Ferroptosis Is Crucial for Cisplatin Induced Sertoli Cell Injury via N6-Methyladenosine Dependent Manner.

PURPOSE: This study aimed to investigate the effect of the N6-methyladenosine (m6A) dependent ferroptosis on cisplatininduced Sertoli cell injury. MATERIALS AND METHODS: A cisplatin exposure mouse model was established by intraperitoneal injection of cisplatin in our study. TM4 cell lines was used for in vitro study. Ferroptosis was detected according to metabolomic analysis and a series of assays, including malondialdehyde, glutathione, and glutathione disulfide concentration detection, 2',7'-dichlorodihydrofluorescein diacetate and BODIPY 581/591 C11 probe detection, and transmission electron microscope imaging. Key ferroptosis-related genes were identified via transcriptomic analysis, western blot and immunohistochemistry. The m6A modification was demonstrated via m6A RNA immunoprecipitation and luciferase reporter assays. Immune cell infiltration was detected by mass cytometry, and verified by flow cytometry and immunofluorescence. RESULTS: Ferroptosis, but not other types of programmed cell death, is a significant phenomenon in cisplatin-induced testis damage and Sertoli cell loss. Ferroptosis induced by cisplatin in Sertoli cell/TM4 cell is GPX4 independent but is regulated by SLC7A11 and ALOX12. Both SLC7A11 and ALOX12 are regulated via m6A dependent manner by METTL3. Furthermore, overexpressed ALOX12-12HETE pathway may result in macrophage polarization and inflammatory response in cisplatin exposure testis. CONCLUSIONS: Cisplatin-induced Sertoli cell injury via ferroptosis and promoted ferroptosis in an m6A dependent manner. m6A modification of both SLC7A11 and ALOX12 mRNA could result in ferroptosis in our in vitro model. Further, overexpressed ALOX12 can cause more production of 12-HETE, which may be responsible for testis inflammation caused by cisplatin.

NCAPG2 promotes prostate cancer malignancy and stemness via STAT3/c-MYC signaling.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related mortality among men worldwide, and its incidence has risen substantially in recent years. Therefore, there is an urgent need to identify novel biomarkers and precise therapeutic targets for managing PCa progression and recurrence. METHODS: We investigated the clinical significance of NCAPG2 in PCa by exploring public datasets and our tissue microarray. Receiver operating characteristic (ROC) curve and survival analyses were performed to evaluate the correlation between NCAPG2 and PCa progression. Cell proliferation, wound healing, transwell, flow cytometry, cell cycle, tumor sphere formation, immunofluorescence (IF), co-immunoprecipitation (co-IP), and chromatin immunoprecipitation (ChIP) assays were conducted to further elucidate the molecular mechanism of NCAPG2 in PCa. Subcutaneous and orthotopic xenograft models were applied to investigate the effects of NCAPG2 on PCa proliferation in vivo. Tandem mass tag (TMT) quantitative proteomics was utilized to detect proteomic changes under NCAPG2 overexpression. RESULTS: NCAPG2 was significantly upregulated in PCa, and its overexpression was associated with PCa progression and unfavorable prognosis. Knockdown of NCAPG2 inhibited the malignant behavior of PCa cells, whereas its overexpression promoted PCa aggressiveness. NCAPG2 depletion attenuated the development and growth of PCa in vivo. TMT quantitative proteomics analyses indicated that c-MYC activity was strongly correlated with NCAPG2 expression. The malignancy-promoting effect of NCAPG2 in PCa was mediated via c-MYC. NCAPG2 could directly bind to STAT3 and induce STAT3 occupancy on the MYC promoter, thus to transcriptionally activate c-MYC expression. Finally, we identified that NCAPG2 was positively correlated with cancer stem cell (CSC) markers and enhanced self-renewal capacity of PCa cells. CONCLUSIONS: NCAPG2 is highly expressed in PCa, and its level is significantly associated with PCa prognosis. NCAPG2 promotes PCa malignancy and drives cancer stemness via the STAT3/c-MYC signaling axis, highlighting its potential as a therapeutic target for PCa.

Deacetylation of Septin4 by SIRT2 (Silent Mating Type Information Regulation 2 Homolog-2) Mitigates Damaging of Hypertensive Nephropathy.

