ARPC1A correlates with poor prognosis in prostate cancer and is up-regulated by glutamine metabolism to promote tumor cell migration, invasion and cytoskeletal changes

Patients and clinical specimens

Twenty pairs of cancerous and matched adjacent noncancerous tissue specimens were obtained from patients diagnosed with PCa between October 2019 and March 2020 in First Affiliated Hospital of Fujian Medical University. Samples used to construct the tissue microarray (TMA) were collected from 301 patients with detailed clinical information who had undergone radical prostatectomy (RP) between January 2011 and December 2018 in our hospital. The clinicopathological characteristics of these patients are shown in Table 1. All patients were followed up until March 2020, and the follow-up period ranged from 1.2 to 9 years after surgery. This study was approved by the Ethics Committee of our hospital. All patients signed informed consent forms before sample collection.

Table 1 Clinicopathological characteristics of 301 patients with PCa from TMA cohortConstruction of the co-expression network and analysis of the relationship between gene modules and disease

The workflow of our study is shown in Fig. 1. We downloaded mRNA expression profiles of PCa patients from the cancer genome atlas (TCGA) database (https://portal.gdc.cancer.gov/). After data cleaning and quality checking, 434 cases of PCa were finally included in this study [12]. The clinicopathological characteristics of these cases are summarized in Table 2. WGCNA was conducted to explore the relationship between gene modules and disease progression. Gene significance (GS) was defined as the log10 transformation of the P-value (GS = lgP) in the linear regression between gene expression and clinical features. Module significance (MS) was defined as the average GS for all genes in a module. The module with the highest absolute MS among all the selected modules for a given clinical feature was considered to be the one related to that feature. For each gene module, the expression patterns of all genes were summarized into a single characteristic expression value, the module eigengene (ME), which was used as the major component in the principal component analysis. We calculated the correlations between MEs and clinical features to identify the most relevant gene module for each given clinical feature. The module that demonstrated the strongest correlation with clinical features of interest was selected for further analysis.

Fig. 1figure 1

Flowchart detailing the study design

Table 2 Clinicopathological characteristics of 434 patients with PCa from TCGA databaseFunctional enrichment analysis

WebGestalt (http://www.webgestalt.org/) and Metascape (http://metascape.org/) are online databases providing a comprehensive set of functional annotation tools for researchers to better understand biological meaning behind large lists of genes [13]. We uploaded genes in the chosen module to perform gene ontology (GO) analysis and pathway enrichment analysis. A P-value < 0.05 was considered statistically significant. Gene set enrichment analysis (GSEA) software was used to explore biological functions related to the candidate gene. The cut-off criteria for GSEA were a nominal P-value < 0.05 and a false discovery rate (FDR) < 0.25.

Protein–protein interaction (PPI) network construction and hub gene selection

Search Tool for the Retrieval of Interacting Genes (STRING) is a biological database for constructing protein–protein interaction (PPI) networks, providing a system-wide view of interactions between each member [14]. Genes of selected modules were mapped to STRING to explore their relationships with each other, and a combined score > 0.4 was set as the cut-off criterion, as described previously [15]. Subsequently, a PPI network was constructed using Cytoscape software. The Centiscape plug-in was used to search for the most important nodes in a network by calculating centrality parameters for each node [15]. The genes with highest degree of connectivity were selected as the hub genes.

Cell lines and cell culture

Four human PCa cell lines, PC-3, DU-145, 22Rv1, and LNCaP, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The BPH-1 human prostate epithelial cell line was purchased from the Procell Life Technology Company (Wuhan, China). All cell lines were authenticated by short tandem repeat genotype analysis. Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) with 10% bovine serum at 37 °C in a humidified atmosphere containing 5% CO2.

