Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure

Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure Fan Wu, Fei Huang, Nili Jiang, Jinfeng Su, Siyi Yao, Boying Liang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3214106/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Few studies are focusing on the mechanism of erastin acts on prostate cancer(PCa) cells, and essential ferroptosis-related genes (FRGs) that can be PCa therapeutic targets are rarely known. In the current study, in vitro assays were performed to evaluate the ferroptotic levels of PCa cells under erastin treatment. RNA-sequecing was used to measure the expression of differentially expressed genes (DEGs) in erastin-induced PCa cells. A series of bioinformatic analyses were applied to analyze the pathways, modules, transcription factors, and expression levels of DEGs. Erastin inhibited the expression of SLC7A11 and cell survivability in LNCaP and PC3 cells. After treatment with erastin, the concentration of malondialdehyde (MDA) and Fe 2+ significantly increased, whereas the glutathione (GSH) and the oxidized glutathione (GSSG) significantly decreased in both cells. A total of 295 overlapping DEGs were screened and identified in two cells under erastin exposure and significantly enriched for association with several pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, et al. For four hub FRGs, TMEFF2 in PCa tissue is higher than in normal tissue and the expression levels of CLU , NRXN3 , and UNC5B were lower in PCa tissue. The expression levels of SLC7A11 and cell survivability were inhibited after the knockdown of TMEFF2 in LNCaP cells but not in PC3 cells. The concentration of Fe 2+ only significantly increased in TMEFF2 downregulated LNCaP cells. This study extends our understanding of the molecular mechanism in erastin-affected PCa cells, and provides potential treatment ideas for PCa therapy. ferroptosis erastin prostate cancer cell TMEFF2 RNA-seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction As a highly lethal cancer, prostate cancer (PCa) ranks second in the incidence of male malignant tumors worldwide [ 1 ]. The androgen deprivation therapy is the clinical treatment for PCa, however, patients receiving this treatment may develop castration-resistant prostate cancer (CRPC) [ 2 ]. Therefore, it is necessary to screen and identify more targeted genes and develop a more effective therapy for PCa. As a unique mode of cell death, ferroptosis is widely divergent from other cell death including apoptosis, autophagy, and necrosis [ 3 ]. Mechanically, a few essential factors or products of lipid peroxidation metabolism such as solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase (GPX4), glutathione (GSH), and malondialdehyde (MDA) are related with ferroptosis [ 4 ].In consideration of PCa with metastatic potential [ 5 ] and cancer cells with metastatic and invasive ability were susceptible to ferroptosis [ 6 ], we hypothesized that targeting certain ferroptosis-related genes (FRGs) may be useful in treating PCa. Increasing number of researchers focused on the relationship between ferroptosis and PCa, Wo et al. identified some FRGs by analyzing the RNA-sequecing (RNA-seq) data from The Cancer Genome Atlas (TCGA) [ 7 ]. Unfortunately, they only included the datasets from the clinical data of PCa patients and did not analyze the data of PCa cells because of the lack of RNA-seq results derived from ferroptotic PCa cells. However, focusing on ferroptosis in PCa cells will provide more comprehensive theoretical basis or treatment strategy for clinical treatment. So we had an idea to obtain the RNA-seq information to get clear about the potential mechanisms of ferroptotic PCa cells. As one of the most famous ferroptosis inducers, erastin is a perfect drug to trigger the ferroptotic progress of cells [ 8 ]. There are few studies and limited information on the ipact of erastin acts on PCa cells. Ghoochani A et al. used cell and animal tests to improve various ferroptosis inducers, including erastin, and remarkably slowed down the PCa progress [ 9 ]. Yang et al. claimed that erastin can downregulate the androgen receptor expression both in PCa cells and animal models [ 10 ]. After analyzing the data of PCa patients in a public database, Wo et al. found and validated several FRGs [ 7 ]. Based on the studies above, we realize that erastin exerts inhibitory effects on PCa cells or tumor growth, and we asked two questions: what are the mechanisms of erastin acts on PCa cells? Which FRGs play an essential role in ferroptosis triggered by erastin? To get clear these questions, erastin was selected as a ferroptosis inducer and to construct the ferroptotic PCa cell model. Moreover, PC3 is an androgen-independent cell and LNCaP is an androgen-dependent cell [ 11 ], so these two different and representative PCa cell lines were selected. In our research, cellular experiments were performed in erastin-induced LNCaP and PC3 cells, and the impact of erastin brings on ferroptotic levels in PCa cells was studied. RNA-seq was applied to the screen and identify the differentially expressed genes (DEGs) in PCa cells under erastin exposure. Ferroptosis-related pathways and FRGs that may play an essential role in erastin-induced PCa cell lines were identified. In addition, we tried to provide ferroptotic clues in terms of pathways, transcription factors (TFs), and modules for two different cells. Several FRGs with clinical significance were analyzed and validated. Hence, our study not only extends our understanding of erastin-affected PCa cells but also provides potential therapeutic targets and ideas for PCa therapy. Materials and methods Cell lines and reagents LNCaP and PC3 cells (a gift from the Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences ) were cultured at 37°C in RPMI-1640 containing 15% and 10% fetal bovine serum (BI, Biological Industries, Co., Ltd., Israel), respectively. The ferroptosis inducer erastin was bought from MCE company (5.0 µM, HY-15763, USA). A virus expressing a short hairpin RNA (shRNA) targeting TMEFF2 was obtained from GenePharma Co., Ltd., for infection. The TMEFF2 targeted shRNA sequence was as follows: 5′-GUGUGAGCAUUCUAUCAAU-3′ [ 12 ]. Stably infected LNCaP and PC3 cells exhibiting TMEFF2 knockdown were obtained by selective screening with puromycin (1 µg/mL; Solarbio, China). Western blot analysis For the Western blot analysis, proteins isolated from the control cell samples (LNCaP and PC3) and from the experimental cell samples (LNCaP_5_0_era and PC3_5_0_era) were collected after 5.0 µM erastin incubated for 12 h. Each protein from these samples was separated in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. These membranes were probed with the primary antibody for SLC7A11(1:250, ab307601, Abcam, USA), or TMEFF2 (1:500, ab133562, Abcam, USA), β-tublin (1:2000, 10094-1-AP, ProteinTech, China). Subsequently, the membranes and the corresponding second antibody (ProteinTech, China) were incubated at room temperature for 1 h before being photographed. Cell survivability assay Herein, 2.5 × 10 3 cells were inoculated in 96-well plates. After 24 h, the 5.0 µM erastin was added to the culture medium. Each well was added with CCK-8 solution (Dojindo, Japan) at 24 h, 48 h, 72 h, and 96 h. The viability of the cell was determined by measuring the absorbance values of the 96-well plates at 450 nm using a quantitative microplate spectrophotometer (BioTek, Winooski, USA). Malondialdehyde (MDA) assay Micro malondialdehyde assay kit (BC0025, Solarbio, China) was used to detect the cellular MDA levels. After 5 × 10 6 cells at the logarithmic growth phase, they were treated with 5 µM erastin for 12 h. These cells were respectively lysed and cracked with extracting solution by using the ultrasonic cell disruptor. The supernatants were obtained after a series of centrifugation and preservation under specific temperatures based on the instruction. Finally, the light absorption value of 532 nm and 600 nm wavelengths of the two groups’ supernatants were detected, calculated, and analyzed. Ferrous ions ( Fe 2+ ) concentration assay The intracellular iron colorimetric assay kit (E1042, Applygen, China) was used to detect the level of Fe 2+ . After cells were treated with 5 µM erastin for 12 h, the cell lysis solution and the shaker were used for 2 h aiming at lysing a total of 2 × 10 6 cells. Moreover, the diluted reagents were mixed, incubated, and centrifuged to acquire a standard solution then subsequently stained with 30 µl Fe 2+ detection reagent for 30 min. The absorption of the reagent at 550 nm wavelength was measured by the quant microplate spectrophotometer. Glutathione (GSH) and oxidized glutathione (GSSG) assay Herein, the control and the experimental group cells after treatment with 5 µM erastin for 12 h, were prepared in a 6-well plate, and collected and homogenized with 150 µl protein removal reagent. Then, the samples were put in the liquid nitrogen and 37°C water bath twice in sequence to rupture the cells. Afterward, cell samples were placed on the ice, then centrifuged to obtain the supernatant. Subsequently, GSH and GSSG Assay Kits (Beyotime, China) were used to measure and analyze the GSH and GSSG levels, and the absorbance at 412 nm wavelength was measured. Sample collection and detection After washing with PBS, the cells were digested and obtained, which were then sent to Novogene, Co., Ltd., China. 1 µg RNA of cells was extracted by the illumina TruSeq RNA Sample Prep Kit (FC-122-1001, illumina, USA) to construct the sequencing libraries. The purified double-stranded cDNA was synthesized by Olygo (dT) reverse transcription, amplified by PCR, and screened with AMPureXP beads to acquire cDNA libraries. RNA-seq was performed on the Illumina NovaSeq 6000 to generate 150 bp paired-end readings, and approximately 8 G of reads per sample were obtained. Data is available in the Gene Expression Omnibus database (GEO submission number: GSE232034). Data processing First, sequencing adapters and low-quality reads were trimmed and removed. Then, Homo_sapiens_Ensemble_94 was selected as the reference genome sequence for high-quality reads. The feature counts were applied to calculate the reads and the fragments per kilobase of the exon model per million mapped fragments (FPKM) for the length of genes. The Pearson correlational and the principal component analyses (PCA) were selected to analyze the sample correlation and the sample clustering, respectively. The R (Version 3.0.3) ggplot2 package was used for visualization. DEGs analysis The DEGs analysis was performed within the experimental group (LNCaP_5_0_era and PC3_5_0_era) and the control group (LNCaP and PC3) using DESeq2 software (1.20.0), and each sample group was performed in triplicate. DESeq2 offers statistical procedures to determine the DEGs by the negative binomial distribution model [ 13 ]. The significant differential expression mean p -value (padj) ≤ 0.05 & |log2(foldchange)| ≥ 2. Functional enrichment analysis of erastin-induced genes For the potential functions and signal pathways of erastin-induced gene-expression changes in PCa cells to be further explored, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome analyses were performed to explore and study the different overlapping DEGs of the LNCaP group (LNCaP_5_0_era and LNCaP) and the PC3 group (PC3_5_0_era and PC3). Screening of hub gene and module analysis A protein-protein interaction (PPI) network obtained through different overlapping DEGs of the LNCaP group (LNCaP_5_0_era and LNCaP) and the PC3 group (PC3_5_0_era and PC3) were submitted to the STRING database [ 14 ]. Cytoscape (version 3.9.1) [ 15 ] was applied to analyze the PPI relationships, and a total score of > 0.9 was selected as the cutoff criterion. In addition, the CytoHubba obtained the top 10 hub genes during the screening process [ 16 ]. The Cytoscape plug-in MCODE was employed to detect the molecular complex and acquire the module of DEGs. Gene set enrichment analysis (GSEA) The GSEA enrichment analysis [ 17 ] was carried out to analyze the GO datasets of the LNCaP group (LNCaP_5_0_era vs LNCaP) and the PC3 group (PC3_5_0_era vs PC3) separately. The significant GSEA results with the stringed threshold of nominal p -value < 0.05, false discovery rate (FDR) q-value 1. Prediction of hub genes’ TFs A total of 10 hub genes were submitted to the NetworkAnalyst platform to predict their TFs. The putative TFs associated with hub genes have been identified and sorted through the mean rank score. Validation for FRGs The Human Protein Atlas database was used to determine the gene expression patterns of interested hubs in tissues. The GEPIA platform was selected to draw a box map of the hub gene with illustrated the expression patterns of tissues [ 18 ]. In addition, this online tool provided the correlation analysis for the hub gene. The online database, GeneMANIA, created an interactive network to predict the tangible relationship between the hub genes [ 19 ]. Western blot, Fe 2+ concentration assay, and CCK8 were performed to assess the correlation between the hub gene and ferroptosis. Results Erastin accelerates ferroptosis of PCa cells LNCaP and PC3 cell lines added with the erastin were phenotypically different in the two cell groups (Fig. 1 A). Erastin repressed the expression of the ferroptosis marker protein SLC7A11 in both PCa cell lines (Fig. 1 B). Thus, we carried out the cell survivability, MDA, Fe 2+ , GSH, and GSSG assay of erastin-induced PCa cells. After 48 h, erastin significantly inhibited the proliferation of LNCaP and PC3 cells in the CCK8 assay ( p < 0.05; Fig. 1 C–D). Results showed that MDA levels under erastin-induced conditions were notably increased in PC3 cells ( p 0.05; Fig. 1 E). Fe 2+ content, one of the key features of ferroptosis, was notably increased with erastin treatment in these two cells ( p < 0.05; Fig. 