Experimental animal

Male Sprague Dawley (SD) rats aged 6 to 7 weeks (200–250 g) came from the Guilin Medical University Experimental Animal Center. They were raised in an SPF laboratory with 20 to 26℃ ventilation and light/dark circulation of 12 h. The animals were acclimatized for one week before the operation and could take standard food and water. The experimental scheme was implemented by the guidelines for the use and care of experimental animals of the European Community and approved by the Animal Ethics Committee of the Guilin Medical University Experimental Animal Center. Every effort was made to minimize the number of animals used and their suffering.

CP/CPPS animal model

SD rats were divided into three groups: blank control group, saline injection group, and carrageenan injection group (normal group, sham group, and model group). First, use 3% pentobarbital sodium salt solution (Sigma-Aldrich) intraperitoneal anesthesia. After complete anesthesia, the limbs and head of the rats were fixed in a supine position, and the abdominal skin was disinfected. Then, make a small incision in the middle of the lower abdomen and gently lift the bladder, visible on both sides of the prostate. In the model group, 50ul 3% carrageenan saline suspension was injected into the dorsal lobe of each rat’s prostate. Rats in the Sham group were injected with the same amount of normal saline into the dorsal lobe of the prostate. The rats in the normal group were not treated. Then, disinfection and suturing. The rats in each group were put back in the cage and fed synchronously for 60 days.

Rat model Sample collection

After 60 days of simultaneous feeding, rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (350 mg/kg body weight). Cut the skin of the lower abdomen of the rat and fully expose the prostate. After carefully removing the prostate, each sample was divided into three parts for preservation. One part was stored at 80 °C for immunoblotting analysis, one part was immersed in RNA protection solution and placed in a new EP tube 24 h later for RNA extraction, and the other part was used to prepare paraffin sections.

Expressed prostatic secretion collection

The Ethics Committee of Guilin Medical College approved this study. The samples were from the outpatients of the Affiliated Hospital of Guilin Medical College and used after informed consent. EPS was collected by gentle prostate massage. EPS were collected and centrifuged to remove cell particles/precipitates and stored at −80 °C until use.

Cell culture, RNA interference, and LPS stimulation

RWPE-1 cells were purchased from the American Type Culture Collection (ATCC), incubated in the atmosphere of 95% air and 5% CO2 at 37 °C, and cultured in Keratinocyte-SFM medium with bovine pituitary extract and human recombinant epidermal growth factor (Thermo).

RWPE-1 cells were transfected with BLNK-knockdown lentivirus (sh18-BLNK, sh19-BLNK, sh20-BLNK) or its negative control (sh-NC), purchased from GK company. Transfect BLNK silencing virus and control lentivirus into RWPE-1 cells according to the manufacturer’s instructions. After 48 h of infection, 1 µg/mL puromycin was added to the medium to observe the expression of green fluorescent protein (GFP), and an RWPE-1 cell line with stable silencing of BLNK was established.

The human prostatitis cell model is induced by RWPE-1 cells with 2.5 ug/mL lipopolysaccharide (LPS) for 2 h.

Histological analyses

The 4% paraformaldehyde-fixed rat prostate tissue was automatically dehydrated and paraffin-embedded. The thickness of the section was four µm. Paraffin sections were dewaxed with xylene and hydrated with alcohol. Subsequently, sections for HE staining analysis were stained with hematoxylin and eosin, dehydrated with alcohol, and transparent with xylene. The pathological changes of prostate tissue were observed under a light microscope. For immunohistochemical analysis, the sections were heated with EDTA buffer for antigen extraction for 15 min. Then, the samples were removed and placed in 0.3% hydrogen peroxide for 30 min to block endogenous peroxidase activity, and the serum was blocked for 20 min. Slide incubated anti-BLNK primary antibody (Sangon Biotech), diluted 1:100, 4℃ overnight, then cultured with biotinylated subcultures for 25 min. After hematoxylin re-staining, the slides were dehydrated, sealed with neutral resin, and observed under a light microscope. Immunohistochemical results were evaluated using an immunoreactive score (IRS). The prostatitis cell model was immobilized with 4% paraformaldehyde and incubated with 1% Triton X-100. The subsequent steps were the same as immunohistochemistry.

