Epithelial cells derived exosomal miR-203a-3p facilitates stromal inflammation of type IIIA chronic prostatitis/chronic pelvic pain syndrome by targeting DUSP5 and increasing MCP-1 generation | Journal of Nanobiotechnology
Collection of human prostatic fluid samples
This study involved twenty prostatic fluid samples from CP/CPPS-A patients diagnosed at the Department of Urology, Southwest Hospital, Army Medical University (Chongqing, China). Additionally, twenty healthy volunteers were randomly recruited from the general population for this study. The inclusion and exclusion criteria for patients with CP/CPPS-A and healthy adult males were implemented as previously reported [21]. Written informed consent was obtained from each participant. The study protocol adhered to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Southwest Hospital of Army Medical University (Ethics approval number: KY201801).
Cell culture and collection of cell supernatant
The human prostate epithelial cell line (PEC: RWPE-1 cell) and human prostate stromal cell line (PSC: WPMY-1 cell) were purchased from the American Type Culture Collection (ATCC, USA) and tested for no mycoplasma contamination. WPMY-1 cells were cultured in DMEM medium (Gibco, USA), supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, USA). RWPE-1 cells were cultured in keratinocyte growth medium supplemented with human recombinant epidermal growth factor (5 ng/mL) and bovine pituitary extract (0.05 mg/mL) (ScienCell, USA). Cells were maintained at 37 °C in a 5% CO2 environment. To collect cell supernatant, RWPE-1 cells were grown to 70% confluence in five 175 cm2 flasks (4–8 × 107 cells). The growth medium was then replaced with an equivalent medium supplemented with exosomes-depleted FBS (obtained by ultracentrifuging FBS supernatant at 100,000×g for 16 h) with corresponding treatment (100 ng/mL LPS, 100 nM miRNA mimics, 100 nM miRNA inhibitors or negative controls). LPS was purchased from Sigma (USA). miR-203a-3p mimics, miR-203a-3p inhibitors, and negative controls were designed and synthesized by Genepharma (China), with target sequences listed in Table S1. After 24 h, the conditioned medium was harvested.
Isolation and characterization of exosomes
Exosomes were isolated by using differential ultracentrifugation [22]. Briefly, the prostatic fluid sample or cell supernatant was first centrifuged at 300×g for 10 min to pellet floating cells, followed by centrifugation at 2,000×g for 10 min to pellet cellular debris. The supernatant was then centrifuged at 10,000×g for 30 min to pellet apoptotic bodies and microparticles larger than 1 μm. The resulting supernatant was filtered through a 0.22 μm filter and ultracentrifuged at 100,000×g for 90 min (Beckman Coulter, USA). The resulting exosomal pellets were pooled, washed with phosphate buffer saline (PBS), and ultracentrifuged again at 100,000×g. The final exosomal pellets were resuspended in 100 µL of PBS and stored at -80 °C. Using this method, exosomes were isolated from RWPE-1 cells, LPS-stimulated RWPE-1 cells, normal prostatic fluids, and CP/CPPS-A prostatic fluids, yielding four distinct exosome types: NC-Exos, LPS-Exos, Normal-Exos, and CP/CPPS-A-Exos. For the detection of exosomal particle size, 100 µL of exosomes was diluted into a final volume of 1 mL and subjected to Nanoparticle Tracking Analysis (NTA) (Malvern, UK). The morphology of the exosomes was observed by Transmission Electron Microscope (TEM) (JEOL, Japan). Western blotting was performed to confirm the presence of exosomal biomarkers such as CD9, CD63, CD81, TSG101, Hsp70, and Calnexin (ab275018, Abcam, UK). The concentration of exosomes was determined by using an Exosome Quantitation Assay Kit (SBI, USA) following the manufacturer’s instructions.
Animal models
The study was conducted with the approval of the Laboratory Animal Welfare and Ethics Committee of Army Medical University (Ethics approval number: AMUWEC2020939) and adhered to the US Public Health Service Policy on Humane Care and Use of Laboratory Animals. Adult male Sprague-Dawley rats, weighing 350 ± 20 g, were procured from the Experimental Animal Center of Army Medical University. The procedure entailed standard hair removal and lower abdominal skin disinfection, followed by a layered incision performed under aseptic surgical conditions to expose the bladder and the prostate situated posteriorly. A sterile micro-syringe was used to inject 20 µL of PBS, prostatic fluid derived exosomes, or RWPE-1 cells derived exosomes into the left and right ventral lobes of the prostate. Untreated rats were used as controls. After the injections, the abdominal incisions were closed in layers. Post-surgery, the rats were allowed unrestricted access to water upon regaining consciousness. One week later, the rats were euthanized, and their serum samples and prostates were collected for further analysis.
