The collection of tissue samples was approved by the Ethics Committee of the First Hospital of China Medical University according to the Declaration of Helsinki, and all participants provided written informed consent (approval number,  2018-190-2). All methods were performed in accordance with the relevant guidelines and regulations.
The prostate cancer mRNA matrix and clinical information were obtained from TCGA (The Cancer Genome Atlas) database (https://www.cancer.gov/) . We considered 547 prostate samples, including 52 normal prostate samples and 495 tumor samples. We downloaded 36 prostate cancer tissues matrix and the clinical information from GSE46602, the platform for which is the GPL570 Affymetrix Human Genome U133 Plus 2.0 Array .
There are few studies on TACR2 at present. To explore the pathways related to TACR2 in prostate cancer, we conducted Gene Set Enrichment Analysis (GSEA) . The GSEA method is used to determine whether a gene set of interest differs significantly in two biological states. We used GSEA to explore functional pathways related to TACR2 expression, grouping according to the median of TACR2 expression. Using a single data set that causes high false-positive results, we performed cross-validation analysis between TCGA-PRAD, and GSE46602.
TACR2 co-expression mRNA analysis
We found that TACR2 was significantly related to the Wnt pathway. We know that the Wnt pathway is involved in a variety of biological processes in the body. To determine which biological processes TACR2 relates to, we used Gene Ontology (GO) (http://geneontology.org/) analysis to illustrate the biological function of TACR2 co-expressed protein-coding genes . We used the weighted gene co-expression network (WGCNA) method to explore the co-expression factors of TACR2 by constructing a scale-free network . The number of genes in the minimum module was defined as 30. By drawing a correlation heat map between the module and TACR2, we determined the module with the strongest correlation with TACR2. We extracted protein-coding genes with Pearson correlation coefficients > 0.4. GO analysis of these genes identified the most relevant biological functions and molecular results of TACR2. We did not directly screen based on the Pearson correlation coefficient of TACR2 and other protein-coding genes; instead, we first determined its co-expression module before the screening. Such a screening strategy assumes that co-expression modules usually have similar biological behaviors. The most relevant co-expression module of TACR2 can be obtained using this method, determining the most relevant biological function.
Immune cell proportion correlation
There is tachykinin-mediated modulation of the immune response . Therefore, we attempted to determine associated immune processes by measuring prostate cancer tissues’ immune cell content. We ran the CIBERSORT algorithm and determined the immune cell proportion based on the bulk tissue gene expression matrices. LM22 is a gene signature matrix that defines 22 immune cell subtypes obtained from a website (https://cibersort.stanford.edu/).
Clinical specimen collection
From January 2020 to December 2020, we performed radical prostatectomy on prostate cancer patients according to EAU GUIDELINES ON PROSTATE CANCER, and 30 pairs of prostate cancer tissue samples and adjacent normal prostate tissue samples (> 1 cm from the tumor) were collected at the First Hospital of China Medical University (Shenyang, China). No patients received endocrine therapy, chemotherapy, or radiotherapy before radical prostatectomy. All tissues were examined histologically. Prostate cancer tissue samples had high-density cancer foci, and no cancer foci were found in the adjacent normal prostate tissue samples. Tissue samples were stored at − 80 °C before use.
Cell lines and reagents
The human prostate cancer cell lines, PC3, DU145, and LNCaP Clone FGC, were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and were STR certified. PCR was used to detect Mycoplasma in the culture medium, and the passage time of the cells was not more than 6 months. The cell lines were cultured in RPMI 1640 (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and grown at 37 °C, 5% CO2. The antibodies included TACR2 (Proteintech, 25270-1-AP), beta-Catenin (Proteintech, 51067-2-AP), GAPDH (Cell Signaling Technology, 5174S), Cyclin D1 (Cell Signaling Technology, 55506S), Lamin B1 (Cell Signaling Technology, 13435S).
Construction of cell lines with TACR2 overexpression
PC3, DU145, and LNCaP cells were seeded in 6-well or other plates and were transfected 24 h later. Genechem (Shanghai, Genechem) was commissioned to design and synthesize the TACR2 overexpression lentiviral vector and the corresponding negative control lentiviral vector. Three cell lines were transfected according to the manufacturer’s protocol. The stable negative control (NC) and TACR2 overexpression (TA-OE) cell lines were established in a puromycin-containing medium.
Western blot analysis
The total proteins were extracted from tissues and cell lines using RIPA lysis buffer (containing protease and phosphatase inhibitors). Nuclear protein was extracted using the Nuclear and Cytoplasmic Protein Extraction kit (Beyotime). Protein concentration was determined with bicinchoninic acid assay (Beyotime Institute of Biotechnology). The denatured protein sample (40 µg/lane) was added to the 10% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis (140 V, 60 min), then separated and transferred to the polyvinylidene fluoride membrane (340 mA, 90 min). Subsequently, membranes were blocked in a sealed container with 5% non-fat milk (37 °C, 1 h) and incubated with anti-TACR2 (1:500), anti-β-catenin (1:5000) or anti-GAPDH (1:1000) primary antibodies overnight in 5% fat-free milk at 4 °C.
The stable cells were seeded on 24-well plates. According to the manufacturer’s instructions, EdU (BeyoClick™, EDU-488, China) reagent was added to the medium at the ratio of 1:1000. According to the manufacturer’s protocol, the culture was continued at 37 °C for 2 h; after labeling, complete the following experimental steps. Finally, the number of proliferating cells was counted under the fluorescence microscope (Olympus Corporation, Japan).
Cell viability assay
The stable PC3, DU145, and LNCaP cells were seeded into 96-well plates at the density of 5 × 103 cells/well. Cell Counting Kit-8 (CCK-8) (Bimake, USA) solution was added to each well to a final concentration of 0.5 mg/ml and incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a plate reader (Model 680; Bio-Rad Laboratories).
Transwell chambers without matrix were used for cell migration tests. We plated 200 µl of serum-free medium containing stable cells (1 × 105) into the upper chambers (Corning, NY, USA) of 24-well plates, and 600 µl medium (10% FBS) was plated into the lower chamber. After 48 h of incubation, the cells under the membrane were fixed with 4% paraformaldehyde for 10 min and stained with crystal violet stain for 10 min. The remaining cells in the upper chamber were removed with cotton swabs. The migration of cells on the surface under the membrane was measured using an inverted light microscope, and the migration efficiency was calculated using ImageJ.
Wound-healing migration assay
Wound-healing tests were used to measure cell migration. Stable PC3 and DU145 cells were seeded on 24-well plates. When 90–100% confluence was achieved, a 200-µl sterile pipette was used to create a linear scratch. After washing with a serum-free medium, the cells were cultured for 48 h. Cell images at 0 and 48 h after injury were recorded under a microscope.
GraphPad Prism version 8.0 (GraphPad Software Inc., USA) was used for statistical analyses. The data of at least three independent experiments were expressed as mean ± standard deviation. The differences between groups were analyzed using Student’s t-test. Statistically, significance was set at P < 0.05. Pearson coefficients > 0.4 were considered significant. We used R-version 3.6.3 for all the R package statistical analyses.