1 F). Given that GSH/GSSG regulates cellular redox homeostasis, the level of GSH and GSSG were measured. The results showed that GSH levels in both cell lines were decreased following erastin treatment ( p < 0.05; Fig. 1 G). Meanwhile, the GSH/GSSG levels were also downregulated in these cell samples ( p < 0.01; Fig. 1 H). Summary of RNA sequencing data RNA sequencing was used to analyze the transcriptome profiling of PCa cells and erastin-induced PCa cells to investigate the molecular mechanisms of ferroptosis inducer erastin affect PCa cells and find more FRGs. Using control cells and erastin-induced PCa cells, each of the four groups had three biological replicates, and 12 sequencing samples were prepared. Three replicates in four groups were consistent with the principal component analysis (Fig. 2 A). Based on correlation analysis results (Fig. 2 B), the correlation coefficient of the samples was between 0.8 and 1, which showed good biological repeatability in these sequencing samples. Erastin-induced DEGs analysis In the RNA-seq assay with padj ≤ 0.05&|log2(foldchange)|≥2 as screening conditions, LNCaP_5_0_Era identified a total of 942 DEGs as compared with LNCaP, including 447 upregulated genes and 495 downregulated genes (Fig. 2 C–D). Then, a total of 5,625 DEGs were screened and recognized between PC3_5_0_Era and PC3, of which 3,704 were upregulated and 1,921 were downregulated (Fig. 2 E–F). A total of 295 overlapping DEGs in two compared groups were screened and recognized via the Venn diagram (Fig. 2 G). Functional enrichment in erastin-induced genes GO, KEGG, and REACTOME functional enrichment analyses were performed to analyze 295 overlapping DEGs to further explore the potential biological behavior of erastin-induced changes in PCa cells. As exhibited by the GO analysis results, the erastin-induced DEGs were primarily related to DNA replication, DNA-dependent DNA replication, and DNA replication initiation for biological processes (BPs), condensed chromosome, chromosomal region, condensed chromosome, centromeric region for molecular functions (MFs), and catalytic activity acting on DNA, DNA helicase activity, and helicase activity for cellular components (CCs) (Fig. 3 A). In contrast, the KEGG analysis indicated that these DEGs mapped to DNA replication, cell cycle, and homologous recombination pathways (Fig. 3 B). Furthermore, REACTOME analysis revealed that these DEGs were associated with DNA strand elongation, cell-cycle checkpoints, activation of the pre-replicative complex, and so on (Fig. 3 C). Pathways and module analysis of two PCa cells An MCODE plug-in was carried out to construct modules representing the crucial clusters to access which related pathways brought more weight into ferroptotic process of PCa cells (Supplementary Fig. 1). Among the LNCaP group, the DEGs in module 1 were selected to conduct the next analysis and found they involved with several prominent signaling pathways (Supplementary Fig. 1A–B). For the PC3 group, enriched pathways including nephron morphogenesis, positive regulation of blood vessel endothelial cell migration, positive regulation of bone mineralization, and regulation of steroid metabolic process were detected in module 1 of PC3 DEGs (Supplementary Fig. 1C–D). GSEA analysis of erastin-induced genes Herein, we have then performed GSEA analysis between erastin treatment and control groups across the four cell lines to reveal more gene set enrichment information that involves the erastin-induced changes in PCa cells. We found that most of those pathways in GSEA were also found in the GO, KEGG, and Reactome functional enrichment analyses, which supports and validates the previous results. However, it was not unexpected to find that erastin-induced PCa DEGs were involved in the regulation of cell death, cellular response to reactive oxygen species, fatty acid biosynthetic process, and activation of MAPK activity pathway, intrinsic apoptotic signaling pathway in response to DNA damage by P53 class mediator and cellular amino acid metabolic process in LNCaP (Fig. 4 A) and PC3 cells (Fig. 4 B). Moreover, we further focused on several pathways expected to be only induced in the PC3 group, including regulation of I-kappab kinase NF-kappab signaling, regulation of JNK cascade, regulation of ERK1 and ERK2 cascade, negative regulation of ERBB signaling pathway, negative regulation of Ras protein signal transduction, and regulation of JAK-STAT cascade (Fig. 4 C). Erastin-induced hub genes and their TFs CytoHubba plug-in was selected to calculate the hub genes of 295 overlapping DEGs to find more potential and pivotal genes related to ferroptosis in PCa cells. Of these, 10 top hub genes (including CLU , IL1B , MET , NRXN3 , PLXNA4 , GAD1 , UNC5B , SLC7A5 , DAPK1 , and TMEFF2 ) were identified in order of degrees (Fig. 5 A). Details and descriptions of 10 hub genes are summarized in Table 1 . Table 1 Top 10 hub genes of erastin-induced LNCaP and PC3 cells Ensemble ID Symbol log2(FC) In LNCaP group FDR Value In LNCaP group log2(FC) In PC3 group FDR Value In PC3 group ENSG00000120885 CLU 2.227676954 4.66194E-14 2.328754927 3.10046E-26 ENSG00000125538 IL1B 2.659181706 0.023399869 -2.354730765 5.37023E-07 ENSG00000105976 MET 2.178704253 5.57265E-06 -2.716782674 4.70167E-33 ENSG00000021645 NRXN3 2.219194193 2.12366E-07 2.442352118 6.87491E-18 ENSG00000221866 PLXNA4 3.383825988 4.60173E-05 5.0250723 0.006594555 ENSG00000128683 GAD1 2.674364639 5.49826E-07 4.298554233 1.7675E-116 ENSG00000107731 UNC5B 2.073123293 4.61178E-09 -2.989199096 3.58257E-30 ENSG00000103257 SLC7A5 -2.079560779 1.34346E-12 -2.392270174 1.17417E-23 ENSG00000196730 DAPK1 2.188978999 6.02751E-14 -5.892959015 8.06199E-77 ENSG00000144339 TMEFF2 -3.727660511 1.65524E-66 4.052271557 1.96873E-07 Given that TFs had a critical influence on gene expression levels, 10 top hub genes were uploaded to the NetworkAnalyst database for TFs prediction. The regulatory network of top TFs (including SP9 , DLX2 , ARX , PEG3 , CSRNP3 , ZNF697 , INSM2 , STAT3 , NR2F1 , FOXL1 , E2F1 , and NFIC ) of the hub genes was obtained and presented (Fig. 5 B). Moreover, the expression levels of these TFs were shown in Fig. 5 C. Among them, PEG3 , CSRNP3 , ZNF697 , NR2F1 , DLX2 , and INSM2 were highly expressed, whereas SP9 , ARX , E2F1 , FOXL1 , STAT3 , and NFIC were lowly expressed in PCa cells after erastin exposure. Therefore, we analyzed their expression patterns in HPA and GEPIA database to investigate whether these hub genes were clinically associated with PCa. In the HPA database, the expression of TMEFF2 in PCa tissue is higher than in normal tissue and the expression levels of CLU , NRXN3 , and UNC5B were lower in PCa tissue by immunohistochemistry (Fig. 5 D). Consistently, TMEFF2 mRNA levels were higher in PCa tissue ( p < 0.05), and CLU , NRXN3 , and UNC5B were downregulated ( p < 0.05) in PCa tissue in the GEPIA database (Fig. 5 E). Prediction and validation Of FRGs The relationships between hub genes’ expression levels and ferroptosis marker SLC7A11 and GPX4 in the GEPIA database were respectively analyzed to investigate whether or not these 4 hub genes were potentially involved with ferroptosis. As expected, the expression patterns of TMEFF2 , CLU , NRXN3 , and UNC5B were positively correlated with SLC7A11 and GPX4 (Fig. 6 A). The gene interactive networks were established to clarify the possible direct relationship between hub genes and ferroptosis markers and identify their potential associations. TMEFF2 , SLC7A11 , and GPX4 showed the complex PPI network with the physical interactions, co-expression, prediction, co-localization, genetic interactions, pathway, and shared protein domains of 77.64%, 8.01%, 5.37%, 3.63%, 2.87%, 1.88%, and 0.60%, respectively (Fig. 6 B). Among these networks, there were direct genetic interactions within TMEFF2 and SLC7A11 . Moreover, TMEFF2 showed direct genetic interactions with ALOX5 , then ALOX5 showed physical interactions with GPX4 . However, CLU , NRXN3 , and UNC5B failed to display such a close relationship with GPX4 as compared with TMEFF2 (data not shown). Therefore, TMEFF was selected as the interesting ferroptosis-related gene for further experiments. In addition, we constructed LNCaP and PC3 cells with the downregulation of TMEFF2 to test whether or not TMEFF2 can be an FRG in the experiment, then detected their cell survivability and Fe 2+ concentration. LNCaP cells with knockdown of TMEFF2 exhibited lower expression of SLC7A11 , while down-regulation of TMEFF2 did not significantly affect the expression level of SLC7A11 in PC3 cells (Fig. 6 C). After TMEFF2 was knocked down, the Fe 2+ in LNCaP cells was remarkably increased ( p < 0.05; Fig. 6 D). By contrast, there was no significant difference between PC3 cells with erastin treatment and the control group (Fig. 6 D). After 48 h, the downregulation of TMEFF2 only significantly reduced the proliferative ability of LNCaP cells ( p < 0.05; Fig. 6 E). Discussion Our study discussed the role of ferroptosis inducer erastin in two PCa cells, LNCaP and PC3. Unsurprisingly, we found that erastin exposure caused an impact on LNCaP and PC3 cells in terms of ferroptosis marker, cell survivability, and the concentration of MDA, Fe 2+ , GSH, and GSSG. In addition, we extracted DEGs of both two PCa cells at the transcriptional level. Subsequently, we identified various pathways, TFs and modules in different PCa cells. Moreover, we identified and validated several erastin-affected FRGs which were potentially meaningful in PCa treatment through a series of bioinformatics analyses and experiments. First, we focused on the identification of FRGs in PCa cells after erastin exposure. From RNA-Seq results, 10 FRGs were identified as the hub genes via overlapping 295 DEGs. TMEFF2 , a type I transmembrane protein with an EGF-like and two follistatin motifs 2 proteins, was selectively expressed in the brain and prostate [ 20 ]. Based on our analysis, TMEFF2 was a co-expressed gene and correlated with GPX4 and SLC7A11 , as determined by GeneMANIA and GEPIA. Despite the absence of direct reports on the connection between TMEFF2 and ferroptosis, a study reported that oxidative stress downregulate the expression of TMEFF2 in PCa [ 21 ]. Moreover, TMEFF2 can influence the proliferative activity of PCa cells [ 22 ], which was consistent with our results. In addition, similar to our results, several researchers reported that the alternative expression levels of TMEFF2 changed the disease stage in PCa tissue of patients and mouse model [ 23 – 25 ]. Therefore, the above reports supported our view that the TMEFF2 is related to ferroptosis and can be the FRG of PCa. It was worth mentioning that the ferroptotic level was significantly affected by knockdown of TMEFF2 in LNCaP cells, but not in PC3 cells. Considering that LNCaP is an androgen-dependent cell and PC3 is an androgen-independent cell[ 11 ]. Furthermore, TMEFF2 has been identified as an androgen-related gene [ 22 ]. And there was a report claimed that knock-down of TMEFF2 could suppressed the androgen-response of LNCaP cells [ 26 ]. Thus, we speculated that TMEFF2 promotes ferroptosis via androgen, which means TMEFF2 only bring an influence on ferroptosis of androgen-sensitive LNCaP cells. Then if TMEFF2 were used as a ferroptotic treatment target in the future, it would be more suitable for PCa patients than CRPC patients. Our results also indicated FRGs such as CLU , NRXN3 , and UNC5B showed different expression levels between PCa and normal tissue, and their expression patterns were positively related to GPX4 and SLC7A11 . Meanwhile, CLU caused ATP degradation which resulted in peroxidation in cells [ 27 ], while the upregulation of CLU inhibited PCa cell death [ 28 ]. Furthermore, NRXN3 is a gene associated with obesity [ 29 ], and we speculated that NRXN3 takes part in the metabolism process of lipids. Moreover, UNC5B is a crucial regulator of ferroptosis in osteosarcoma cells [ 30 ]. Thus, these four hub DEGs identified in LNCaP and PC3 cells under erastin exposure exhibit to play an essential role in ferroptotic process of PCa cells, and maybe the potential treatment targets of PCa. Subsequently, we obtained a TFs network for the 10 DEGs in PCa cells after erastin treatment. In the top TFs, STAT3 and E2F1 were differently expressed in erastin-induced PCa cells, and accumulating evidence indicated that they were related to ferroptosis. STAT3 , signal transduction and activators of transcription 3, the ferroptosis was stimulated in PCa cells with knockdown of STAT3 [ 31 ]. Mechanistically, STAT3 binds to GPX4 and regulates its expression in pancreatic cancer cells [ 32 ]. E2F1 , E2F transcription factor 1, can be one of the ferroptosis-related gene prognostic indexes (FRGPI) to predict disease-free survival (DFS) for PCa patients’ radical prostatectomy, PCa patients with high FRGPI had worse DFS than patients with low FRGPI [ 33 ]. Hence, we speculated that these TFs associated with ferroptosis also participate to the regulation of hub genes’ expression to affect the ferroptosis in PCa cells. In addition to hub DEGs and their TFs, several pathways play an essential role in ferroptotic cells. Based on the GO functional analysis, the DEGs were primarily enriched in DNA replication, Chromosome segregation, and DNA helicase activity pathway. These three pathways were closely related to DNA replication, meanwhile, DNA replication is the crucial link to cell growth. Our findings indicated that erastin remarkably reduced the proliferative activity of PCa cells. In addition, Fe 2+ is one of the essential factors of DNA replication, which is vital for cell survivability [ 34 ]. Consequently, we believed that erastin primarily transforms PCa cell proliferation by altering DNA replication. Except for DNA replication, KEGG analysis revealed that these DEGs were also associated with steroid hormone biosynthesis, MARK signal pathway, and