Cell transfection
The full-length coding sequence of human DUSP5 was synthesized and cloned into the pEGFP-N1 expression vector (Tsingke, China). DUSP5 siRNAs were designed and synthesized by Genepharma (China), with target sequences listed in Table S1. For functional analysis of miR-203a-3p, the transfection was conducted using complexes of Lipofectamine 3000 (Invitrogen, USA) and miR-203a-3p inhibitors/mimics or negative controls, as per the manufacturer’s instructions. To overexpress DUSP5, WPMY-1 cells were infected with either the control pEGFP-N1 expression vector or the DUSP5-expressing pEGFP-N1 expression vector. For loss-of-function studies, WPMY-1 cells were transfected with DUSP5-specific siRNA or scrambled control oligonucleotides (si-NC).
Detection of the uptake of exosomes
To analyze the cellular uptake of exosomes, WPMY-1 cells were seeded in a 20 mm dish (Corning, USA) at a density of 1 × 105 cells/dish. Upon reaching a confluency of 30–50%, the cells were incubated with PKH67-labeled (Sigma, USA) LPS-Exos and CP/CPPS-A-Exos in DMEM medium at 37 °C for 0, 1, 2, and 4 h, respectively. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde, and stained with DiR (MCE, USA) to label the cell membrane. The nuclei were stained with DAPI (Invitrogen, USA) following the manufacturer’s instructions. All images were captured using a confocal microscope (ZEISS, Germany). To analyze the effects of exosomes on cells, WPMY-1 cells were seeded in a 6-well plate at a density of 4 × 105 cells/well. Once the cells reached a confluency of 70–80%, they were incubated with exosomes derived from RWPE-1 cells and prostatic fluids at a concentration of 40 µg/mL.
miRNA expression profile chip of exosomal miRNA
Exosomes isolated from prostatic fluid samples were sent to Shanghai Genechem Company and underwent Affymetrix miRNA 4.0 microarray analysis for miRNA profiling. The significance thresholds for differentially expressed miRNA were set at adjusted P value < 0.01 and fold change > 2.
Real-time quantitative PCR
Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, USA). Reverse transcription of miRNAs or mRNAs was carried out using the Hairpin-it™ miRNAs RT-PCR Quantitation Kit (Genepharma, China) or PrimeScript™ RT reagent Kit (TaKaRa, Japan), respectively. The levels of miRNAs and mRNAs were then measured by using real-time quantitative PCR (qPCR), which was performed using SYBR Green dye (TaKaRa, Japan). The relative expression level of miRNAs or mRNAs was calculated by normalizing to the level of U6 or GAPDH, respectively. The relative expression level was computed using the 2−ΔΔCT method. The specific primer sequences used for the qPCR reactions are listed in Table S2 and all primers were synthesized by Tsingke Biotech Company (China).
Western blotting assay
Protein extraction was performed by using RIPA lysis buffer (Beyotime, China). The protein concentration was determined by using the BCA Protein Assay Reagent (Beyotime, China). For electrophoresis, 30 µg of protein was used. The primary antibodies used for the detection of specific proteins included the following: DUSP5 (sc-393801, Santa Cruz, USA), ERK1/2 (9102S, CST, USA), pERK1/2 (4370S, CST, USA), MCP-1 (26161-1-AP, Proteintech, China), GAPDH (60004-1-Ig, Proteintech, China). HRP-conjugated anti-mouse (SA00001-1) and anti-rabbit (SA00001-2) IgG secondary antibodies were purchased from Proteintech. The detection of the antibody-protein complexes was performed using the ECL chemiluminescence kit (Beyotime, China). The image analysis was performed using ImageJ (ImageJ 1.8, NIH, USA).
ELISA assay
The serum and cell supernatant samples were collected from rats, mice, and cells, followed by 1,000 rpm for 20 min to isolate the supernatant. The concentration of MCP-1, IL-6, IL-15 (LiankeBio, China), CXCL1, CCL3, CCL11, IL-7, IL-17 A, IL-31 (NeoBioscience, China), and CXCL12 (JianglaiBio, China) were detected by using the corresponding ELISA kits following the manufacturer’s instructions.
Prediction of miRNA’s target genes and analysis of KEGG signaling pathway
Several bioinformatics tools were used to predict the miRNA target genes of interest. These tools included TarBase v.8 (http://www.microrna.gr/tarbase), miRDB (https://mirdb.org/index.html), DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php), and mirtargetlink2.0 (https://ccb-compute.cs.uni-saarland.de/mirtargetlink2/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to analyze miRNA pathway enrichment using DIANA-miRPath v3.0 software. The significance threshold for KEGG analysis was set at P < 0.05.
Dual-luciferase reporter assay
The 3’UTR full-length cDNA of human DUSP5 was synthesized and cloned into the pmiRGLO luciferase vectors as Wild-type DUSP5 (WT), which contained the putative miR-203a-3p target binding sequence, and one of two mutated versions (MUT1 or MUT2) with altered bases in the binding site respectively (Tsingke, China). Transfection of these constructs into cells was performed using Lipofectamine 3000 reagent, following the manufacturer’s protocol. Cells were lysed after transfection 48 h, and luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, USA), following the manufacturer’s instructions. The firefly luciferase activity was normalized to Renilla luciferase activity for each sample. The transfection experiments were performed in triplicate.