BACKGROUND: Hypertension can lead to podocyte damage and subsequent apoptosis, eventually resulting in glomerulosclerosis. Although alleviating podocyte apoptosis has clinical significance for the treatment of hypertensive nephropathy, an effective therapeutic target has not yet been identified. The function of septin4, a proapoptotic protein and an important marker of organ damage, is regulated by post-translational modification. However, the exact role of septin4 in regulating podocyte apoptosis and its connection to hypertensive renal damage remains unclear. METHODS: We investigated the function and mechanism of septin4 in hypertensive nephropathy to discover a theoretical basis for targeted treatment. Mouse models including Rosa 26 (Gt(ROSA)26Sor)-SIRT2 (silent mating type information regulation 2 homolog-2)-Flag-TG (transgenic) (SIRT2-TG) mice SIRT2-knockout, and septin4-K174Q mutant mice, combined with proteomic and acetyl proteomics analysis, followed by multiple molecular biological methodologies, were used to demonstrate mechanisms of SIRT2-mediated deacetylation of septin4-K174 in hypertensive nephropathy. RESULTS: Using transgenic septin4-K174Q mutant mice treated with the antioxidant Tempol, we found that hyperacetylation of the K174 site of septin4 exacerbates Ang II (angiotensin II)- induced hypertensive renal injury resulting from oxidative stress. Proteomics and Western blotting assays indicated that septin4-K174Q activates the cleaved-PARP1 (poly [ADP-ribose] polymerase family, member 1)-cleaved-caspase3 pathway. In septin4-knockdown human renal podocytes, septin4-K174R, which mimics deacetylation at K174, rescues podocyte apoptosis induced by Ang II. Immunoprecipitation and mass spectrometry analyses identified SIRT2 as a deacetylase that interacts with the septin4 GTPase domain and deacetylates septin4-K174. In Sirt2-deficient mice and SIRT2-knockdown renal podocytes, septin4-K174 remains hyperacetylated and exacerbates hypertensive renal injury. By contrast, in Rosa26-Sirt2-Flag (SIRT2-TG) mice and SIRT2-knockdown renal podocytes reexpressing wild-type SIRT2, septin4-K174 is hypoacetylated and mitigates hypertensive renal injury. CONCLUSIONS: Septin4, when activated through acetylation of K174 (K174Q), promotes hypertensive renal injury. Septin4-K174R, which mimics deacetylation by SIRT2, inhibits the cleaved-PARP1-cleaved-caspase3 pathway. Septin4-K174R acts as a renal protective factor, mitigating Ang II-induced hypertensive renal injury. These findings indicate that septin4-K174 is a potential therapeutic target for the treatment of hypertensive renal injury.

RUNX2 interacts with SCD1 and activates Wnt/ß-catenin signaling pathway to promote the progression of clear cell renal cell carcinoma.

BACKGROUND: Previous studies have demonstrated that Runt-associated transcription factor 2 (RUNX2) serves as the main transcription factor for osteoblast differentiation and chondrocyte maturation. RUNX2 is related to a variety of tumors, particularly tumor invasion and metastasis, while the expression and molecular mechanisms of RUNX2 in clear cell renal cell carcinoma (ccRCC) keep to be determined. Stearyl CoA desaturase 1 (SCD1), an endoplasmic reticulum fatty acid desaturase, transfers saturated fatty acids to monounsaturated fatty acids, is expressed highly in numerous malignancies. METHODS: The Cancer Genome Atlas (TCGA) datebase and Western blot was used to analyzed the mRNA and protein levels of the target gene in ccRCC tissues and adjacent tissues. The proliferation ability of ccRCC cells was tested by colony forming and EdU assay. The migration ability of cells was detected by transwell assay. Immunoprecipitation was utilized to detect protein-protein interaction. Cycloheximide chase assay was used to measure the half-life of SCD1 protein. RESULTS: In this study, the expressions of RUNX2 and SCD1 are increased in ccRCC tissues as well as ccRCC cell lines. Both RUNX2 and SCD1 could promote proliferation and migration in ccRCC cells. Furthermore, RUNX2 could physically interact with SCD1. In addition, the functional degradation and the inactivation of Wnt/ß-catenin signaling pathway triggered by the downregulation of RUNX2 could be partly offset by the overexpression of SCD1. CONCLUSION: The findings indicate that the RUNX2/SCD1 axis may act as a potential therapeutic target via the Wnt/ß-catenin signaling pathway of ccRCC.

Flavokawain A is a natural inhibitor of PRMT5 in bladder cancer.