RNA-seq data analysis

Each group of the CP/CPPS rat model was mixed with nine rat samples and divided into the Sham group and Model group. Each group of cell models contained three biological replicates for RNA-seq. After total RNA was extracted by TRIzol, the library and sequenced were conduct by Annaroad Gene Technology (Beijing)Co., Ltd and NovogeneCo.,Ltd, and used Illumina sequencing. Fragments Per Kilobase of exon model per Million mapped fragments were used to determine and standardize gene expression. The DEseq2 package identified differential expression. After statistical analysis, CP/CPPS rat model differentially expressed (DE) genes were determined according to fold change > 1.5 or fold change < 0.67 and p-value < 0.05, and Cell models differentially expressed (DE) genes were determined according to fold change > 2 or fold change < 0.5 and p-value < 0.05.

RT-qPCR

Up to 1 µg of total RNA reversed transcribed by using the PrimeScript RT reagent Kit with gDNA Eraser (Takara) was added to SYBR green master mix (Roche) with the Realplex (Eppendorf) according to the manufacturer’s instructions for Complementary DNA (cDNA) synthesis. Relative messenger RNA (mRNA and lncRNA) levels were determined using the 2 − ΔΔCt interpretation and normalized by ACTB. The primers used are listed in Table S1.

GO and KEGG analyses

All differentially expressed mRNAs and lncRNAs were analyzed by David for GO enrichment analysis and KEGG pathway function analysis. P < 0.05 indicated the significant enrichment of differentially expressed genes.

Western blot

Western blot protocols have been previously described [19]. Protein levels weredetermined by bicinchoninic acid (BCA) protein assay. Primary antibodies against BLNK (Biotechn) and β-actin (Abcam) were used. Immunoreactive bands were visualized using the BeyoECL Plus kit (Beyotime). Human prostatic fluid samples were diluted with RIPA buffer at a ratio of 1:3 and denatured with SDS-PAGE protein loading buffer at 100 °C for 10 min, followed by equal volume Western blot.

Cell proliferation assay

For 5-ethynyl-2’-deoxyuridine (EdU) staining, the cells were seeded on a circular glass cell slide in a 96-well plate for 24 h. Then, the cells were stained using the Yefuor 594 EdU imaging kit (UElandy) according to the manufacturer’s instructions. Cell DNA content was detected by fluorescence microscope.

Flow cytometry

Cell cycle analysis: Each group of cells (1 × 106 cells) was fixed overnight at 4 °C with pre-cooled 75% ethanol. Subsequently, cells were washed twice with PBS and resuspended with 500 µL propidium iodide (PI) (Becton) at room temperature. After 15 min, the cells were loaded into a flow cytometer (Calibur) for analysis. The results were evaluated using FlowJo software.

Cell apoptosis analysis: Allophycocyanin-conjugated annexin V and 7-amino actinomycin D (BD) were used to analyze the cell apoptosis rate. Each group of cells was washed twice with PBS and treated with 0.25% pancreatin (without EDTA) to facilitate digestion. After adjusting the cell density to 1 × 105/mL, the cells were incubated with fluorescent antibodies at room temperature for 25 min, then pelleted and resuspended. The samples were analyzed using a flow cytometer and the FlowJo software.

LncRNA Identification

The potential candidate lncRNA consists of two or more exons; the transcript length exceeds 200 nt. CNCI, CPC, CPAT, and PFAM were employed for lncRNA identification. Finally, four connectivity analysis methods were utilized to identify lncRNAs for subsequent analysis.

LncRNA-mRNA Co-expression network analysis

The function of lncRNAs was analyzed by predicting differentially expressed lncRNA target genes. Cis and Trans target analysis, respectively. Cis analysis is to screen the protein-coding genes at the adjacent positions of lncRNA (upstream and downstream 50Kb) as target genes. Trans analysis was based on the correlation coefficient between lncRNA and mRNA expression, and the absolute value of the correlation coefficient was corr ≥ 0.97. The co-expression network of lncRNA-mRNA interactions was visualized using Cytoscape software.

Molecular docking

The structures of BLNK and its interacting proteins were downloaded from the RCSB Protein Data Bank (PDB) database (https://www.rcsb.org/). SYBYL-X 1.2 software prepared PDB files, including removing foreign water molecules, adding hydrogen molecules, and adjusting pH-sensitive protonation. Protein-protein docking was performed using the docking program HEX 8.0.0, and the average docking energy between some proteins and the BLNK complex was calculated. The KFC2 server (https://mitchell-web.ornl.gov/) calculates the protein-protein binding interface hotspots.

Statistical analyses

All data were presented as mean ± standard deviation (s.d.), and the student’s t-test was used to analyze the difference between the two groups. Correlations among data were analyzed using Spearman’s correlation analysis. The software used was SPSS version 27.0 and GraphPad Prism version 8.0. P < 0.05 was considered statistically significant.