Public database analysis
In the present study, the corresponding transcriptome data and clinical information of The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) were acquired from the UCSC Xena database platform (http://xena.ucsc.edu/). This dataset is comprised of 496 PCa samples (includes 488 primary localized PCa and 8 metastatic PCa) and 52 adjacent normal tissues (ANT). Subsequently, the expression profile of GSE21034 PCa mRNA sequencing dataset submitted by Taylor BS et al. was downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/), which contains 150 PCa samples (131 primary localized PCa and 19 metastatic PCa) and 29 normal tissues (Taylor et al., 2010). The “limma” R package was utilized to identify the differentially expressed genes (DEGs) in database (Ritchie et al., 2015). For TCGA dataset, DEGs were recruited with |log2FC| value > 1 and FDR < 0.05, but for GSE21034 dataset, DEGs were recruited with |log2FC| value > 0.59 and FDR < 0.05. A Venn diagram was constructed to identify the overlapping DEGs. The key genes in our subsequent studies have complete clinical data, including clinicopathologic parameters and follow-up information that match the gene expression. Because that patient mortality rate in the database is too low, we chose the BCR as the endpoint event of prognosis index.
Patients And Tissue Specimens
Total of 86 PCa patients’ tissue specimen were collected at our institution from January 2009 to December 2019. All of these patients underwent surgical resection or biopsy, and none of them had received hormonal treatment, chemotherapy, or preoperative radiotherapy. Among them 50 patients had primarily localized disease and 36 cases had distal metastases. All enrolled patients underwent follow-up through telephone. This study was approved by the Ethics Committee of the First Affiliated Hospital for Guangzhou Medical University, and all patients have signed informed consents.
Cell Lines And Cell Transfection
The normal human prostate epithelial cell line RWPE-1 and human PCa cell lines LNCAP, PC-3 and DU145 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Human PCa cell line C4-2, 22RV1 and human embryonic kidney cell 293 T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines had authenticated and were cultured as described previously (Zhao et al., 2021).
The timely and stable transfected cell lines were constructed to study the biological function of NETO2. We used the small interfering RNA (siRNA) targeting NETO2 (GenePharma, Shanghai, China) to silence endogenous NETO2 (named si-NETO2). Cells were transfected with Lipofectamine 3000 according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). In addition, to establish stable overexpression or knockdown of NETO2 (named NETO2 or shNETO2) in PCa cells, lentivirus (LANDM BIOTECH, Guangzhou, China) were synthesized and used to infect the cell. Stable cell lines were selected for 10 days with Puromycin (2µg/mL). The sequence information of siRNAs and shRNAs are listed in Supplementary Table S1. The transfection efficiency was identified by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, and the most efficient sequence was used in the following experiments.
Rna Extraction And Qrt-pcr
Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, CA, USA). All-in-One First-Strand cDNA Synthesis kit (GeneCopoeia, Guangzhou, China) was used to reverse transcribe RNA into cDNA. PCR for quantification of gene expression was performed with SYBR green Premix Ex Taq II (Takara) on a CFX-96 system (Bio-Rad, Hercules, CA). The PCR thermal cycling conditions consisted of an initial denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 58°C for 20 s, and a 30 s extension at 72°C. GAPDH was used as internal control. Relative expression was determined by 2−△△Ct method. The sequence information of primers are listed in Supplementary Table S1.
Protein Extraction And Western Blotting
Total proteins were extracted from cells with RIPA lysis buffer (KeyGEN, KGP703) containing PMSF (1%), and its concentration was quantified using the BCA protein assay regents (#23225, Thermo Pierce, Rockford, IL, USA). The procedure of western blot was conducted as previously described (Zhao et al., 2021). Primary antibodies used in the study include: anti-NETO2, anti-GAPDH, anti-P53, anti-P21 and anti-P16 purchased from Abcam. Detection was achieved in Odyssey CLX Two-color infrared laser imaging system (LI-COR Biosciences, Nebraska, USA). Densitometric analysis of the bands was performed using ImageJ software.
Cell Proliferation Assays
The proliferation ability of PCa cells was measured by MTS assay, 5-ethynyl‐2′‐deoxyuridine (EdU) assay and colony formation assay, respectively. For MTS assay, the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, USA) was used according to the manufacturer’s instructions. Briefly, transfected PCa cells were seeded into 96-well plates (5,000 cells per well in 200 µl of complete culture medium). Following culture for 24, 48, 72, 96, and 120 h, cells in each well were incubated with 10 µl of MTS reagent for 4 h, and the optical density (OD) value of each well was then measured at 490 nm. For EdU assay and colony formation assay, the process was performed as previously described (Liu et al., 2021).