Short interfering RNA (siRNA) and ARPC1A knockdown

Three siRNAs were designed for ARPC1A knockdown. The sequences were as follows: 5′-ACGGACACAUCACAGGUAUUGTT-3′, 5′-GCGAUUUCAUUCCAUUCUUGATT-3′ and 5′-GCCUAUGUCUGGAGUCAGAAATT-3′. Cells were seeded in 6-well plates (Corning Costar, Corning, USA) at a density of 3 × 105 cells per well. When the cells had reached 70% confluency, they were transfected with siRNA using Lipofectamine 3000 reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. After verifying the efficiency of knockdown, two efficient siRNAs were selected for subsequent experiments.

Establishment of stable cell lines overexpressing ARPC1A

Lentivirus for ARPC1A overexpression and control lentivirus were purchased from GeneChem Corporation (Shanghai, China). Transfection was performed according to the manufacturer’s instructions. Virally-transduced cells were cultured for at least one week in the presence of puromycin (2 μg/ml; Gibco) to select stable clones. At the indicated time points, the cells were harvested for subsequent experiments.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from PCa cell lines and tissue specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized using the PrimeScript RT reagent kit (Takara, Osaka, Japan). qRT-PCR was performed using the SYBR Green assay (Takara) and an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, USA). Primer sequences used were as follows: ARPC1A, forward primer: 5′-ATTGCCCTCAGTCCCAATAATCA-3′, reverse primer: 5′-CAAGTGACAATGCGGTCGC-3′; Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward primer: 5′-TGACTTCAACAGCGACACCCA-3′, reverse primer: 5′-CACCCTGTTGCTGTAGCCAAA-3′.

Western blotting (WB)

Total protein extracts from tissues and cells were prepared using RIPA lysis buffer (Beyotime, Haimen, China) supplemented with PMSF (Beyotime). Sample protein concertation was measured using the bicinchoninic acid kit (Beyotime). Proteins were separated on 8%–12% polyacrylamide gels by electrophoresis and subsequently transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with primary anti-ARPC1A (1:1000; Abcam, Cambridge, UK) or anti-GAPDH (1:2000; Abcam) antibodies overnight at 4 °C. After washing with Tris-buffered saline/0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated goat anti‐rabbit IgG antibody (1:5000; MDL Biotechnology, Beijing, China) for 2 h at room temperature and immunoreactive bands then detected using an enhanced chemiluminescence reagent.

Immunohistochemistry

Immunohistochemistry was performed on the TMA according to the manufacturer’s recommended protocols. Briefly, the TMA slides were deparaffinized and rehydrated, and then endogenous tissue peroxides were quenched by incubation with 0.3% H2O2 for 30 min. For antigen retrieval, the slides were boiled in sodium citrate buffer (10 mmol/L, pH 6.0) in a pressure cooker for 7 min. Subsequently, nonspecific binding was blocked with 5% normal goat serum, and then the slides were incubated with primary anti-ARPC1A antibody (1:200, Abcam) overnight at 4 °C, and then with anti-rabbit secondary antibody (Zhongshan Biotech, Zhongshan, China). Immunoreactivity was visualized using diaminobenzidine as the chromogen substrate. The slides were counterstained with hematoxylin, then dehydrated and mounted with glass coverslips according to a standard laboratory protocol. A previously described semi-quantitative analysis method based on staining intensity and the proportion of positive cells was used to derive the total ARPC1A immunostaining score[16]. Staining intensity (I) was recorded as 0, absent; 1, weak; 2, moderate; 3, strong, whereas the proportion (P) of positive cells was recorded as 0, < 5%; 1, 5% to 25%; 2, 26% to 50%; 3, 51% to 75%; and 4, > 75%. A score for each histological grade (H-score) was determined as the summary of intensity and proportion H-score = I × P according to a previous study. The final ARPC1A expression score was calculated using the value of the percent positivity score multiplied by the staining intensity score as “-” (score, 0–1), “ + ” (score, 2–3), “ +  + ” (score, 4–5), and “ +  +  + ” (score ≥ 6) (Fig. 4A).

