Kit (Tiangen) according to the manufacturer’s instructions. Quantitative real-time PCR were performed using the Super Real PreMix Plus SYBR Green (Tiangen) on a QuantStudio 6 Flex system. The relative quantification of genes was calculated using 2-∆∆Ct method.
Western blot
Cells were lysed with RIPA buffer (Beyotime, Shanghai, China), and total protein was obtained. The protein concentration was measured using the BCA protein assay kit (Tiangen). After processing with loading buffer, proteins (50 µg) were subjected to SDS/PAGE (10-12% gels), and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The blot was blocked with 5% nonfat milk for 2 h at room temperature and incubated primary antibodies overnight at 4℃. After washing, blots were incubated with appropriate HRP-conjugated secondary antibody for 2 h. The protein bands were visualized revealed by chemiluminescence using an ECL detection kit (Millipore).