P53 signal pathway. Steroid hormone biosynthesis is in charge of ferroptosis induction in adrenocortical carcinoma cells [ 35 ]. Considering our TFs network, it can be speculated that erastin affects MAPK/ERK to regulate STAT3 , then STAT3 is a TF to change the expression patterns of hub genes CLU in PCa cells. Furthermore, STAT3 participate into P53/SLC7A11 pathway in osteosarcoma cells [ 36 ]. In our results, SLC7A11 was also downregulated by erastin exposure. We speculated that STAT3 not only affects the expression level of CLU but also changes the P53 signal pathway to alter the expression of SLC7A11 . Meanwhile, REACTOME enrichment analysis suggested that DEGs were enriched in cell cycle checkpoints and activation of E2F1 target genes at the G1/S pathway. Experimental evidence showed that erastin caused a change in the proportion of PCa cells in certain phases [ 10 ]. Based on our analysis, E2F1 may be the TFs of hub genes TMEFF2 , thus, E2F1 could affect the transcript level of TMEFF2 , which may involve the cell cycle of PCa cells. Therefore, erastin may affect the expression patterns of hub genes and their TFs through regulate various signal pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, to transform the cell growth and cell cycle of PCa cells. Interestingly, we found the erastin-induced PC3 group owned more DEGs than erastin-induced LNCaP groups. In our module analysis, the top 1 module of the PC3 group had more direct ferroptosis-related pathways, such as positive regulation of bone mineralization and regulation of steroid metabolic process, than the LNCaP groups. Therefore, as to these two PCa cell lines, we suggested that PC3 cells are much more sensitive to ferroptosis inducer erastin than LNCaP cells. Subsequently, we wanted to know why CRPC cell lines PC3 exhibited more susceptibility to erastin. GSEA analysis was applied to study various cellular signal pathways, which other functions enrichment did not identify. As expected, several cell death-related pathways were enriched, and the PC3 group displayed more and several unique signaling pathways in GSEA, such as negative regulation of ERBB signaling pathway, negative regulation of Ras protein signal transduction, and regulation of JAK-STAT cascade. These signaling pathways are all associated with ferroptosis in various cells. A complex suppressed the ERBB signal pathway to inhibit ferroptosis in vivo and in vitro [ 37 ]. Moreover, the RAS/MAPK pathway may regulated oxidative stress and the related ferroptosis pathways in glioblastoma [ 38 ]. Furthermore, IFNγ enhanced the JAK-STAT signal pathway to activate ferroptosis in hepatocellular carcinoma cells [ 39 ]. Unfortunately, there were lack of direct reports about the relationship between these signal pathways and ferroptosis of PCa. Hence, erastin transformed more crucial signal pathways, which led to PC3 being more sensitive to erastin. Moreover, considering that PC3 is an androgen-independent cell and LNCaP is an androgen-sensitive cell [ 11 ], we speculated that CRPC patients may be more susceptible to targeting gene therapy related to ferroptosis than PCa patients. This current study has some limitations. Firstly, two PCa cell lines, LNCaP and PC3, were selected to carry out the experiment. If we included more PCa cells, the results will be more comprehensive. Secondly, although we verified the FRGs in cell experiments and in public databases, our further study will be confirmed in animal experiments and clinical practices. Conclusion This study verified that erastin successfully brought about a significant effect on the proliferation and ferroptotic index of LNCaP and PC3 cells. In addition, we carried out RNA-seq and annotated 295 overlapping DEGs after erastin treatment in these two cells. Functional enrichment analysis showed that erastin may be act on PCa cells by participating in a variety of pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, et al. Our results also indicated several FRGs such as TMEFF2 , CLU , NRXN3 , and UNC5B showed different expression levels between PCa and normal tissue. The potential TFs, STAT3 and E2F1 , may take part in regulating the ferroptotic process of these FRGs. We also found downregulation of TMEFF2 only promote ferroptosis in androgen-sensitive LNCaP cells but not in androgen-independent PC3 cells. Our finding elucidate potential molecular mechanisms in the subsequent ferroptotic studies of PCa cells, and suggest the novel therapeutic targets and strategy for PCa. Declarations Author contributions FW and FH performed data analysis and drafted the manuscript. FW, NJ, BL and QZ performed the experiments. JFS, YY, WL and TY prepared figures and tables. SZ and QZ secured research funding supports. All authors contributed to the article and approved the submitted version. Funding This work was supported by grants from the National Natural Science Foundation of China (No. 32260164), Natural Science Foundation of Guangxi Zhuang Autonomous Region (No.2020GXNSFBA297150, No.2023GXNSFAA026080). Data availability The original contributions presented in the study are publicly available. This data can be found here: GSE232034 datasets downloaded from GEO database. Competing Interests All authors have no relevant fnancial or nonfnancial interests to disclose. Ethical approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. References Konoshenko MY, Bryzgunova OE, Laktionov PP (2021) miRNAs and androgen deprivation therapy for prostate cancer. Biochim et Biophys acta Reviews cancer 1876(2):188625. 10.1016/j.bbcan.2021.188625 Ge R, Wang Z, Montironi R, Jiang Z, Cheng M, Santoni M et al (2020) Epigenetic modulations and lineage plasticity in advanced prostate cancer. Annals of oncology: official journal of the European Society for Medical Oncology 31(4):470–479. 10.1016/j.annonc.2020.02.002 Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE et al (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060–1072. 10.1016/j.cell.2012.03.042 Xie Y, Hou W, Song X, Yu Y, Huang J, Sun X et al (2016) Ferroptosis: process and function. Cell Death Differ 23(3):369–379. 10.1038/cdd.2015.158 Johnson RP, Ratnacaram CK, Kumar L, Jose J (2022) Combinatorial approaches of nanotherapeutics for inflammatory pathway targeted therapy of prostate cancer. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy. 64:100865. 10.1016/j.drup.2022.100865 Yee PP, Wei Y, Kim SY, Lu T, Chih SY, Lawson C et al (2020) Neutrophil-induced ferroptosis promotes tumor necrosis in glioblastoma progression. Nat Commun 11(1):5424. 10.1038/s41467-020-19193-y Wo Q, Liu Z, Hu L (2022) Identification of Ferroptosis-Associated Genes in Prostate Cancer by Bioinformatics Analysis. Front Genet 13:852565. 10.3389/fgene.2022.852565 Zhang H, Deng T, Liu R, Ning T, Yang H, Liu D et al (2020) CAF secreted miR-522 suppresses ferroptosis and promotes acquired chemo-resistance in gastric cancer. Mol Cancer 19(1):43. 10.1186/s12943-020-01168-8 Ghoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD et al (2021) Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. Cancer Res 81(6):1583–1594. 10.1158/0008-5472.Can-20-3477 Yang Y, Liu T, Hu C, Xia H, Liu W, Chen J et al (2021) Ferroptosis inducer erastin downregulates androgen receptor and its splice variants in castration–resistant prostate cancer. Oncol Rep 45(4). 10.3892/or.2021.7976 You Z, Dong Y, Kong X, Zhang Y, Vessella RL, Melamed J (2007) Differential expression of IL-17RC isoforms in androgen-dependent and androgen-independent prostate cancers. Neoplasia (New York NY) 9(6):464–470. 10.1593/neo.07109 Xie S, Zhang Y, Peng T, Guo J, Cao Y, Guo J et al (2021) TMEFF2 promoter hypermethylation is an unfavorable prognostic marker in gliomas. Cancer Cell Int 21(1):148. 10.1186/s12935-021-01818-x Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. 10.1186/s13059-014-0550-8 Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P et al (2011) The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39(Database issue):D561–D568. 10.1093/nar/gkq973 Smoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T, Cytoscape (2011) 2.8: new features for data integration and network visualization. Bioinformatics (Oxford, England). ;27(3):431-2. doi: 10.1093/bioinformatics/btq675 Chin CH, Chen SH, Wu HH, Ho CW, Ko MT, Lin CY (2014) cytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Syst Biol 8(Suppl 4):S11. 10.1186/1752-0509-8-s4-s11 Powers RK, Goodspeed A, Pielke-Lombardo H, Tan AC, Costello JC (2018) GSEA-InContext: identifying novel and common patterns in expression experiments. Bioinformatics 34(13):i555–i64. 10.1093/bioinformatics/bty271 Tang Z, Li C, Kang B, Gao G, Li C, Zhang Z (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45(W1):W98–W102. 10.1093/nar/gkx247 Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res W214–W220 38(Web Server issue). 10.1093/nar/gkq537 Masood M, Grimm S, El-Bahrawy M, Yague E (2020) TMEFF2: A Transmembrane Proteoglycan with Multifaceted Actions in Cancer and Disease. Cancers (Basel) 12(12). 10.3390/cancers12123862 Gawel-Beben K, Ali N, Ellis V, Velasco G, Poghosyan Z, Ager A et al (2018) TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer. Cell Biol Int 42(3):273–280. 10.1002/cbin.10832 Gery S, Sawyers CL, Agus DB, Said JW, Koeffler HP (2002) TMEFF2 is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cells. Oncogene 21(31):4739–4746. 10.1038/sj.onc.1205142 Afar DE, Bhaskar V, Ibsen E, Breinberg D, Henshall SM, Kench JG et al (2004) Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer. Mol Cancer Ther 3(8):921–932 Glynne-Jones E, Harper ME, Seery LT, James R, Anglin I, Morgan HE et al (2001) TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence. Int J Cancer 94(2):178–184. 10.1002/ijc.1450 Corbin JM, Overcash RF, Wren JD, Coburn A, Tipton GJ, Ezzell JA et al (2016) Analysis of TMEFF2 allografts and transgenic mouse models reveals roles in prostate regeneration and cancer. Prostate 76(1):97–113. 10.1002/pros.23103 Corbin JM, Georgescu C, Wren JD, Xu C, Asch AS, Ruiz-Echevarría MJ (2021) Seed-mediated RNA interference of androgen signaling and survival networks induces cell death in prostate cancer cells. Mol therapy Nucleic acids 24:337–351. 10.1016/j.omtn.2021.03.002 Materia S, Cater MA, Klomp LW, Mercer JF, La Fontaine S (2011) Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B. J Biol Chem 286(12):10073–10083. 10.1074/jbc.M110.190546 Zhang Y, Lv X, Chen L, Liu Y (2022) The role and function of CLU in cancer biology and therapy. Clin Exp Med. 10.1007/s10238-022-00885-2 Prats-Puig A, Grau-Cabrera P, Riera-Perez E, Cortes-Marina R, Fortea E, Soriano-Rodriguez P et al (2013) Variations in the obesity genes FTO, TMEM18 and NRXN3 influence the vulnerability of children to weight gain induced by short sleep duration. Int J Obes (Lond) 37(2):182–187. 10.1038/ijo.2012.27 Zheng D, Xia K, Wei Z, Wei Z, Guo W (2022) Identification of a novel gene signature with regard to ferroptosis, prognosis prediction, and immune microenvironment in osteosarcoma. Front Genet 13:944978. 10.3389/fgene.2022.944978 Ji J, Li H, Wang W, Yuan B, Shen T (2022) ARPC1A is regulated by STAT3 to inhibit ferroptosis and promote prostate cancer progression. Hum Cell 35(5):1591–1601. 10.1007/s13577-022-00754-w Zhang W, Gong M, Zhang W, Mo J, Zhang S, Zhu Z et al (2022) Thiostrepton induces ferroptosis in pancreatic cancer cells through STAT3/GPX4 signalling. Cell Death Dis 13(7):630. 10.1038/s41419-022-05082-3 Wang Y, Fan J, Chen T, Xu L, Liu P, Xiao L et al (2022) A novel ferroptosis-related gene prognostic index for prognosis and response to immunotherapy in patients with prostate cancer. Front Endocrinol (Lausanne) 13:975623. 10.3389/fendo.2022.975623 Chen L, Cai Q, Yang R, Wang H, Ling H, Li T et al (2023) GINS4 suppresses ferroptosis by antagonizing p53 acetylation with Snail. Proc Natl Acad Sci USA 120(15):e2219585120. 10.1073/pnas.2219585120 Weigand I, Schreiner J, Rohrig F, Sun N, Landwehr LS, Urlaub H et al (2020) Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis 11(3):192. 10.1038/s41419-020-2385-4 Luo Y, Gao X, Zou L, Lei M, Feng J, Hu Z (2021) Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells. Oxid Med Cell Longev 2021:1783485. 10.1155/2021/1783485 Zhao X, Zhang J, Zhang W, Guo Z, Wei W, Wang X et al (2023) A chiral fluorescent Ir(iii) complex that targets the GPX4 and ErbB pathways to induce cellular ferroptosis. Chem Sci 14(5):1114–1122. 10.1039/d2sc06171f Wu J, Zhang T, Xiong H, Zeng L, Wang Z, Peng Y et al (2022) Tumor-Infiltrating CD4(+) Central Memory T Cells Correlated with Favorable Prognosis in Oral Squamous Cell Carcinoma. J Inflamm Res 15:141–152. 10.2147/jir.S343432 Kong R, Wang N, Han W, Bao W, Lu J (2021) IFNgamma-mediated repression of system xc(-) drives vulnerability to induced ferroptosis in hepatocellular carcinoma cells. J Leukoc Biol 110(2):301–314. 