Cell proliferation, migration and invasion assays

Cell proliferation was evaluated using a CCK-8 assay Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Briefly, cells were seeded in 96-well plates (Corning Costar) at a density of 3 × 103 cells per well and cultured for 24, 48, 72, 96 or 120 h. CCK-8 reagent (10 μl) was added to each well and the cells then incubated for 2 h before the absorbance at 450 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

The migration and invasion of cells were evaluated using a Transwell assay (Corning Costar). Briefly, 3 × 104 cells resuspended in 2000 μl of serum-free medium were added to the upper chamber of a Transwell system with an 8 μm pore membrane. The chamber was uncoated (for the migration assay) or coated with Matrigel (BD Biocoat, Bedford, MA, USA; for the invasion assay). The lower chamber contained 300 μl medium containing 10% fetal bovine serum. Cells were allowed to migrate or invade for 24 h, and then the cells that had not penetrated the membrane were removed with a cotton swab. The cells on the lower surface of the membrane were subsequently fixed and stained, and cells then counted in five randomly selected fields under a light microscope.

Additionally, wound healing assays were conducted to evaluate cell migration. Cells were cultured for 24 h to achieve confluent monolayer cultures. A pipette tip was then used to manually scratch the cell monolayers to generate a uniform wound. We photographed the wounds at 0 h, 16 h, 24 h, and 48 h, then drew two parallel lines at the edges of the cells and measured the distance between them. Migration rate was used to assessed the the migration of cells quantitatively.

Flow cytometry analysis

Cells were seeded in 6-well plates and cultured for 24 h. Next, cells were harvested and fixed with 70% ethanol at 4 °C overnight. The fixed cells were then washed, stained with propidium iodide and filtered through a 70-micron cell strainer immediately prior to analysis by flow cytometry, which was carried out on a FACScan flow cytometer (ACEA Biosciences, San Diego, CA, USA).

Immunofluorescence analysis

Immunofluorescence analysis was performed according to the procedures described in a previous study [17, 18]. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, stained with Fluorescein isothiocyanate (FITC)‐conjugated phalloidin (1:100; MDL Biotechnology) to label the filamentous actin cytoskeleton, and then counterstained with 4,6‐diamidino‐2‐phenylindole to label cell nuclei (Invitrogen). The cells were then imaged using an epifluorescence microscope to observe the morphology of pseudopodia.

In vivo experiments

A total of 26 male BALB/c nude mice (8 weeks, 20–22 g) were used in present study. To establish subcutaneous xenograft tumor model, 2 × 106 PC-3 cells were injected on sides of the flank of mice (n = 8 per group). 28 days after injection, the mice were sacrificed the size and the weight of tumors were measured. To establish pulmonary metastasis model, 2 × 106 PC-3 cells were inoculated via caudal vein (n = 10 per group). After 28 days, the fluorescent signal of pulmonary metastases was detected and analyzed using an IVIS Lumina II In Vivo Imaging System (Perkin Elmer) with Live Imaging Acquisition and Analysis software. Then the lungs of mice were removed and HE staining was performed to check the number of metastatic nodules. All the protocols of animal experiments have been approved by the Animal Care Committee of Fujian Medical University.

Statistical analysists

Statistical analyses were conducted using SPSS version 22.0 software (SPSS, Chicago, IL, USA) and GraphPad 5.0 software (GraphPad Software, San Diego, CA, USA). Univariate and multivariate analyses were conducted using the Cox proportional hazard regression model. Survival curves were plotted using the Kaplan–Meier method and log-rank test. We used the “Surv_cutpoint” function of “Survival” R package (version 3.2.13, by Terry M Therneau, performed on R version 3.5.1) to get the best cutpoint of ARPC1A expression for the TMA cohort, which can maximize the difference of survival curves between subgroups. Comparison between two groups was performed using a Student t test. Comparison between paired samples was performed using a paired t test. P < 0.05 was considered statistically significant.

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