In silico analysis of DDX49 expression

To explore the correlation between DDX49 expression and PCa, six cohorts were enrolled from TCGA (https://portal.gdc.cancer.gov/repository) and GEO databases (https://www.ncbi.nlm.nih.gov/geo/), including TCGA-PRAD, GSE29079, GSE25183, GSE46602, GSE38241, and GSE69223. From these, the association between different DDX49 expression levels and recurrence-free survival (RFS) in PCa patients was assessed. In addition, the correlation of DDX49 expression with apoptosis-related genes was assessed based on TCGA cohorts via a visualization tool, GEPIA (http://gepia.cancer-pku.cn/) [18]. We also identified genes correlated with DDX49 expression from the STRING database, which predicted protein–protein interactions [19]. Subsequently, all genes correlated with DDX49 were analysed for cellular processes and pathways through Gene Ontology (GO) term enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Pathway enrichment analyses

Patients in the TCGA-PRAD cohort were classified into high-DDX49 and low-DDX49 groups in accordance with the mean DDX49 expression. The R package “limma” was employed for screening differentially expressed genes (DEGs) between groups with the absolute value of fold-change > 0.4 and p adjusted value < 0.01 as cut-offs [20]. GO and KEGG enrichment analyses were conducted with the R package “org.Hs.eg.db” and further annotated via the R package “clusterProfiler”. The top five GO terms and the top KEGG term were listed and visualized by the R package “enrichplot” [21, 22].

Cell culture

We purchased human cell lines, including RWPE-1, PC-3, LNCaP, and C4-2, from the Shanghai Cell Bank (Shanghai, China). Keratinocyte serum-free medium (K-SFM) supplemented with human recombinant epidermal growth factor (EGF) and bovine pituitary extract (BPE) was adopted for culturing RWPE-1 cells. PC-3, LNCaP, and C4-2 cells were cultured with RPMI 1640 media, 10% foetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin–streptomycin liquid (all from Gibco®, Shanghai, China), respectively. We cultivated cells at 37 °C and 5% CO2 under a humidified environment.

Quantitative reverse transcription–polymerase chain reaction (qRT‒PCR)

TRIzol reagent (Invitrogen, Shanghai, China) was adopted for isolating total RNA from RWPE-1, PC-3, LNCaP, and C4-2 cells. In addition, the primers used to test the expression of DDX49 were as follows; forward: 5′-ATGAGCACGAGGACTGGTC-3′ and reverse: 5′-GCGGCAAAGCGTTCTTTCT-3′; for the GAPDH control, forward: 5′-TGACTTCAACAGCGACACCCA-3′ and reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′. Thermocycling conditions included 94 °C for 3 min, then 22 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and then 72 °C for 5 min. All results were evaluated three times.

Construction of and infection with shDDX49 lentivirus

The DDX49 ORF was targeted to express shRNA using pGCL-GFP-lentivirus (GenBank no. NM_003410) (shDDX49 lentivirus). A nontargeting sequence was used for the lentivirus-negative control (shCtrl) (Shanghai Genechem Co. Ltd, Shanghai, China). Experiment template: 5′-GCCTGAGAATGATCATGGA-3′.

We chose PC-3 as the candidate for treatment with shDDX49 lentivirus. According to the MOI of lentivirus, 5 µl of shDDX49 or shCtrl lentivirus was added to each well of PC-3 cells cultured in 6-well plates. After the first 72 h of infection, we observed green fluorescence.

Cell growth assay

We adopted the Celigo® Image Cytometer to automatically identify the intensity and distribution of fluorescence in the PC-3 cells after treatment with shCtrl or shDDX49 lentivirus. After treatment with lentivirus for ten days, PC-3 cells in different groups were inoculated on 96-well plates at 2000 cells per well, and in the following five days, Celigo® Image Cytometers were adopted to record the cell status each day.

The MTT assay is another way to evaluate cell growth inhibition caused by the lack of DDX49. PC-3 cells in different treatment groups were inoculated on six 96-well plates at 2000 cells per well, and we collected and detected the activity of cells on the 1st, 2nd, 3rd, 4th, and 5th days. First, the cells were washed three times in cold PBS, and a 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added to every well to reach a final concentration of 0.5 mg/ml. Then, the cells were cultured in MTT solution at 37 °C for 4 h, and the solution was discarded. Finally, formazan salt was solubilized in 100 ml of dimethyl sulfoxide (DMSO) for 10 min. In addition, the OD was measured at 490 nm per well.

Cell cycle assay

We employed the flow cytometry system to assess the different proportions of cell cycle stages among the shCtrl and shDDX49 groups. After treatment with different lentiviruses, we collected the cells at 1, 2, 3, 4, or 5 days. Cells were rinsed twice in cold PBS, fixed with 0.5 ml of 70% ethanol, and cultured for 1 h at 4 °C. Then, 50 µg/ml propidium iodide (Sigma‒Aldrich) was used to stain these cells. The cells were filtered by a 300-mesh filter, and then we recorded the cell cycle phase of each cell through the stained nuclei using a BD FACSCalibur flow cytometer (BD Biosciences, USA).

Analysis of stress and apoptosis

To evaluate whether the lack of DDX49 affects the apoptosis pathway, the PathScan® Stress and Apoptosis Signalling Antibody Array Kit was used. This kit tested the phosphorylation statuses of ERK1/2, Akt, Bad, HSP27, Smad2, p53, p38 MAPK, SAPK/JNK, Chk1, Chk2, IkBa, eIF2a, and TAK1, the cleaved levels of PARP, cleaved caspase-3, and cleaved caspase-7, and the total expression levels of IkBa, survivin, and α-tubulin. First, after 48 h of transfection with shDDX49 or shCtrl lentivirus, we collected and washed the PC-3 cells twice with cold PBS. The cell protein concentrations were regulated to 0.2–1.0 mg/ml. After treatment with blocking buffer for 15 min, all 50–75-µl cell protein samples were introduced into every tube and cultured at 4 °C overnight. On the second day, the cell protein solution was erased, and the plate was rinsed three times for 5 min each and then treated with 75 µl of 1 × antibody while gently shaking for 1 h at room temperature. Then, 75 µl of 1 × HRP-streptavidin was added for 30 min. Finally, the plate was washed three times and then exposed and visualized on the CLINX ChemiScope 5300 machine.

Statistical analysis

To explore the association of DDX49 expression with clinicopathological characteristics, Fisher’s exact test and Pearson’s chi-squared test were conducted, while Student’s t-test was adopted to assess the differences between groups. Recurrence-free survival (RFS) was analysed with the Kaplan–Meier method, and differences were assessed with the log-rank test. Multivariate recurrence-associated analysis was carried out with logistic regression analysis. The relative risks of PCa recurrence were denoted as adjusted hazard ratios and corresponding 95% confidence intervals (95% CIs). The statistical data are displayed as the mean ± SD. For all analyses, p < 0.05 indicated statistical significance. In addition, SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) was involved in statistical analyses.