BACKGROUND: Protein arginine methyltransferases (PRMTs) regulate protein biological activity by modulating arginine methylation in cancer and are increasingly recognized as potential drug targets. Inhibitors targeting PRMTs are currently in the early phases of clinical trials and more candidate drugs are needed. Flavokawain A (FKA), extracted from kava plant, has been recognized as a potential chemotherapy drug in bladder cancer (BC), but its action mechanism remains unclear. METHODS: We first determined the role of a type II PRMT, PRMT5, in BC tissue samples and performed cytological experiments. We then utilized bioinformatics tools, including computational simulation, virtual screening, molecular docking, and energy analysis, to identify the potential use of PRMT5 inhibitors for BC treatment. In vitro and in vivo co-IP and mutation assays were performed to elucidate the molecular mechanism of PRMT5 inhibitor. Pharmacology experiments like bio-layer interferometry, CETSA, and pull-down assays were further used to provide direct evidence of the complex binding process. RESULTS: Among PRMTs, PRMT5 was identified as a therapeutic target for BC. PRMT5 expression in BC was correlated with poor prognosis and manipulating its expression could affect cancer cell growth. Through screening and extensive experimental validation, we recognized that a natural product, FKA, was a small new inhibitor molecule for PRMT5. We noticed that the product could inhibit the action of BC, in vitro and in vivo, by inhibiting PRMT5. We further demonstrated that FKA blocks the symmetric arginine dimethylation of histone H2A and H4 by binding to Y304 and F580 of PRMT5. CONCLUSIONS: In summary, our research strongly suggests that PRMT5 is a potential epigenetic therapeutic target in bladder cancer, and that FKA can be used as a targeted inhibitor of PRMT5 for the treatment of bladder cancer.

An RNA-binding protein-related risk signature can predict the prognosis and tumor immunity of patients with testicular germ cell tumors.

BACKGROUND: The functions of RNA-binding proteins (RBPs) in the occurrence and development of tumors remain largely unexplored. We established a risk signature based on RBPs to predict the prognosis, tumor-related immunity, and treatment benefits of patients with testicular germ cell tumors (TGCTs). METHODS: A risk signature was built based on RBPs closely related to survival obtained from TGCT data in The Cancer Genome Atlas (TCGA) database. The ability of the signature to predict prognosis was analyzed by survival curves and Cox regression. The risk signature was validated using the Gene Expression Omnibus (GEO) database. The connection between tumor immunity and the risk score was evaluated. Risk score-related drug sensitivity and biofunctions were also explored. RESULTS: A risk signature including four selected RBP genes (PARP12, USB1, POLR2E and EED) was established. The prognosis of high-risk TGCT patients was worse than that of low-risk TGCT patients. The risk score was considered a critical factor closely related to prognosis, as determined via Cox regression, and was also closely associated with multiple characteristics of tumor immunity, chemotherapy drugs and biofunctions. CONCLUSION: The established risk signature including four selected RBPs in TGCTs could predict the prognosis, tumor-related immunity and treatment benefits of patients with TGCTs. Utilization of this signature could help clinicians make personalized treatment decisions.

An HDAC9-associated immune-related signature predicts bladder cancer prognosis.

BACKGROUND: The close relationship between histone deacetylase 9 (HDAC9) and immunity has attracted attention. We constructed an immune signature for HDAC9, a vital epigenetic modification, to predict the survival status and treatment benefits in bladder cancer (BC). METHODS: An exhaustive analysis of HDAC9 and immunology via the tumor and immune system interaction database (TISIDB) was performed, and an immune prognostic risk signature was developed based on genes enriched in the top five immune-related pathways under high HDAC9 status. Comprehensive analysis of survival curves and Cox regression were used to estimate the effectiveness of the risk signature. The relationship between immunological characteristics and the risk score was evaluated, and the mechanisms were also explored. RESULTS: In the TISIDB, HDAC9 was closely related to various immunological characteristics. The risk signature was obtained based on genes related to prognosis enriched in the top five immune-related pathways under high HDAC9 status. The survival rate of the high-risk BC patients was poor. The risk score was closely related to multiple immunological characteristics, drug sensitivity, immunotherapy benefits and biofunctions. CONCLUSION: An immune-related prognostic signature established for HDAC9 expression status could independently predict the prognosis of BC patients. The use of this signature could help clinicians make personalized treatment decisions.

METTL14-mediated N6-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma.

BACKGROUND: Integrin ß4 (ITGB4) participates in tumorigenesis and progression of several malignancies, but its role and related mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear. METHODS: Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were used to detect mRNA and protein levels of relevant genes. Biological functions of ITGB4 and methyltransferase-like 14 (METTL14) were determined by in vitro and in vivo experiments. The levels of N6-methyladenosine (m6A) in ccRCC tissues and adjacent normal tissues were calculated via total RNA m6A quantification assay. The m6A modification of ITGB4 was demonstrated via m6A RNA immunoprecipitation (MeRIP), RIP and luciferase reporter assays. RESULTS: ITGB4 was significantly overexpressed in ccRCC tissues and high level of ITGB4 predicted poor prognosis as well as metastasis. Functionally, ITGB4 stimulated ccRCC cell migration and invasion in vitro and metastasis in vivo with epithelial-mesenchymal transition (EMT) strengthened. Mechanically, the total levels of m6A were reduced in ccRCC tissues. METTL14, a favorable factor for ccRCC patients' prognosis, facilitated m6A modification on ITGB4 3'UTR and subsequently accelerated ITGB4 mRNA degradation, leading to its declined expression. Furthermore, the METTL14-mediated inhibition of ITGB4 expression was dependent on the YTH domain family protein 2 (YTHDF2), which acted as an m6A reader to bind to ITGB4 mRNA and to promote its decay. In addition, we demonstrated that knockdown of METTL14 promoted ccRCC cell migration, invasiveness and metastasis as well as stimulating the EMT process and the PI3K/AKT signal by overexpressing ITGB4. CONCLUSION: Our study reveals that METTL14 inhibits ITGB4 expression via m6A modification to attenuate metastasis and EMT of ccRCC cells, suggesting the METTL14/ITGB4 axis as a potential prognostic biomarker and therapeutic target for ccRCC. Video Abstract.