10.1002/JLB.3MA1220-815RRR Additional Declarations No competing interests reported. Supplementary Files supplementaryfig1.tif Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3214106","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":222515236,"identity":"97a9d597-03ea-4240-8282-c97b840ad985","order_by":0,"name":"Fan Wu","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Wu","suffix":""},{"id":222515237,"identity":"13c408cb-be0c-4933-a325-5aa2ce065220","order_by":1,"name":"Fei Huang","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Huang","suffix":""},{"id":222515238,"identity":"0ed3c586-3094-4de5-9632-87d167086cf7","order_by":2,"name":"Nili Jiang","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nili","middleName":"","lastName":"Jiang","suffix":""},{"id":222515239,"identity":"ab8ed07a-cb5e-40f7-80f3-bfdc275ef1e6","order_by":3,"name":"Jinfeng Su","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Su","suffix":""},{"id":222515240,"identity":"f2b745f4-5a8d-4e02-ac9b-2028bbbd1ff3","order_by":4,"name":"Siyi Yao","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siyi","middleName":"","lastName":"Yao","suffix":""},{"id":222515241,"identity":"35fae700-c475-4730-a05d-7341b6a66046","order_by":5,"name":"Boying Liang","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Boying","middleName":"","lastName":"Liang","suffix":""},{"id":222515242,"identity":"3b5dc72a-2b6d-4916-a6c9-96757ebb4679","order_by":6,"name":"Wen Li","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Li","suffix":""},{"id":222515243,"identity":"fcb297f1-c052-4252-b6b7-316ad845b20b","order_by":7,"name":"Tengyue Yan","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tengyue","middleName":"","lastName":"Yan","suffix":""},{"id":222515244,"identity":"b7723093-ae3f-498f-b678-27d129a41996","order_by":8,"name":"Sufang Zhou","email":"","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sufang","middleName":"","lastName":"Zhou","suffix":""},{"id":222515245,"identity":"416b78ad-edb4-48bf-bb54-ac2b1ae57795","order_by":9,"name":"Qingniao Zhou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIie3PIQvCUBDA8RuC6eQ1eUNhX+HEOtxX2RBMC0bj4Ikm+8R9CKPxHsLSxCpoEKyGgXWgolFxWzO8P1y7H8cBmEz/mLQiQAIUQjHnkxqka8dpoOOsEnkOArjEYX/bmlYQoqNm+XXsInCWcysCR7T5N7ETrZYJjdBSizXbG+gtV/5vQodANZC22IDdmnsZ+HQsId6b3LEJ4ZmDWQVC8kUYEUJgXYXIk1ZWQkOUMiUdZbL8F7GaX+BaDDxvry63YuI6olNCPq7WWzeZTCbT9x49hUd4w36NFQAAAABJRU5ErkJggg==","orcid":"","institution":"Guangxi Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qingniao","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2023-07-28 18:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3214106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3214106/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":41032044,"identity":"ea927470-3767-441e-9912-a40f7fb7dc8b","added_by":"auto","created_at":"2023-08-03 17:47:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2907997,"visible":true,"origin":"","legend":"\u003cp\u003eErastin inhibits cell proliferation and induces ferroptosis in LNCaP and PC3 cells.\u003cstrong\u003eA\u003c/strong\u003e Phenotypic characterization of LNCaP and PC3 groups; “DMSO”, cells induced by DMSO as negative control; “Erastin”, cells induced by erastin as experiment group. \u003cstrong\u003eB\u003c/strong\u003e Expression of the SLC7A11 was suppressed by erastin treatment in LNCaP and PC3. Proliferative levels of LNCaP(\u003cstrong\u003eC\u003c/strong\u003e) and PC3(\u003cstrong\u003eD\u003c/strong\u003e) cells after erastin treatment. The relative MDA(\u003cstrong\u003eE\u003c/strong\u003e), Fe\u003csup\u003e2+\u003c/sup\u003e(\u003cstrong\u003eF\u003c/strong\u003e), GSH(\u003cstrong\u003eG\u003c/strong\u003e), and GSH/GSSG(\u003cstrong\u003eH\u003c/strong\u003e) levels of LNCaP and PC3 cells treated with erastin were detected. \u0026nbsp;Three independent experiments were performed with triplicate wells. Representative experiments and images are shown. Statistical significance was calculated using an \u003ca href=\"javascript:;\"\u003eindependent-sample\u003c/a\u003e t-test. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/7ee4ad89621957697291f145.png"},{"id":41032045,"identity":"da11bea9-5e90-4068-ab2b-2da0399d9e26","added_by":"auto","created_at":"2023-08-03 17:47:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2370832,"visible":true,"origin":"","legend":"\u003cp\u003eDEGs identification of LNCaP and PC3 cells after erastin exposure. \u003cstrong\u003eA\u003c/strong\u003e Principal component analysis (PCA) of RNA-seq. \u003cstrong\u003eB\u003c/strong\u003e Correlation analysis, the results were consistent with PCA. \u003cstrong\u003eC \u003c/strong\u003eVolcano plot analysis identifies the DEGs of erastin-induced LNCaP groups; red and green dots represent 447 and 495 upregulated and downregulated genes, respectively. \u003cstrong\u003eD\u003c/strong\u003eHeatmap of LNCaP groups after erastin exposure. \u003cstrong\u003eE\u003c/strong\u003e Volcano plot analysis identifies the DEGs of PC3 groups; red and green dots represent 3,704 and 1,921 upregulated and downregulated genes, respectively. \u003cstrong\u003eF\u003c/strong\u003eHeatmap for PC3 groups after erastin exposure. \u003cstrong\u003eG\u003c/strong\u003e Venn diagram of erastin-induced DEGs.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/c6b552c83ebc5de17c5851ef.png"},{"id":41031429,"identity":"b4426d9e-46c1-4039-a2c1-eb511becf86d","added_by":"auto","created_at":"2023-08-03 17:39:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1925391,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis for the DEGs of erastin-induced LNCaP and PC3 cells. \u003cstrong\u003eA\u003c/strong\u003e GO functional analysis. BP, CC, and MF represent biological processes, cellular components, and molecular functions, respectively. \u003cstrong\u003eB\u003c/strong\u003eKEGG pathway analysis. \u003cstrong\u003eC\u003c/strong\u003eRECTOME functional analysis.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/0f5bb443e08ea1d4da7e380a.png"},{"id":41031431,"identity":"f31dec78-7162-4a3f-b7f1-d6f2b118ab41","added_by":"auto","created_at":"2023-08-03 17:39:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6670361,"visible":true,"origin":"","legend":"\u003cp\u003eGSEA analysis based on the RNA-seq datasets. GSEA analysis shows enrichment of the response genes in both LNCaP group (\u003cstrong\u003eA\u003c/strong\u003e) and PC3 group (\u003cstrong\u003eB\u003c/strong\u003e) under erastin exposure. \u003cstrong\u003eC\u003c/strong\u003e GSEA analysis of enrichment of the response genes, which only appeared in erastin-induced PC3 group. NES, normalized enrichment score; nominal \u003cem\u003ep\u003c/em\u003e-value, and FDR, false discovery rate, and q-value were determined by the GSEA software.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/24a92be488711073299e10eb.png"},{"id":41031433,"identity":"6d792c61-05b2-4b1c-961c-74fee4342447","added_by":"auto","created_at":"2023-08-03 17:39:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6212400,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction and expression of TFs targeting hub genes and the clinical characteristics of the essential hub genes. \u003cstrong\u003eA\u003c/strong\u003e Top 10 hub DEGs of LNCaP and PC3 cells group with erastin treatment. \u003cstrong\u003eB\u003c/strong\u003e Interaction network of hub genes and their TFs. \u003cstrong\u003eC\u003c/strong\u003e The heatmap of the expression of the TFs was revealed by RNA-seq. The expression levels of \u003cem\u003eTMEFF2\u003c/em\u003e, \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eUNC5B \u003c/em\u003ein normal tissues and prostate cancer tissue from the HPA database (\u003cstrong\u003eD\u003c/strong\u003e) and GEPIA database (\u003cstrong\u003eE\u003c/strong\u003e). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/e0cc45df2ba98921f2ab495c.png"},{"id":41031430,"identity":"be727e6a-44f1-4868-b28f-2eff37a859eb","added_by":"auto","created_at":"2023-08-03 17:39:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3196441,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation analysis and validation of FRGs. \u003cstrong\u003eA\u003c/strong\u003e Correlation analysis of between four DEGs and \u003cem\u003eSLC7A11 \u003c/em\u003eand \u003cem\u003eGPX4\u003c/em\u003e. \u003cstrong\u003eB\u003c/strong\u003e Gene co-expression network analysis of \u003cem\u003eTMEFF2\u003c/em\u003e,\u003cem\u003e SLC7A11\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003eGPX4\u003c/em\u003e. \u003cstrong\u003eC\u003c/strong\u003e Expression of the \u003cem\u003eSLC7A11 \u003c/em\u003ewas suppressed by downregulating \u003cem\u003eTMEFF2 \u003c/em\u003ein LNCaP cells but not in PC3 cells. The relative Fe\u003csup\u003e2+\u003c/sup\u003e(\u003cstrong\u003eD\u003c/strong\u003e) and cell growth (\u003cstrong\u003eE\u003c/strong\u003e) of LNCaP and PC3 cells with downregulation of \u003cem\u003eTMEFF2 \u003c/em\u003ewere detected.Three independent experiments were performed with triplicate wells. Representative experiments and images are shown. Statistical significance was calculated using an \u003ca href=\"javascript:;\"\u003eindependent-sample\u003c/a\u003e t-test. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/fdaa1281c9bd42edf049e047.png"},{"id":41032519,"identity":"c6340b44-5c31-4503-8b9e-da5fd05cb805","added_by":"auto","created_at":"2023-08-03 18:03:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2926249,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/34bdb8ed-d7d2-4225-954d-1964a23758d9.pdf"},{"id":41031436,"identity":"0f4678bc-a961-4482-91bc-320a5f7ce4b0","added_by":"auto","created_at":"2023-08-03 17:39:11","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":5714730,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3214106/v1/affdd5dfce0dab63a21ba487.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a highly lethal cancer, prostate cancer (PCa) ranks second in the incidence of male malignant tumors worldwide [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e]. The androgen deprivation therapy is the clinical treatment for PCa, however, patients receiving this treatment may develop castration-resistant prostate cancer (CRPC) [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, it is necessary to screen and identify more targeted genes and develop a more effective therapy for PCa.\u003c/p\u003e\n\u003cp\u003eAs a unique mode of cell death, ferroptosis is widely divergent from other cell death including apoptosis, autophagy, and necrosis [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mechanically, a few essential factors or products of lipid peroxidation metabolism such as solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase (GPX4), glutathione (GSH), and malondialdehyde (MDA) are related with ferroptosis [\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e].In consideration of PCa with metastatic potential [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e] and cancer cells with metastatic and invasive ability were susceptible to ferroptosis [\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e], we hypothesized that targeting certain ferroptosis-related genes (FRGs) may be useful in treating PCa. Increasing number of researchers focused on the relationship between ferroptosis and PCa, Wo et al. identified some FRGs by analyzing the RNA-sequecing (RNA-seq) data from The Cancer Genome Atlas (TCGA) [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unfortunately, they only included the datasets from the clinical data of PCa patients and did not analyze the data of PCa cells because of the lack of RNA-seq results derived from ferroptotic PCa cells. However, focusing on ferroptosis in PCa cells will provide more comprehensive theoretical basis or treatment strategy for clinical treatment. So we had an idea to obtain the RNA-seq information to get clear about the potential mechanisms of ferroptotic PCa cells.\u003c/p\u003e\n\u003cp\u003eAs one of the most famous ferroptosis inducers, erastin is a perfect drug to trigger the ferroptotic progress of cells [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. There are few studies and limited information on the ipact of erastin acts on PCa cells. Ghoochani A et al. used cell and animal tests to improve various ferroptosis inducers, including erastin, and remarkably slowed down the PCa progress [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. Yang et al. claimed that erastin can downregulate the androgen receptor expression both in PCa cells and animal models [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. After analyzing the data of PCa patients in a public database, Wo et al. found and validated several FRGs [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Based on the studies above, we realize that erastin exerts inhibitory effects on PCa cells or tumor growth, and we asked two questions: what are the mechanisms of erastin acts on PCa cells? Which FRGs play an essential role in ferroptosis triggered by erastin? To get clear these questions, erastin was selected as a ferroptosis inducer and to construct the ferroptotic PCa cell model. Moreover, PC3 is an androgen-independent cell and LNCaP is an androgen-dependent cell [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e], so these two different and representative PCa cell lines were selected.\u003c/p\u003e\n\u003cp\u003eIn our research, cellular experiments were performed in erastin-induced LNCaP and PC3 cells, and the impact of erastin brings on ferroptotic levels in PCa cells was studied. RNA-seq was applied to the screen and identify the differentially expressed genes (DEGs) in PCa cells under erastin exposure. Ferroptosis-related pathways and FRGs that may play an essential role in erastin-induced PCa cell lines were identified. In addition, we tried to provide ferroptotic clues in terms of pathways, transcription factors (TFs), and modules for two different cells. Several FRGs with clinical significance were analyzed and validated. Hence, our study not only extends our understanding of erastin-affected PCa cells but also provides potential therapeutic targets and ideas for PCa therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCell lines and reagents\u003c/h2\u003e\n \u003cp\u003eLNCaP and PC3 cells (a gift from the Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences ) were cultured at 37\u0026deg;C in RPMI-1640 containing 15% and 10% fetal bovine serum (BI, Biological Industries, Co., Ltd., Israel), respectively. The ferroptosis inducer erastin was bought from MCE company (5.0 \u0026micro;M, HY-15763, USA). A virus expressing a short hairpin RNA (shRNA) targeting \u003cem\u003eTMEFF2\u003c/em\u003e was obtained from GenePharma Co., Ltd., for infection. The \u003cem\u003eTMEFF2\u003c/em\u003e targeted shRNA sequence was as follows: 5\u0026prime;-GUGUGAGCAUUCUAUCAAU-3\u0026prime; [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e]. Stably infected LNCaP and PC3 cells exhibiting \u003cem\u003eTMEFF2\u003c/em\u003e knockdown were obtained by selective screening with puromycin (1 \u0026micro;g/mL; Solarbio, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eWestern blot analysis\u003c/h2\u003e\n \u003cp\u003eFor the Western blot analysis, proteins isolated from the control cell samples (LNCaP and PC3) and from the experimental cell samples (LNCaP_5_0_era and PC3_5_0_era) were collected after 5.0 \u0026micro;M erastin incubated for 12 h. Each protein from these samples was separated in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. These membranes were probed with the primary antibody for SLC7A11(1:250, ab307601, Abcam, USA), or TMEFF2 (1:500, ab133562, Abcam, USA), \u0026beta;-tublin (1:2000, 10094-1-AP, ProteinTech, China). Subsequently, the membranes and the corresponding second antibody (ProteinTech, China) were incubated at room temperature for 1 h before being photographed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eCell survivability assay\u003c/h2\u003e\n \u003cp\u003eHerein, 2.