Isolation of exosomes from mice prostates
Briefly, the prostate tissues were harvested from normal C57BL/6 mice (male, 25–30 g). Then, the tissues were cut into small pieces less than 0.1 cm3 and cultured in serum-free RPMI-1640 culture medium containing collagenase D and DNase I (Sigma, USA) for 45 min with gentle rotation [23]. After incubation, the supernatant was collected and filtered through a 70 μm filter. The resulting supernatant was collected for exosomes as before.
Biodistribution of exosomes in vivo and ex vivo
The in vivo biodistribution study was conducted on normal C57BL/6 mice (male, 25–30 g). Mice were intravenously injected with 100 µL of PKH67-labeled exosomes (100 µg) derived from mouse prostate tissues, prostatic fluids from CP/CPPS-A patients, and healthy adult males, as well as RWPE-1 cells, respectively. In vivo, fluorescent signals emitted by the exosomes were detected 24 h post-injection employing the AniView100 system (Biolight, China). After imaging, all mice were sacrificed and major organs including the kidney, heart, liver, lung, spleen, reproductive system, and prostate were harvested. The ex vivo fluorescent signals within these organs were then measured to assess the distribution of the exosomes. All procedures were conducted under dark conditions.
Preparation and characterization of exosomes loaded with miR-203a-3p antagomirs
Exosomes were electroporated with miRNAs (Cy5-miR-203a-3p antagomirs, miR-203a-3p antagomirs, or the negative controls) purchased from GenePharma, with target sequences listed in Table S1. The electroporation was performed at 400 V/125 µF in a 0.4 cm electroporation cuvette (Bio-Rad, USA). The ratio of exosomes to miRNAs was approximately 3 µg of exosomes to 1 pmol of miRNAs. After electroporation, any unloaded miRNAs that were attached to the surface of the exosomes were removed by performing another round of exosome isolation using the ultracentrifugation method. To quantify the amount of miR-203a-3p antagomirs that were successfully loaded into the exosomes, Cy5-tagged antagomirs were used. The fluorescent signal of the Cy5-tagged antagomirs was evaluated using a microplate reader from Thermo Fisher Scientific.
Flow cytometry
Exosomes were attached to 4 μm aldehyde/sulfate latex beads (Invitrogen, USA) by mixing 30 µg exosomes with 10 µL beads for 15 min at room temperature with continuous rotation. Subsequently, the bead-exosomes suspension was diluted to 1 mL PBS and rotated at room temperature for 30 min. The reaction was stopped by adding 100 mM glycine and 2% bovine serum albumin (BSA) (Beyotime, China) in PBS, followed by rotating at room temperature for 30 min. Exosomes-bound beads were then washed once in 2% BSA and centrifuged for 1 min at 10,000 rpm. Beads were blocked with 10% BSA at room temperature for 30 min with rotation, followed by a second wash in 2% BSA and centrifugation for 1 min at 10,000 rpm. Subsequently, the beads were incubated with an Alexa-488-tagged anti-CD9 antibody (312104, Biolegend, USA) for 30 min at 4 °C with rotation. After incubation, the beads were centrifuged for 1 min at 10,000 rpm, the supernatant was discarded, and the beads were washed in 2% BSA and centrifuged for 1 min at 10,000 rpm. The percentage of positive beads (those bound to CD9-positive exosomes) was calculated relative to the total number of beads analyzed per sample (10,000 events).
Analysis of anti-inflammatory efficacy of miR-203a-3p antagomirs loaded exosomes in vivo
To assess the anti-inflammatory effects of miR-203a-3p antagomirs loaded exosomes, a mouse model of chronic prostatitis was established. This was achieved by injecting 3% carrageenan (Sigma, USA) into the ventral lobes of the prostate gland. Subsequently, the mouse models were then randomly divided into three groups. Each group was intravenously injected with 100 µL of one of the following: sterile PBS, exosomes loaded with miR-203a-3p negative control antagomirs (Exo-antagomirs NC), or exosomes loaded with miR-203a-3p antagomirs (Exo-miR-203a-3p antagomirs). These injections were administered every 3 days, with a total of 3 times injections per mouse. Additionally, a group of mice without treatment was designated as the control group. Following the various treatment regimens, we collected both serum sample and prostate from each mouse for further analysis.
Hematoxylin and eosin staining and immunofluorescence
Paraffin sections of rat and mouse prostate (n = 3) were used for hematoxylin and eosin (H&E) staining and immunofluorescence. Histological scoring of H&E-stained sections of prostates was assessed by pathologists who were blinded to the study. In brief, histological slides were evaluated by using a 6-point scoring system (Table S3). For immunofluorescence, MCP-1 (26161-1-AP, Proteintech, China) was used.
Statistical analysis
All statistical analyses were performed using the software GraphPad Prism (version 8.00, GraphPad Software Inc, USA). For comparisons between two data groups, unpaired t-tests were used. For multi-group data comparisons, either one-way or two-way Analysis of Variance (ANOVA) followed by the Tukey HSD test was used. One-way ANOVA was used to compare the means of three or more independent groups, while two-way ANOVA was used when there were two independent variables. A P value less than 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. And with P values greater than or equal to 0.05 (ns ≥ 0.05) indicating a lack of statistical significance.