All small interfering RNA (siRNA) duplexes were purchased from Gemma (Suzhou, China). The siRNA sequences were as follows: Nontargeting control siRNA (siNegative), 5'-UUCUCCGAACGUGUCACGUTT-3’ (sense); siRNA targeting DDX17 (siDDX17-3), 5'- GCACCCAUCCUUAUUGCUATT -3’ (sense).
Cell culture and transfection
A549 cells (a human LUAD cell line) obtained from the China Center for Type Culture Collection (CCTCC; Wuhan, China) were cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C in 5% CO2. A549 cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 48 h of transfection, cells were harvested for quantitative reverse transcription–polymerase chain reaction (RT–qPCR) analysis.
Assessment of gene expression
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Complementary DNA (cDNA) was synthesized by standard procedures, and RT–qPCR was performed using Bestar SYBR Green RT–PCR Master Mix (DBI Bioscience, Shanghai, China) in a Bio–Rad S1000 thermal cycler. Detailed primer sequences are listed in Table 1. The relative expression level of each mRNA transcript was normalized to the GAPDH mRNA level and calculated by the 2-ΔΔCT method. Statistical comparisons were made using paired Student’s t test in GraphPad Prism 7.0 software (Graphpad Prism software, San Diego, CA, USA).
Cell proliferation assay
Cell proliferation was examined using a Cell Counting Kit-8 (CCK-8; Dojindo, Co., Shanghai, China). In brief, A549 cells were seeded at a density of 6000 cells/well in 96-well culture plates. Cell-free wells were used as blank controls (blank), and cells treated with an equal volume of phosphate-buffered saline (PBS) were used as controls (control). After 24, 48 and 72 h of siRNA transfection, CCK-8 solution (20 μl) was added to the culture medium and incubated for an additional 3 h. Finally, 50 μl of stop solution was added to each well, and the OD450 values were measured with a PerkinElmer EnVision plate reader. The cell proliferation rate was calculated according to the following formula: proliferation rate = (experimental OD value − blank OD value)/(control OD value − blank OD value) × 100%.
Annexin V apoptosis assay
An Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) detection kit (Shanghai Yeasen Corporation, Shanghai, China) was used to detect apoptosis according to the manufacturer’s instructions. Specifically, A549 cells were cultured in six-well plates for 24 h. and were then transfected with siRNA for 24, 48, or 72 h. Cells were treated with an equal volume of PBS as a control. The treated and control cells were mixed gently, and 5 μl of Annexin V-FITC reagent and 10 μl of 20 μg/ml PI reagent were then added and incubated at room temperature for 10-15 min in the dark. Following incubation, 400 µl of binding buffer was added, mixed, and placed on ice. Flow cytometric analysis was used to detect apoptosis within 1 h. Cells positive for Annexin V-FITC or negative for PI fluorescence were considered apoptotic cells.
Acquisition of RNA-seq data from TCGA
For RNA-seq, LUAD gene-level expression data (526 tumor samples and 59 normal samples) from TCGA were downloaded from the UCSC Xena hub (http://xena.ucsc.edu). Splice junction data in BED format for 518 LUAD tumor and 59 normal samples from TCGA were downloaded from the Genomic Data Commons (GDC) portal for identification of ASEs 41.
RNA extraction and sequencing
Total RNA was extracted with TRIzol (Ambion, Life Technologies, Carlsbad, CA, USA). RQ1 DNase (Promega, Madison, WI, USA) was used to remove any remaining DNA from the total RNA. RNA quality and quantity were assessed by measuring the absorbance ratio at 260 nm/280 nm (A260/A280) using a Nanodrop One spectrophotometer (Thermo Scientific, San Jose, CA, USA). RNA integrity was further verified by electrophoresis on a 1.5% agarose gel.
Equal amounts of total RNA (1 μg) from each sample were used for RNA-seq library preparation with a KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (#KK8544). Polyadenylated mRNAs were purified with VAHTS mRNA Capture Beads (N401-01). Purified mRNAs were fragmented and were then converted into double-stranded cDNA. Next, the cDNA was subjected to end repair, poly(A) tailing and ligation with a Diluted Roche Adaptor Kit (KK8726). After ligation, the cDNA was fragmented into 300–500 bp fragments. Finally, the DNA fragments were amplified, purified, quantified, and stored at -80°C until used for sequencing. The dUTP-labeled second strand was not amplified, providing strand specificity.
For high-throughput sequencing, libraries were prepared according to the manufacturer's instructions and subjected to 150 nt paired-end sequencing using an Illumina NovaSeq 6000 system.
Cleaning and alignment of raw RNA-seq data
First, raw reads containing more than 2 N bases were discarded. The adaptors were removed from the raw reads, and low-quality bases were trimmed using FASTX-Toolkit (Version 0.0.13). In addition, short reads containing fewer than 16 nt were discarded. Subsequently, clean reads were aligned to the GRCh38 genome by TopHat2, allowing no more than 4 mismatches 65. Only the uniquely mapped reads were used to calculate the gene read numbers and fragments per kilobase of transcript per million fragments mapped (FPKM) values 66.
Analysis of differentially expressed genes (DEGs)
The R Bioconductor package edgeR (Robinson, McCarthy et al. 2010) was utilized to identify the DEGs. The cutoff criteria for identifying DEGs were set as a p value <0.01 and a fold change>1.5 or < 2/3.
Analysis of AS
The ABLas pipeline was applied to define and quantify the ASEs and regulated ASEs (RASEs) between the samples according to previously described methods 67,68. In brief, ten types of ASEs were identified by the ABLas pipeline based on the splice junction reads: exon skipping (ES), alternative 5' splice site selection (A5SS), alternative 3' splice site selection (A3SS), intron retention (IR), mutually exclusive exons (MXE), mutually exclusive 5'UTRs (5pMXE), mutually exclusive 3'UTRs (3pMXE), cassette exon (CassetteExon), A3SS&ES and A5SS&ES.
For analysis of RBP-regulated ASEs, Student’s t test was applied to evaluate the significance of the alteration ratio of ASEs. A false discovery rate (FDR) of 0.05 was set as the significance cutoff.
Validation of DEGs and ASEs by RT–qPCR
To confirm the results of RNA-seq-based identification of DEGs in A549 cells, some DEGs were selected for verification by RT–qPCR. Information on the primers is shown in Table 1. A549 cells transfected with DDX17 siRNA were harvested after 48 h for RT–qPCR analysis. cDNA was synthesized from RNA using M-MLV Reverse Transcriptase (Vazyme). RT–qPCR was carried out on a StepOne RealTime PCR System using HieffTM qPCR SYBR® Green Master Mix (Low Rox Plus; Shanghai Yeasen Corporation, Shanghai, China). The thermal cycling conditions used for PCR were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing and extension at 60°C for 30 sec. PCR amplification was performed in triplicate for each sample. The RNA expression levels of all genes were normalized to the expression level of GAPDH.
Functional enrichment analysis
To comprehensively assess the biological functions of DEGs, Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed with the KOBAS 2.0 server 69. The enrichment of each term was determined using the hypergeometric test and Benjamini–Hochberg procedure to control the FDR.