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells were inoculated in 96-well plates. After 24 h, the 5.0 \u0026micro;M erastin was added to the culture medium. Each well was added with CCK-8 solution (Dojindo, Japan) at 24 h, 48 h, 72 h, and 96 h. The viability of the cell was determined by measuring the absorbance values of the 96-well plates at 450 nm using a quantitative microplate spectrophotometer (BioTek, Winooski, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eMalondialdehyde (MDA) assay\u003c/h2\u003e\n \u003cp\u003eMicro malondialdehyde assay kit (BC0025, Solarbio, China) was used to detect the cellular MDA levels. After 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells at the logarithmic growth phase, they were treated with 5 \u0026micro;M erastin for 12 h. These cells were respectively lysed and cracked with extracting solution by using the ultrasonic cell disruptor. The supernatants were obtained after a series of centrifugation and preservation under specific temperatures based on the instruction. Finally, the light absorption value of 532 nm and 600 nm wavelengths of the two groups\u0026rsquo; supernatants were detected, calculated, and analyzed.\u003c/p\u003e\n \u003ch2\u003eFerrous ions (\u003cstrong\u003eFe\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e) concentration assay\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe intracellular iron colorimetric assay kit (E1042, Applygen, China) was used to detect the level of Fe\u003csup\u003e2+\u003c/sup\u003e. After cells were treated with 5 \u0026micro;M erastin for 12 h, the cell lysis solution and the shaker were used for 2 h aiming at lysing a total of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells. Moreover, the diluted reagents were mixed, incubated, and centrifuged to acquire a standard solution then subsequently stained with 30 \u0026micro;l Fe\u003csup\u003e2+\u003c/sup\u003e detection reagent for 30 min. The absorption of the reagent at 550 nm wavelength was measured by the quant microplate spectrophotometer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eGlutathione (GSH) and oxidized glutathione (GSSG) assay\u003c/h2\u003e\n \u003cp\u003eHerein, the control and the experimental group cells after treatment with 5 \u0026micro;M erastin for 12 h, were prepared in a 6-well plate, and collected and homogenized with 150 \u0026micro;l protein removal reagent. Then, the samples were put in the liquid nitrogen and 37\u0026deg;C water bath twice in sequence to rupture the cells. Afterward, cell samples were placed on the ice, then centrifuged to obtain the supernatant. Subsequently, GSH and GSSG Assay Kits (Beyotime, China) were used to measure and analyze the GSH and GSSG levels, and the absorbance at 412 nm wavelength was measured.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSample collection and detection\u003c/h2\u003e\n \u003cp\u003eAfter washing with PBS, the cells were digested and obtained, which were then sent to Novogene, Co., Ltd., China. 1 \u0026micro;g RNA of cells was extracted by the illumina TruSeq RNA Sample Prep Kit (FC-122-1001, illumina, USA) to construct the sequencing libraries. The purified double-stranded cDNA was synthesized by Olygo (dT) reverse transcription, amplified by PCR, and screened with AMPureXP beads to acquire cDNA libraries. RNA-seq was performed on the Illumina NovaSeq 6000 to generate 150 bp paired-end readings, and approximately 8 G of reads per sample were obtained. Data is available in the Gene Expression Omnibus database (GEO submission number: GSE232034).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eData processing\u003c/h2\u003e\n \u003cp\u003eFirst, sequencing adapters and low-quality reads were trimmed and removed. Then, Homo_sapiens_Ensemble_94 was selected as the reference genome sequence for high-quality reads. The feature counts were applied to calculate the reads and the fragments per kilobase of the exon model per million mapped fragments (FPKM) for the length of genes. The Pearson correlational and the principal component analyses (PCA) were selected to analyze the sample correlation and the sample clustering, respectively. The R (Version 3.0.3) ggplot2 package was used for visualization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003eDEGs analysis\u003c/h2\u003e\n \u003cp\u003eThe DEGs analysis was performed within the experimental group (LNCaP_5_0_era and PC3_5_0_era) and the control group (LNCaP and PC3) using DESeq2 software (1.20.0), and each sample group was performed in triplicate. DESeq2 offers statistical procedures to determine the DEGs by the negative binomial distribution model [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. The significant differential expression mean \u003cem\u003ep\u003c/em\u003e-value (padj)\u0026thinsp;\u0026le;\u0026thinsp;0.05 \u0026amp; |log2(foldchange)| \u0026ge; 2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eFunctional enrichment analysis of erastin-induced genes\u003c/h2\u003e\n \u003cp\u003eFor the potential functions and signal pathways of erastin-induced gene-expression changes in PCa cells to be further explored, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome analyses were performed to explore and study the different overlapping DEGs of the LNCaP group (LNCaP_5_0_era and LNCaP) and the PC3 group (PC3_5_0_era and PC3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eScreening of hub gene and module analysis\u003c/h2\u003e\n \u003cp\u003eA protein-protein interaction (PPI) network obtained through different overlapping DEGs of the LNCaP group (LNCaP_5_0_era and LNCaP) and the PC3 group (PC3_5_0_era and PC3) were submitted to the STRING database [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Cytoscape (version 3.9.1) [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e] was applied to analyze the PPI relationships, and a total score of \u0026gt;\u0026thinsp;0.9 was selected as the cutoff criterion. In addition, the CytoHubba obtained the top 10 hub genes during the screening process [\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]. The Cytoscape plug-in MCODE was employed to detect the molecular complex and acquire the module of DEGs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eGene set enrichment analysis (GSEA)\u003c/h2\u003e\n \u003cp\u003eThe GSEA enrichment analysis [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e] was carried out to analyze the GO datasets of the LNCaP group (LNCaP_5_0_era vs LNCaP) and the PC3 group (PC3_5_0_era vs PC3) separately. The significant GSEA results with the stringed threshold of nominal \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, false discovery rate (FDR) q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.25, and |normalized enrichment score (NES)| \u0026gt;1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003ePrediction of hub genes\u0026rsquo; TFs\u003c/h2\u003e\n \u003cp\u003eA total of 10 hub genes were submitted to the NetworkAnalyst platform to predict their TFs. The putative TFs associated with hub genes have been identified and sorted through the mean rank score.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eValidation for FRGs\u003c/h2\u003e\n \u003cp\u003eThe Human Protein Atlas database was used to determine the gene expression patterns of interested hubs in tissues. The GEPIA platform was selected to draw a box map of the hub gene with illustrated the expression patterns of tissues [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, this online tool provided the correlation analysis for the hub gene. The online database, GeneMANIA, created an interactive network to predict the tangible relationship between the hub genes [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. Western blot, Fe\u003csup\u003e2+\u003c/sup\u003e concentration assay, and CCK8 were performed to assess the correlation between the hub gene and ferroptosis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eErastin accelerates ferroptosis of PCa cells\u003c/h2\u003e\n \u003cp\u003eLNCaP and PC3 cell lines added with the erastin were phenotypically different in the two cell groups (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eA). Erastin repressed the expression of the ferroptosis marker protein SLC7A11 in both PCa cell lines (Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eB). Thus, we carried out the cell survivability, MDA, Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, and GSSG assay of erastin-induced PCa cells. After 48 h, erastin significantly inhibited the proliferation of LNCaP and PC3 cells in the CCK8 assay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eC\u0026ndash;D). Results showed that MDA levels under erastin-induced conditions were notably increased in PC3 cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eE). On the contrary, erastin did not trigger a remarkable difference in MDA levels in LNCaP cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eE). Fe\u003csup\u003e2+\u003c/sup\u003e content, one of the key features of ferroptosis, was notably increased with erastin treatment in these two cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eF). Given that GSH/GSSG regulates cellular redox homeostasis, the level of GSH and GSSG were measured. The results showed that GSH levels in both cell lines were decreased following erastin treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eG). Meanwhile, the GSH/GSSG levels were also downregulated in these cell samples (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan\u003e1\u003c/span\u003eH).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eSummary of RNA sequencing data\u003c/h2\u003e\n \u003cp\u003eRNA sequencing was used to analyze the transcriptome profiling of PCa cells and erastin-induced PCa cells to investigate the molecular mechanisms of ferroptosis inducer erastin affect PCa cells and find more FRGs. Using control cells and erastin-induced PCa cells, each of the four groups had three biological replicates, and 12 sequencing samples were prepared. Three replicates in four groups were consistent with the principal component analysis (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eA). Based on correlation analysis results (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eB), the correlation coefficient of the samples was between 0.8 and 1, which showed good biological repeatability in these sequencing samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eErastin-induced DEGs analysis\u003c/h2\u003e\n \u003cp\u003eIn the RNA-seq assay with padj\u0026thinsp;\u0026le;\u0026thinsp;0.05\u0026amp;|log2(foldchange)|\u0026ge;2 as screening conditions, LNCaP_5_0_Era identified a total of 942 DEGs as compared with LNCaP, including 447 upregulated genes and 495 downregulated genes (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eC\u0026ndash;D). Then, a total of 5,625 DEGs were screened and recognized between PC3_5_0_Era and PC3, of which 3,704 were upregulated and 1,921 were downregulated (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eE\u0026ndash;F). A total of 295 overlapping DEGs in two compared groups were screened and recognized via the Venn diagram (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003eG).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eFunctional enrichment in erastin-induced genes\u003c/h2\u003e\n \u003cp\u003eGO, KEGG, and REACTOME functional enrichment analyses were performed to analyze 295 overlapping DEGs to further explore the potential biological behavior of erastin-induced changes in PCa cells. As exhibited by the GO analysis results, the erastin-induced DEGs were primarily related to DNA replication, DNA-dependent DNA replication, and DNA replication initiation for biological processes (BPs), condensed chromosome, chromosomal region, condensed chromosome, centromeric region for molecular functions (MFs), and catalytic activity acting on DNA, DNA helicase activity, and helicase activity for cellular components (CCs) (Fig. \u003cspan\u003e3\u003c/span\u003eA). In contrast, the KEGG analysis indicated that these DEGs mapped to DNA replication, cell cycle, and homologous recombination pathways (Fig. \u003cspan\u003e3\u003c/span\u003eB). Furthermore, REACTOME analysis revealed that these DEGs were associated with DNA strand elongation, cell-cycle checkpoints, activation of the pre-replicative complex, and so on (Fig. \u003cspan\u003e3\u003c/span\u003eC).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003ePathways and module analysis of two PCa cells\u003c/h2\u003e\n \u003cp\u003eAn MCODE plug-in was carried out to construct modules representing the crucial clusters to access which related pathways brought more weight into ferroptotic process of PCa cells (Supplementary Fig.\u0026nbsp;1). Among the LNCaP group, the DEGs in module 1 were selected to conduct the next analysis and found they involved with several prominent signaling pathways (Supplementary Fig.