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Identification of novel key genes associated with the metastasis of prostate cancer based on bioinformatics prediction and validation | Cancer Cell International | Full Text
Materials and methods . Data collection and processing . The transcriptome expression profiles and corresponding clinical information of prostate tissue samples were downloaded from TCGA database ( https://portal.gdc.cancer.gov/ ). To screen the DEGs responsible for PCa metastasis, the prostate samples were divided into primary prostate adenocarcinoma samples (n?=?310) and metastatic prostate adenocarcinoma samples (n?=?73). Before the analysis of TCGA data, transcriptome data (FPKM values) were transformed into TPM values [ 15 ]. After normalization, a comparison between primary and metastatic samples was conducted using the limma package from R software. DEGs were then identified based on the thresholds as log 2 (fold change)>?1.0 and adjusted P value (adj.P.Val)?Transcriptional expression analysis . Based on the LASSO logistic regression, the expression of DEGs in primary and metastatic PCa was conducted using the ggbeeswarm package from R software. After identifying the potential key genes, the transcriptional levels of these genes were analyzed using the online UALCAN ( http://ualcan.path.uab.edu/ ) database. UALCAN is an online resource for gene expression and survival analyses in various tumors, providing easy access to public transcriptional expression data, including TCGA [ 16 ]. In the present study, UALCAN database was applied to analyze the transcripts of these key genes in normal, primary, and metastatic prostate adenocarcinoma tissues. P value was shown in the webpage, and P?statistically significant. Clinical prostate sample collection . Paraffin-embedded prostate tissues of primary and metastatic PCa were obtained from patients who had undergone surgery between 2018 and 2020 at Zhejiang Provincial People’s Hospital (Hangzhou, China). The tissues were fixed in paraffin and stored in the pathology department of Zhejiang Provincial People’s Hospital. A total of three primary prostate tissue samples and three metastatic prostate tissue samples were obtained, and the corresponding clinical information were listed in Additional file 1 : Table S1. This study was approved by the Institutional Review Board of Zhejiang Provincial People’s Hospital (IRB-2021019). Immunohistochemical staining . Paraffin-embedded prostate sections were deparaffinized with xylene and rehydrated with ethanol. Then the sections were treated with 1?mM EDTA to retrieve the antigen and preincubated with 5% goat serum in TBS to decrease non-specific binding. Next, the sections were incubated with the following primary antibodies: anti-ISG15 (Cat. 15981-1-AP, 1:200), anti-CST2 (Cat. 19935-1-AP, 1:20) (Proteintech, Wuhan, China), anti-DNAH8 (Cat. HPA028447, 1:250, Sigma, Oakville, Canada). After incubation with horseradish peroxidase secondary antibodies, the prostate sections were measured using DAB (Beyotime, Hangzhou, China) and counterstained with hematoxylin. The IHC score included immune-positive rate and the staining intensities of each sample. In the present study, the protein expressing score of all the slices were analyzed by the pathologist. The immune-positive rate was graded from 0 to 4, where less than 5% of cells stained was scored as 0; 6–25% was scored as 1; 26–50% was scored as 2; 51–75% was scored as 3; and 76–100% was scored as 4. The staining intensities were graded from 0 to 3, where 0 was defined as negative; 1 as weak; 2 as moderate; and 3 as strong. The protein score of ISG15, CST2 and DNAH8 was calculated as the product of intensity and positive rate, which ranged from 0 to 12. Survival analysis . Survival analysis of the key genes in patients with prostate adenocarcinoma was conducted using the GEPIA database ( http://gepia.cancer-pku.cn/ ) [ 17 ]. GEPIA is an online tool used to perform the differential expression analysis, correlation analysis, and survival analysis based on the TCGA and GTEx projects. In this study, survival curves including overall survival (OS) and disease-free survival (DFS) were generated based on high and low expression of key genes. The cutoff value was set as quartile, and P?statistically significant. Gene set enrichment analysis . To investigate the signaling pathways of the key genes participating in the metastasis of prostate adenocarcinoma, GSEA was performed using R software (version 4.0.3) and clusterProfiler package (version 3.18.0). The clusterProfiler package was used for biological-term classification and enrichment analysis of gene clusters [ 18 ]. For GSEA, the KEGG enrichment maps were generated based on high and low expression of key genes. Cell culture . Human PCa cell lines 22Rv1 and PC3 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 22Rv1 and PC3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Sangon Biotech, Shanghai, China) and maintained at 37?