Cell Migration And Invasion Assays
The migration and invasion ability of PCa cells were measured by wound-healing assays and transwell assay. The detailed procedure was conducted as described in our previous research (Liu et al., 2021;Zhao et al., 2021). It should be emphasized that, in the transwell assay, we simulated the invasion or migration of tumor cells by adding or not adding matrigel (BD Biosciences) in advance in the permeable support chamber (Corning Incorporated, Corning, NY, USA).
Thirty 4–5 weeks old male BALB/c nude mice were purchased from the Experimental Animal Center of Guangdong Province (Guangzhou, China). The animals were fed as described previously (Zhao et al., 2021). All procedures related to the experimental animals were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Guangzhou Medical University.
To evaluate the effects of NETO2 on tumor growth, mice were randomly divided into six groups (NC/NETO2/shNETO2 group, n = 5/group) and each mouse was subcutaneously injected with concentrated stable PCa cells 5×106 to establish xenograft tumors. The tumor sizes were monitored weekly. After 4 weeks, the mice were sacrificed by cervical dislocation, and the tumors were dissected and weighed. The tissue was fixed and embedded in paraffin wax for histological examination and immunohistochemical (IHC) assay.
Histological And Immunohistochemical Assessment
To study tissue morphology and protein expression, histological and IHC analysis were performed in mice xenografts and clinical PCa samples. The procedure of H&E and IHC were carried out as described previously (Li et al., 2017). Primary antibodies used in IHC include anti-NETO2 (Abcam) and anti-KI67 (Servicebio). When the experiment complete, slices were scanned by PathScope digital scanner (Gene Tech, Shanghai, China), and the protein expression was quantified according to the protocol described previously (Li et al., 2017). For NETO2 IHC score, we comprehensively evaluated the protein staining intensity of each microscopic field and the proportion of positive staining cell, then the final IHC score is obtained by multiplying the results of the two indicators (Xiang et al., 2020). The IHC score ranged from 1 to 9, where < 6 was classified as the low-expression group, and ≥ 6 was classified as the high-expression group. The final score of all specimens were used for statistical analysis.
Gene Expression Profiling And Pathway Analyses
NETO2-overexpression (PC3-NETO2) and NETO2-knockdown (PC3-shNETO2) PC3 cells were used for gene expression profiling. RNA was isolated using RNeasy Mini kit (QIAGEN). Microarray processing was performed by SINOTECH GENOMICS (Shanghai, China) using GeneChip PrimeView Human Gene Expression Array (Affymetrix). Differentially expressed genes between stable transfected and control cells were calculated by “edgeR” package, and then selected by Q < 0.05 and absolute fold change (FC) value > 1.5. In order to analyze the biological functions involved in differential genes, Gene Ontology (GO, http://pgrc.ipk-gatersleben.de/misa/) enrichment analysis was performed. Then, the pathway enrichment analysis was performed by using KEGG (http://emboss.sourceforge.net/) database. The order of signaling pathways is based on the number of differential genes enriched in the pathway.
The cells were cultured in six-well plates until 90% confluence and then fixed and subjected to β-Galactosidase staining using a Senescence β-Galactosidase Staining Kit (Solarbio, China) following the manufacturer’s instructions. After staining for 12h, the cells were observed and recorded under microscope. The field of vision was randomly selected from three independent experiments.
The SPSS V18.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0 software (San Diego, CA, USA) were used to perform statistical analyses. The association between NETO2 expression and PCa clinicopathologic characteristics was assessed by chi-squared test. Univariate and multivariate Cox regression models were constructed to estimate the hazard ratios (HRs) of independent factors affecting the overall survival in patients with PCa. Survival curves were plotted using Kaplan-Meier’s method and compared between groups by the log-rank test. X-tile program was used to determine the cut-off values which optimized the significance of the split between Kaplan-Meier survival curves (Camp et al., 2004). For continuous variables, data presented as the means ± SD. Comparison between groups was carried out using the Student's t-test, paired t-test or one-way ANOVA. P < 0.05 was considered statistically significant.