\u0026nbsp;1A\u0026ndash;B). For the PC3 group, enriched pathways including nephron morphogenesis, positive regulation of blood vessel endothelial cell migration, positive regulation of bone mineralization, and regulation of steroid metabolic process were detected in module 1 of PC3 DEGs (Supplementary Fig.\u0026nbsp;1C\u0026ndash;D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eGSEA analysis of erastin-induced genes\u003c/h2\u003e\n \u003cp\u003eHerein, we have then performed GSEA analysis between erastin treatment and control groups across the four cell lines to reveal more gene set enrichment information that involves the erastin-induced changes in PCa cells. We found that most of those pathways in GSEA were also found in the GO, KEGG, and Reactome functional enrichment analyses, which supports and validates the previous results. However, it was not unexpected to find that erastin-induced PCa DEGs were involved in the regulation of cell death, cellular response to reactive oxygen species, fatty acid biosynthetic process, and activation of MAPK activity pathway, intrinsic apoptotic signaling pathway in response to DNA damage by P53 class mediator and cellular amino acid metabolic process in LNCaP (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eA) and PC3 cells (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eB). Moreover, we further focused on several pathways expected to be only induced in the PC3 group, including regulation of I-kappab kinase NF-kappab signaling, regulation of JNK cascade, regulation of ERK1 and ERK2 cascade, negative regulation of ERBB signaling pathway, negative regulation of Ras protein signal transduction, and regulation of JAK-STAT cascade (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003eC).\u003c/p\u003e\n \u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003eErastin-induced hub genes and their TFs\u003c/h2\u003e\n \u003cp\u003eCytoHubba plug-in was selected to calculate the hub genes of 295 overlapping DEGs to find more potential and pivotal genes related to ferroptosis in PCa cells. Of these, 10 top hub genes (including \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eIL1B\u003c/em\u003e, \u003cem\u003eMET\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, \u003cem\u003ePLXNA4\u003c/em\u003e, \u003cem\u003eGAD1\u003c/em\u003e, \u003cem\u003eUNC5B\u003c/em\u003e, \u003cem\u003eSLC7A5\u003c/em\u003e, \u003cem\u003eDAPK1\u003c/em\u003e, and \u003cem\u003eTMEFF2\u003c/em\u003e) were identified in order of degrees (Fig. \u003cspan\u003e5\u003c/span\u003eA). Details and descriptions of 10 hub genes are summarized in Table \u003cspan\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eTop 10 hub genes of erastin-induced LNCaP and PC3 cells\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEnsemble ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSymbol\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elog2(FC) In LNCaP group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFDR Value In LNCaP group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003elog2(FC) In PC3 group\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFDR Value In PC3 group\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000120885\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCLU\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.227676954\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.66194E-14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.328754927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.10046E-26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000125538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIL1B\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.659181706\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023399869\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.354730765\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.37023E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000105976\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMET\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.178704253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.57265E-06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.716782674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.70167E-33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000021645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNRXN3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.219194193\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.12366E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.442352118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.87491E-18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000221866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePLXNA4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.383825988\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.60173E-05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.0250723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.006594555\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000128683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eGAD1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.674364639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.49826E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.298554233\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7675E-116\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000107731\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eUNC5B\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.073123293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.61178E-09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.989199096\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.58257E-30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000103257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eSLC7A5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.079560779\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.34346E-12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.392270174\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.17417E-23\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000196730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDAPK1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.188978999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.02751E-14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-5.892959015\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.06199E-77\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eENSG00000144339\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTMEFF2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.727660511\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.65524E-66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.052271557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.96873E-07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eGiven that TFs had a critical influence on gene expression levels, 10 top hub genes were uploaded to the NetworkAnalyst database for TFs prediction. The regulatory network of top TFs (including \u003cem\u003eSP9\u003c/em\u003e, \u003cem\u003eDLX2\u003c/em\u003e, \u003cem\u003eARX\u003c/em\u003e, \u003cem\u003ePEG3\u003c/em\u003e, \u003cem\u003eCSRNP3\u003c/em\u003e, \u003cem\u003eZNF697\u003c/em\u003e, \u003cem\u003eINSM2\u003c/em\u003e, \u003cem\u003eSTAT3\u003c/em\u003e, \u003cem\u003eNR2F1\u003c/em\u003e, \u003cem\u003eFOXL1\u003c/em\u003e, \u003cem\u003eE2F1\u003c/em\u003e, and \u003cem\u003eNFIC\u003c/em\u003e) of the hub genes was obtained and presented (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eB). Moreover, the expression levels of these TFs were shown in Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eC. Among them, \u003cem\u003ePEG3\u003c/em\u003e, \u003cem\u003eCSRNP3\u003c/em\u003e, \u003cem\u003eZNF697\u003c/em\u003e, \u003cem\u003eNR2F1\u003c/em\u003e, \u003cem\u003eDLX2\u003c/em\u003e, and \u003cem\u003eINSM2\u003c/em\u003e were highly expressed, whereas \u003cem\u003eSP9\u003c/em\u003e, \u003cem\u003eARX\u003c/em\u003e, \u003cem\u003eE2F1\u003c/em\u003e, \u003cem\u003eFOXL1\u003c/em\u003e, \u003cem\u003eSTAT3\u003c/em\u003e, and \u003cem\u003eNFIC\u003c/em\u003e were lowly expressed in PCa cells after erastin exposure.\u003c/p\u003e\n \u003cp\u003eTherefore, we analyzed their expression patterns in HPA and GEPIA database to investigate whether these hub genes were clinically associated with PCa. In the HPA database, the expression of \u003cem\u003eTMEFF2\u003c/em\u003e in PCa tissue is higher than in normal tissue and the expression levels of \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e were lower in PCa tissue by immunohistochemistry (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eD). Consistently, \u003cem\u003eTMEFF2\u003c/em\u003e mRNA levels were higher in PCa tissue (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e were downregulated (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in PCa tissue in the GEPIA database (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003eE).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\"\u003e\n \u003ch2\u003ePrediction and validation Of FRGs\u003c/h2\u003e\n \u003cp\u003eThe relationships between hub genes\u0026rsquo; expression levels and ferroptosis marker \u003cem\u003eSLC7A11\u003c/em\u003e and \u003cem\u003eGPX4\u003c/em\u003e in the GEPIA database were respectively analyzed to investigate whether or not these 4 hub genes were potentially involved with ferroptosis. As expected, the expression patterns of \u003cem\u003eTMEFF2\u003c/em\u003e, \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e were positively correlated with \u003cem\u003eSLC7A11\u003c/em\u003e and \u003cem\u003eGPX4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eA). The gene interactive networks were established to clarify the possible direct relationship between hub genes and ferroptosis markers and identify their potential associations. \u003cem\u003eTMEFF2\u003c/em\u003e, \u003cem\u003eSLC7A11\u003c/em\u003e, and \u003cem\u003eGPX4\u003c/em\u003e showed the complex PPI network with the physical interactions, co-expression, prediction, co-localization, genetic interactions, pathway, and shared protein domains of 77.64%, 8.01%, 5.37%, 3.63%, 2.87%, 1.88%, and 0.60%, respectively (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eB). Among these networks, there were direct genetic interactions within \u003cem\u003eTMEFF2\u003c/em\u003e and \u003cem\u003eSLC7A11\u003c/em\u003e. Moreover, \u003cem\u003eTMEFF2\u003c/em\u003e showed direct genetic interactions with \u003cem\u003eALOX5\u003c/em\u003e, then \u003cem\u003eALOX5\u003c/em\u003e showed physical interactions with \u003cem\u003eGPX4\u003c/em\u003e. However, \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e failed to display such a close relationship with \u003cem\u003eGPX4\u003c/em\u003e as compared with \u003cem\u003eTMEFF2\u003c/em\u003e (data not shown). Therefore, \u003cem\u003eTMEFF\u003c/em\u003e was selected as the interesting ferroptosis-related gene for further experiments.\u003c/p\u003e\n \u003cp\u003eIn addition, we constructed LNCaP and PC3 cells with the downregulation of \u003cem\u003eTMEFF2\u003c/em\u003e to test whether or not \u003cem\u003eTMEFF2\u003c/em\u003e can be an FRG in the experiment, then detected their cell survivability and Fe\u003csup\u003e2+\u003c/sup\u003e concentration. LNCaP cells with knockdown of \u003cem\u003eTMEFF2\u003c/em\u003e exhibited lower expression of \u003cem\u003eSLC7A11\u003c/em\u003e, while down-regulation of \u003cem\u003eTMEFF2\u003c/em\u003e did not significantly affect the expression level of \u003cem\u003eSLC7A11\u003c/em\u003e in PC3 cells (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eC). After \u003cem\u003eTMEFF2\u003c/em\u003e was knocked down, the Fe\u003csup\u003e2+\u003c/sup\u003e in LNCaP cells was remarkably increased (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eD). By contrast, there was no significant difference between PC3 cells with erastin treatment and the control group (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eD). After 48 h, the downregulation of \u003cem\u003eTMEFF2\u003c/em\u003e only significantly reduced the proliferative ability of LNCaP cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003eE).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study discussed the role of ferroptosis inducer erastin in two PCa cells, LNCaP and PC3. Unsurprisingly, we found that erastin exposure caused an impact on LNCaP and PC3 cells in terms of ferroptosis marker, cell survivability, and the concentration of MDA, Fe\u003csup\u003e2+\u003c/sup\u003e, GSH, and GSSG. In addition, we extracted DEGs of both two PCa cells at the transcriptional level. Subsequently, we identified various pathways, TFs and modules in different PCa cells. Moreover, we identified and validated several erastin-affected FRGs which were potentially meaningful in PCa treatment through a series of bioinformatics analyses and experiments.