°C in a 5% CO 2 incubator. Construction of shISG15 and shCST2 PCa cell line . Lentivirus encoding the shRNAs were designed and synthesized by HANBIO (Shanghai, China) and the sequences of shRNAs were listed in Additional file 1 : Table S2. According to the manufacturer’s instructions, 22Rv1 and PC3 cells were infected with lentivirus expressing Luc-shCtrl, Luc-shISG15 or Luc-shCST2 for 48?h and selected with puromycin (Cat. ant-pr-1, InvivoGen, USA) at a concentration of 4??g/mL. RNA extraction and qRT-PCR . After generation of the indicated stable cell line, PCa cells were collected and total RNA was extracted using the AxyPrep Multisource RNA Miniprep Kit (Cat. AP-MN-MS-RNA-250, Union City, USA). cDNA was generated by using HiScript II Q RT SuperMix (Vazyme, Cat. R222-01, Jiangsu, China). Next, TB Green Premix Ex Taq II (Takara, Cat. RR820Q, Kusatsu, Japan) was used to prepare the quantitative polymerase chain reaction amplification reaction. The mRNA expression of target genes was determined by qRT-PCR using the LightCycler480 II system (Roche, IN, USA) and quantified by the 2 ?ΔΔCt method. Primers for qRT-PCR were purchased from Sangon Biotech (Shanghai, China) and are listed in Additional file 1 : Table S3. Western blot . Cells were collected and lysed in RIPA buffer containing 1?mM PMSF (Beyotime, Cat. ST506, Shanghai, China). Protein samples were denatured at 100?°C for 10?min and then separated by 10% SDS-PAGE. After being transferred to a PVDF membrane (Millipore, Cat. IPVH00010, Bedford, USA), 5% skim milk in TBST was used to block non-specific binding for 2?h at room temperature. Then the membrane was incubated with the following primary antibodies: anti-CST2 (Cat. 19935-1-AP, 1:1000), anti-ISG15 (Cat. 15981-1-AP, 1:250), anti-E-cadherin (Cat. 20874-1-AP, 1:1000), anti-Twist1 (Cat. 13099-1-AP, 1:500), anti-Vimentin (Cat. 10366-1-AP, 1:10,000), and anti-GAPDH (Cat. 60004-1-Ig, 1:10000) (Proteintech, Wuhan, China) at 4?°C overnight. Next day, membranes were washed with TBST for three times and incubated with the secondary antibodies conjugated with horseradish peroxidase, goat anti-rabbit (Cat.70-GAR0072, 1:5000) or goat anti-mouse (Cat.70-GAM0072, 1:5000) (Lianke Multi Sciences, China), at room temperature for 1?h. ECL (Cat.20-500-120, Beit HaEmek, Israel) was used to detect the binding antibodies by ChemiDoc? XRS + System (Bio-Rad, USA). Wound healing assay . After the stable PCa cells grown to be confluent, a micropipette tip was applied to make a cross wound. Then cells were washed with PBS for three times and cultured in serum-free RPMI-1640 medium and Mitomycin C (5??g/mL, Cat. M5353, Sigma, Oakville, Canada). Photographs were taken by microscopy at once and after wounding for 24?h. Transwell assay . Cell migration and invasion assays were conducted using 24-well transwell chambers (8??m pore size, Corning, Cat. 8432, USA). For a cell invasion assay, the inserts were precoated with 250?μg/mL Matrigel (BD Bioscience, Cat. 356234, USA) on the upper surface and incubated at 37?°C for 2?h. For migration and invasion assays, about 1?×?10 5 cells were suspended in 0.2?mL serum-free RPMI-1640 medium containing 5??g/mL Mitomycin C and added to the inserts. The lower compartment of the chamber was filled with 0.6?mL of RPMI-1640 containing 10% fetal bovine serum. After incubation for 24?h, the cells on the upper membrane were carefully removed using a cotton bud. Cells that invaded the chamber membranes were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Five microscopic views of 100?×?magnification of each insert was selected randomly under a phase-contrast microscope (Olympus, Japan) and counted using ImageJ 1.52a (Wayne Rasband, USA). In vivo animal study . 4–6?weeks old BALB/c nude male mice (20?±?2?g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang Provincial People’s Hospital (A202100033). Mice were kept at least one week before cell implantation. For metastatic in vivo model, BALB/c nude mice were randomized into six groups: 22Rv1-Luc-shCtrl, 22Rv1-Luc-shISG15, 22Rv1-Luc-shCST2, PC3-Luc-shCtrl, PC3-Luc-shISG15, and PC3-Luc-shCST2. Intravenously (I.V.) injection was performed in this study as previously reported [ 19 ]. Briefly, each group of PCa cells (5?×?10 5 ) were resuspended in 100 ?L PBS and slowly injected into the tail vain of the mice. After injection, the IVIS imaging system (IVIS Lumina Series III, Perkin Elmer) was used to monitor and visualize the distribution of tumor cells. D-luciferin (Cat. 7903, DAKEWE, Shenzhen, China) at 150?mg/kg was injected in mice 10?min prior to IVIS spectrum imaging. Tumor burden was measured based on total photons per second with background subtraction per region of interest (ROI). Statistical analyses . All data are expressed as the mean?±?SD. Statistical comparisons between the two groups were determined by Student’s unpaired two-tailed t test. P?statistically significant. .
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