\u003c/p\u003e \u003cp\u003eFirst, we focused on the identification of FRGs in PCa cells after erastin exposure. From RNA-Seq results, 10 FRGs were identified as the hub genes via overlapping 295 DEGs. \u003cem\u003eTMEFF2\u003c/em\u003e, a type I transmembrane protein with an EGF-like and two follistatin motifs 2 proteins, was selectively expressed in the brain and prostate [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Based on our analysis, \u003cem\u003eTMEFF2\u003c/em\u003e was a co-expressed gene and correlated with \u003cem\u003eGPX4\u003c/em\u003e and \u003cem\u003eSLC7A11\u003c/em\u003e, as determined by GeneMANIA and GEPIA. Despite the absence of direct reports on the connection between \u003cem\u003eTMEFF2\u003c/em\u003e and ferroptosis, a study reported that oxidative stress downregulate the expression of \u003cem\u003eTMEFF2\u003c/em\u003e in PCa [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, \u003cem\u003eTMEFF2\u003c/em\u003e can influence the proliferative activity of PCa cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which was consistent with our results. In addition, similar to our results, several researchers reported that the alternative expression levels of \u003cem\u003eTMEFF2\u003c/em\u003e changed the disease stage in PCa tissue of patients and mouse model [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, the above reports supported our view that the \u003cem\u003eTMEFF2\u003c/em\u003e is related to ferroptosis and can be the FRG of PCa. It was worth mentioning that the ferroptotic level was significantly affected by knockdown of \u003cem\u003eTMEFF2\u003c/em\u003e in LNCaP cells, but not in PC3 cells. Considering that LNCaP is an androgen-dependent cell and PC3 is an androgen-independent cell[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, \u003cem\u003eTMEFF2\u003c/em\u003e has been identified as an androgen-related gene [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. And there was a report claimed that knock-down of \u003cem\u003eTMEFF2\u003c/em\u003e could suppressed the androgen-response of LNCaP cells [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, we speculated that \u003cem\u003eTMEFF2\u003c/em\u003e promotes ferroptosis via androgen, which means \u003cem\u003eTMEFF2\u003c/em\u003e only bring an influence on ferroptosis of androgen-sensitive LNCaP cells. Then if \u003cem\u003eTMEFF2\u003c/em\u003e were used as a ferroptotic treatment target in the future, it would be more suitable for PCa patients than CRPC patients.\u003c/p\u003e \u003cp\u003eOur results also indicated FRGs such as \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e showed different expression levels between PCa and normal tissue, and their expression patterns were positively related to \u003cem\u003eGPX4\u003c/em\u003e and \u003cem\u003eSLC7A11\u003c/em\u003e. Meanwhile, \u003cem\u003eCLU\u003c/em\u003e caused ATP degradation which resulted in peroxidation in cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], while the upregulation of \u003cem\u003eCLU\u003c/em\u003e inhibited PCa cell death [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, \u003cem\u003eNRXN3\u003c/em\u003e is a gene associated with obesity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and we speculated that \u003cem\u003eNRXN3\u003c/em\u003e takes part in the metabolism process of lipids. Moreover, \u003cem\u003eUNC5B\u003c/em\u003e is a crucial regulator of ferroptosis in osteosarcoma cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, these four hub DEGs identified in LNCaP and PC3 cells under erastin exposure exhibit to play an essential role in ferroptotic process of PCa cells, and maybe the potential treatment targets of PCa.\u003c/p\u003e \u003cp\u003eSubsequently, we obtained a TFs network for the 10 DEGs in PCa cells after erastin treatment. In the top TFs, \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eE2F1\u003c/em\u003e were differently expressed in erastin-induced PCa cells, and accumulating evidence indicated that they were related to ferroptosis. \u003cem\u003eSTAT3\u003c/em\u003e, signal transduction and activators of transcription 3, the ferroptosis was stimulated in PCa cells with knockdown of \u003cem\u003eSTAT3\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Mechanistically, \u003cem\u003eSTAT3\u003c/em\u003e binds to \u003cem\u003eGPX4\u003c/em\u003e and regulates its expression in pancreatic cancer cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eE2F1\u003c/em\u003e, E2F transcription factor 1, can be one of the ferroptosis-related gene prognostic indexes (FRGPI) to predict disease-free survival (DFS) for PCa patients\u0026rsquo; radical prostatectomy, PCa patients with high FRGPI had worse DFS than patients with low FRGPI [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Hence, we speculated that these TFs associated with ferroptosis also participate to the regulation of hub genes\u0026rsquo; expression to affect the ferroptosis in PCa cells.\u003c/p\u003e \u003cp\u003eIn addition to hub DEGs and their TFs, several pathways play an essential role in ferroptotic cells. Based on the GO functional analysis, the DEGs were primarily enriched in DNA replication, Chromosome segregation, and DNA helicase activity pathway. These three pathways were closely related to DNA replication, meanwhile, DNA replication is the crucial link to cell growth. Our findings indicated that erastin remarkably reduced the proliferative activity of PCa cells. In addition, Fe\u003csup\u003e2+\u003c/sup\u003e is one of the essential factors of DNA replication, which is vital for cell survivability [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Consequently, we believed that erastin primarily transforms PCa cell proliferation by altering DNA replication. Except for DNA replication, KEGG analysis revealed that these DEGs were also associated with steroid hormone biosynthesis, MARK signal pathway, and P53 signal pathway. Steroid hormone biosynthesis is in charge of ferroptosis induction in adrenocortical carcinoma cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Considering our TFs network, it can be speculated that erastin affects MAPK/ERK to regulate \u003cem\u003eSTAT3\u003c/em\u003e, then \u003cem\u003eSTAT3\u003c/em\u003e is a TF to change the expression patterns of hub genes \u003cem\u003eCLU\u003c/em\u003e in PCa cells. Furthermore, \u003cem\u003eSTAT3\u003c/em\u003e participate into P53/SLC7A11 pathway in osteosarcoma cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our results, \u003cem\u003eSLC7A11\u003c/em\u003e was also downregulated by erastin exposure. We speculated that \u003cem\u003eSTAT3\u003c/em\u003e not only affects the expression level of \u003cem\u003eCLU\u003c/em\u003e but also changes the P53 signal pathway to alter the expression of \u003cem\u003eSLC7A11\u003c/em\u003e. Meanwhile, REACTOME enrichment analysis suggested that DEGs were enriched in cell cycle checkpoints and activation of E2F1 target genes at the G1/S pathway. Experimental evidence showed that erastin caused a change in the proportion of PCa cells in certain phases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Based on our analysis, \u003cem\u003eE2F1\u003c/em\u003e may be the TFs of hub genes \u003cem\u003eTMEFF2\u003c/em\u003e, thus, \u003cem\u003eE2F1\u003c/em\u003e could affect the transcript level of \u003cem\u003eTMEFF2\u003c/em\u003e, which may involve the cell cycle of PCa cells. Therefore, erastin may affect the expression patterns of hub genes and their TFs through regulate various signal pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, to transform the cell growth and cell cycle of PCa cells.\u003c/p\u003e \u003cp\u003eInterestingly, we found the erastin-induced PC3 group owned more DEGs than erastin-induced LNCaP groups. In our module analysis, the top 1 module of the PC3 group had more direct ferroptosis-related pathways, such as positive regulation of bone mineralization and regulation of steroid metabolic process, than the LNCaP groups. Therefore, as to these two PCa cell lines, we suggested that PC3 cells are much more sensitive to ferroptosis inducer erastin than LNCaP cells. Subsequently, we wanted to know why CRPC cell lines PC3 exhibited more susceptibility to erastin. GSEA analysis was applied to study various cellular signal pathways, which other functions enrichment did not identify. As expected, several cell death-related pathways were enriched, and the PC3 group displayed more and several unique signaling pathways in GSEA, such as negative regulation of ERBB signaling pathway, negative regulation of Ras protein signal transduction, and regulation of JAK-STAT cascade. These signaling pathways are all associated with ferroptosis in various cells. A complex suppressed the ERBB signal pathway to inhibit ferroptosis \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Moreover, the RAS/MAPK pathway may regulated oxidative stress and the related ferroptosis pathways in glioblastoma [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, IFNγ enhanced the JAK-STAT signal pathway to activate ferroptosis in hepatocellular carcinoma cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Unfortunately, there were lack of direct reports about the relationship between these signal pathways and ferroptosis of PCa. Hence, erastin transformed more crucial signal pathways, which led to PC3 being more sensitive to erastin. Moreover, considering that PC3 is an androgen-independent cell and LNCaP is an androgen-sensitive cell [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], we speculated that CRPC patients may be more susceptible to targeting gene therapy related to ferroptosis than PCa patients.\u003c/p\u003e \u003cp\u003eThis current study has some limitations. Firstly, two PCa cell lines, LNCaP and PC3, were selected to carry out the experiment. If we included more PCa cells, the results will be more comprehensive. Secondly, although we verified the FRGs in cell experiments and in public databases, our further study will be confirmed in animal experiments and clinical practices.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study verified that erastin successfully brought about a significant effect on the proliferation and ferroptotic index of LNCaP and PC3 cells. In addition, we carried out RNA-seq and annotated 295 overlapping DEGs after erastin treatment in these two cells. Functional enrichment analysis showed that erastin may be act on PCa cells by participating in a variety of pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, et al. Our results also indicated several FRGs such as \u003cem\u003eTMEFF2\u003c/em\u003e, \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e showed different expression levels between PCa and normal tissue. The potential TFs, \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eE2F1\u003c/em\u003e, may take part in regulating the ferroptotic process of these FRGs. We also found downregulation of \u003cem\u003eTMEFF2\u003c/em\u003e only promote ferroptosis in androgen-sensitive LNCaP cells but not in androgen-independent PC3 cells. Our finding elucidate potential molecular mechanisms in the subsequent ferroptotic studies of PCa cells, and suggest the novel therapeutic targets and strategy for PCa.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e FW and FH performed data analysis and drafted the manuscript. FW, NJ, BL and QZ performed the experiments. JFS, YY, WL and TY prepared figures and tables. SZ and QZ secured research funding supports. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003eThis work was supported by grants from the National Natural Science Foundation of China (No. 32260164), Natural Science Foundation of Guangxi Zhuang Autonomous Region (No.2020GXNSFBA297150, No.2023GXNSFAA026080).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u003c/strong\u003eThe original contributions presented in the study are publicly available. This data can be found here: GSE232034 datasets downloaded from GEO database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eAll authors \u0026nbsp; have no relevant fnancial or nonfnancial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKonoshenko MY, Bryzgunova OE, Laktionov PP (2021) miRNAs and androgen deprivation therapy for prostate cancer. Biochim et Biophys acta Reviews cancer 1876(2):188625. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbcan.2021.188625\u003c/span\u003e\u003cspan address=\"10.1016/j.bbcan.2021.188625\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGe R, Wang Z, Montironi R, Jiang Z, Cheng M, Santoni M et al (2020) Epigenetic modulations and lineage plasticity in advanced prostate cancer. Annals of oncology: official journal of the European Society for Medical Oncology 31(4):470\u0026ndash;479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.annonc.2020.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.annonc.2020.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE et al (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149(5):1060\u0026ndash;1072. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cell.2012.03.042\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2012.03.042\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Y, Hou W, Song X, Yu Y, Huang J, Sun X et al (2016) Ferroptosis: process and function. Cell Death Differ 23(3):369\u0026ndash;379. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/cdd.2015.158\u003c/span\u003e\u003cspan address=\"10.1038/cdd.2015.158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson RP, Ratnacaram CK, Kumar L, Jose J (2022) Combinatorial approaches of nanotherapeutics for inflammatory pathway targeted therapy of prostate cancer. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy. 64:100865. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drup.2022.100865\u003c/span\u003e\u003cspan address=\"10.1016/j.drup.2022.100865\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYee PP, Wei Y, Kim SY, Lu T, Chih SY, Lawson C et al (2020) Neutrophil-induced ferroptosis promotes tumor necrosis in glioblastoma progression. Nat Commun 11(1):5424. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-020-19193-y\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-19193-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWo Q, Liu Z, Hu L (2022) Identification of Ferroptosis-Associated Genes in Prostate Cancer by Bioinformatics Analysis. Front Genet 13:852565. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fgene.2022.852565\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.852565\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Deng T, Liu R, Ning T, Yang H, Liu D et al (2020) CAF secreted miR-522 suppresses ferroptosis and promotes acquired chemo-resistance in gastric cancer. Mol Cancer 19(1):43. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12943-020-01168-8\u003c/span\u003e\u003cspan address=\"10.1186/s12943-020-01168-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhoochani A, Hsu EC, Aslan M, Rice MA, Nguyen HM, Brooks JD et al (2021) Ferroptosis Inducers Are a Novel Therapeutic Approach for Advanced Prostate Cancer. Cancer Res 81(6):1583\u0026ndash;1594. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.Can-20-3477\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.Can-20-3477\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Liu T, Hu C, Xia H, Liu W, Chen J et al (2021) Ferroptosis inducer erastin downregulates androgen receptor and its splice variants in castration\u0026ndash;resistant prostate cancer. Oncol Rep 45(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/or.2021.7976\u003c/span\u003e\u003cspan address=\"10.3892/or.2021.7976\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYou Z, Dong Y, Kong X, Zhang Y, Vessella RL, Melamed J (2007) Differential expression of IL-17RC isoforms in androgen-dependent and androgen-independent prostate cancers. Neoplasia (New York NY) 9(6):464\u0026ndash;470. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1593/neo.07109\u003c/span\u003e\u003cspan address=\"10.1593/neo.07109\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie S, Zhang Y, Peng T, Guo J, Cao Y, Guo J et al (2021) TMEFF2 promoter hypermethylation is an unfavorable prognostic marker in gliomas. Cancer Cell Int 21(1):148. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12935-021-01818-x\u003c/span\u003e\u003cspan address=\"10.1186/s12935-021-01818-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13059-014-0550-8\u003c/span\u003e\u003cspan address=\"10.1186/s13059-014-0550-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P et al (2011) The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39(Database issue):D561\u0026ndash;D568. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkq973\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkq973\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmoot ME, Ono K, Ruscheinski J, Wang PL, Ideker T, Cytoscape (2011) 2.8: new features for data integration and network visualization. Bioinformatics (Oxford, England). ;27(3):431-2. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/btq675\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/btq675\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChin CH, Chen SH, Wu HH, Ho CW, Ko MT, Lin CY (2014) cytoHubba: identifying hub objects and sub-networks from complex interactome. BMC Syst Biol 8(Suppl 4):S11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1752-0509-8-s4-s11\u003c/span\u003e\u003cspan address=\"10.1186/1752-0509-8-s4-s11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowers RK, Goodspeed A, Pielke-Lombardo H, Tan AC, Costello JC (2018) GSEA-InContext: identifying novel and common patterns in expression experiments. Bioinformatics 34(13):i555\u0026ndash;i64. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/bioinformatics/bty271\u003c/span\u003e\u003cspan address=\"10.1093/bioinformatics/bty271\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Z, Li C, Kang B, Gao G, Li C, Zhang Z (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45(W1):W98\u0026ndash;W102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkx247\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkx247\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res W214\u0026ndash;W220 38(Web Server issue). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/nar/gkq537\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkq537\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasood M, Grimm S, El-Bahrawy M, Yague E (2020) TMEFF2: A Transmembrane Proteoglycan with Multifaceted Actions in Cancer and Disease. Cancers (Basel) 12(12). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers12123862\u003c/span\u003e\u003cspan address=\"10.3390/cancers12123862\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGawel-Beben K, Ali N, Ellis V, Velasco G, Poghosyan Z, Ager A et al (2018) TMEFF2 shedding is regulated by oxidative stress and mediated by ADAMs and transmembrane serine proteases implicated in prostate cancer. Cell Biol Int 42(3):273\u0026ndash;280. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cbin.10832\u003c/span\u003e\u003cspan address=\"10.1002/cbin.10832\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGery S, Sawyers CL, Agus DB, Said JW, Koeffler HP (2002) TMEFF2 is an androgen-regulated gene exhibiting antiproliferative effects in prostate cancer cells. Oncogene 21(31):4739\u0026ndash;4746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/sj.onc.1205142\u003c/span\u003e\u003cspan address=\"10.1038/sj.onc.1205142\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAfar DE, Bhaskar V, Ibsen E, Breinberg D, Henshall SM, Kench JG et al (2004) Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer. Mol Cancer Ther 3(8):921\u0026ndash;932\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlynne-Jones E, Harper ME, Seery LT, James R, Anglin I, Morgan HE et al (2001) TENB2, a proteoglycan identified in prostate cancer that is associated with disease progression and androgen independence. Int J Cancer 94(2):178\u0026ndash;184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijc.1450\u003c/span\u003e\u003cspan address=\"10.1002/ijc.1450\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorbin JM, Overcash RF, Wren JD, Coburn A, Tipton GJ, Ezzell JA et al (2016) Analysis of TMEFF2 allografts and transgenic mouse models reveals roles in prostate regeneration and cancer. Prostate 76(1):97\u0026ndash;113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/pros.23103\u003c/span\u003e\u003cspan address=\"10.1002/pros.23103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorbin JM, Georgescu C, Wren JD, Xu C, Asch AS, Ruiz-Echevarr\u0026iacute;a MJ (2021) Seed-mediated RNA interference of androgen signaling and survival networks induces cell death in prostate cancer cells. Mol therapy Nucleic acids 24:337\u0026ndash;351. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.omtn.2021.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.omtn.2021.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMateria S, Cater MA, Klomp LW, Mercer JF, La Fontaine S (2011) Clusterin (apolipoprotein J), a molecular chaperone that facilitates degradation of the copper-ATPases ATP7A and ATP7B. J Biol Chem 286(12):10073\u0026ndash;10083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M110.190546\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M110.190546\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Lv X, Chen L, Liu Y (2022) The role and function of CLU in cancer biology and therapy. Clin Exp Med. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10238-022-00885-2\u003c/span\u003e\u003cspan address=\"10.1007/s10238-022-00885-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrats-Puig A, Grau-Cabrera P, Riera-Perez E, Cortes-Marina R, Fortea E, Soriano-Rodriguez P et al (2013) Variations in the obesity genes FTO, TMEM18 and NRXN3 influence the vulnerability of children to weight gain induced by short sleep duration. Int J Obes (Lond) 37(2):182\u0026ndash;187. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ijo.2012.27\u003c/span\u003e\u003cspan address=\"10.1038/ijo.2012.27\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng D, Xia K, Wei Z, Wei Z, Guo W (2022) Identification of a novel gene signature with regard to ferroptosis, prognosis prediction, and immune microenvironment in osteosarcoma. Front Genet 13:944978. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fgene.2022.944978\u003c/span\u003e\u003cspan address=\"10.3389/fgene.2022.944978\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJi J, Li H, Wang W, Yuan B, Shen T (2022) ARPC1A is regulated by STAT3 to inhibit ferroptosis and promote prostate cancer progression. Hum Cell 35(5):1591\u0026ndash;1601. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s13577-022-00754-w\u003c/span\u003e\u003cspan address=\"10.1007/s13577-022-00754-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Gong M, Zhang W, Mo J, Zhang S, Zhu Z et al (2022) Thiostrepton induces ferroptosis in pancreatic cancer cells through STAT3/GPX4 signalling. Cell Death Dis 13(7):630. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-022-05082-3\u003c/span\u003e\u003cspan address=\"10.1038/s41419-022-05082-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Fan J, Chen T, Xu L, Liu P, Xiao L et al (2022) A novel ferroptosis-related gene prognostic index for prognosis and response to immunotherapy in patients with prostate cancer. Front Endocrinol (Lausanne) 13:975623. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2022.975623\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2022.975623\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Cai Q, Yang R, Wang H, Ling H, Li T et al (2023) GINS4 suppresses ferroptosis by antagonizing p53 acetylation with Snail. Proc Natl Acad Sci USA 120(15):e2219585120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2219585120\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2219585120\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeigand I, Schreiner J, Rohrig F, Sun N, Landwehr LS, Urlaub H et al (2020) Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis 11(3):192. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41419-020-2385-4\u003c/span\u003e\u003cspan address=\"10.1038/s41419-020-2385-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo Y, Gao X, Zou L, Lei M, Feng J, Hu Z (2021) Bavachin Induces Ferroptosis through the STAT3/P53/SLC7A11 Axis in Osteosarcoma Cells. Oxid Med Cell Longev 2021:1783485. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2021/1783485\u003c/span\u003e\u003cspan address=\"10.1155/2021/1783485\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao X, Zhang J, Zhang W, Guo Z, Wei W, Wang X et al (2023) A chiral fluorescent Ir(iii) complex that targets the GPX4 and ErbB pathways to induce cellular ferroptosis. Chem Sci 14(5):1114\u0026ndash;1122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d2sc06171f\u003c/span\u003e\u003cspan address=\"10.1039/d2sc06171f\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Zhang T, Xiong H, Zeng L, Wang Z, Peng Y et al (2022) Tumor-Infiltrating CD4(+) Central Memory T Cells Correlated with Favorable Prognosis in Oral Squamous Cell Carcinoma. J Inflamm Res 15:141\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/jir.S343432\u003c/span\u003e\u003cspan address=\"10.2147/jir.S343432\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong R, Wang N, Han W, Bao W, Lu J (2021) IFNgamma-mediated repression of system xc(-) drives vulnerability to induced ferroptosis in hepatocellular carcinoma cells. J Leukoc Biol 110(2):301\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/JLB.3MA1220-815RRR\u003c/span\u003e\u003cspan address=\"10.1002/JLB.3MA1220-815RRR\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ferroptosis, erastin, prostate cancer, cell, TMEFF2, RNA-seq","lastPublishedDoi":"10.21203/rs.3.rs-3214106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3214106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFew studies are focusing on the mechanism of erastin acts on prostate cancer(PCa) cells, and essential ferroptosis-related genes (FRGs) that can be PCa therapeutic targets are rarely known. In the current study, in vitro assays were performed to evaluate the ferroptotic levels of PCa cells under erastin treatment. RNA-sequecing was used to measure the expression of differentially expressed genes (DEGs) in erastin-induced PCa cells. A series of bioinformatic analyses were applied to analyze the pathways, modules, transcription factors, and expression levels of DEGs. Erastin inhibited the expression of \u003cem\u003eSLC7A11\u003c/em\u003e and cell survivability in LNCaP and PC3 cells. After treatment with erastin, the concentration of malondialdehyde (MDA) and Fe\u003csup\u003e2+\u003c/sup\u003e significantly increased, whereas the glutathione (GSH) and the oxidized glutathione (GSSG) significantly decreased in both cells. A total of 295 overlapping DEGs were screened and identified in two cells under erastin exposure and significantly enriched for association with several pathways, including DNA replication, steroid hormone biosynthesis, and cell cycle, et al. For four hub FRGs, \u003cem\u003eTMEFF2\u003c/em\u003e in PCa tissue is higher than in normal tissue and the expression levels of \u003cem\u003eCLU\u003c/em\u003e, \u003cem\u003eNRXN3\u003c/em\u003e, and \u003cem\u003eUNC5B\u003c/em\u003e were lower in PCa tissue. The expression levels of \u003cem\u003eSLC7A11\u003c/em\u003e and cell survivability were inhibited after the knockdown of \u003cem\u003eTMEFF2\u003c/em\u003e in LNCaP cells but not in PC3 cells. The concentration of Fe\u003csup\u003e2+\u003c/sup\u003e only significantly increased in \u003cem\u003eTMEFF2\u003c/em\u003e downregulated LNCaP cells. This study extends our understanding of the molecular mechanism in erastin-affected PCa cells, and provides potential treatment ideas for PCa therapy.\u003c/p\u003e","manuscriptTitle":"Identification of ferroptosis related genes and pathways in prostate cancer cells under erastin exposure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-03 17:39:05","doi":"10.21203/rs.3.rs-3214106/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8392454a-4f8b-4c9b-98ca-14e059a5b645","owner":[],"postedDate":"August 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-08-03T17:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-03 17:39:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3214106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3214106","identity":"rs-3214106","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}