[Corrigendum] Knockdown of SNHG15 suppresses renal cell carcinoma proliferation and EMT by regulating the NF­κB signaling pathway.

Subsequently to the publication of the above article, the authors have realized that, on p. 390, the data selected for the siRNA­1 and siRNA­2 experiments for the ACHN and 786-O cell lines concerning both the invasion and the migration assays in Fig. 4B were selected inappropriately. Furthermore, after having inspected the published version of Fig. 5, the authors have realized that, for the immunofluorescence experiments shown in Fig. 5D, the first 'Merged' pictures for the first two columns of the ACHN cell line were accidentally published in the wrong order. The corrected versions of Figs. 4, and 5, including all the correct data for Figs. 4B and 5D, are shown on the next three pages. The authors confirm that these data continue to support the main conclusions presented in their paper, and are grateful to the Editor of International Journal of Oncology for granting them this opportunity to publish a Corrigendum. They also apologize to the readership for any inconvenience caused. [International Journal of Oncology 53: 384­394, 2018; DOI: 10.3892/ijo.2018.4395].

The potential role of c-MYC and polyamine metabolism in multiple drug resistance in bladder cancer investigated by metabonomics.

Bladder cancer has a high incidence worldwide accompanies by high recurrent rate after treatment. The emergence of primary or acquired chemotherapy resistance leads to poor efficacy in many cases. To explore the underlying mechanisms of drug resistance, we firstly established a drug-resistant cell model T24/THP by repeated exposure of T24 cells to pirarubicin (THP) whose concentration increases gradually. Non-targeted metabolomics was performed to identify metabolic changes and key metabolism pathways variance in T24/THP cells. Pathway enrichment analysis demonstrated that the arginine and proline metabolic pathway was the most significantly changed pathway, where two representative members of polyamine, putrescine and spermidine were remarkably down regulated in T24/THP. Subsequent experiments further confirmed that ornithine decarboxylase (ODC1) and spermidine synthase (SRM), the key enzymes involved in the synthesis of these compounds, also showed a stable low expression in T24/THP. However, knocking down of ODC1 and SRM sensitized cells to chemotherapy treatment while overexpression of these two enzymes enhances chemotherapy resistance. This leaded to the point that ODC1 and SRM themselves are more likely to promote the drug resistance, which appears to contradict their low expression in T24/THP. We hypothesize that their diminished levels were due to the declined activity of genes upstream. According to this line of thought, we found that c-MYC was also down-regulated in T24/THP and its content could be significantly affected by drug administration. In addition, c-MYC could not only regulate the expression levels of ODC1 and SRM but also influence drug resistance in T24/THP. In conclusion, alterations in gene expression of ODC1 and SRM in drug resistance cell line is probably mediated by some upstream regulators rather than antineoplastic agents alone. Exploration of upstream signals and research on detailed regulatory mechanism, thereby understanding the actual role of c-MYC and polyamine in response to chemotherapy, can become a potential field direction to overcome drug resistance in bladder cancer.

The Sigma-2 Receptor/TMEM97 Agonist PB28 Suppresses Cell Proliferation and Invasion by Regulating the PI3K-AKT-mTOR Signalling Pathway in Renal Cancer.

Sigma-2 receptor/TMEM97 is overexpressed in many tumours, and sigma-2 receptor ligands are under investigation for cancer therapy. We intended to evaluate the effect of PB28 on renal cancer in proliferation, migration and invasion in vitro and in vivo. Invasive renal cancer cell lines treated with PB28 (or sigma-2 receptor antagonist 1) were subjected to cell proliferation, migration and invasion assays. The therapeutic effect of PB28 was performed on nude mice. Western blot for proteins in the PI3K-AKT-mTOR signalling pathway was conducted. A CCK-8 assay was used to examine the effect of the combination of PB28 and cisplatin on renal cancer cells. Significant inhibitory effects were observed on proliferation, migration and invasion of 786-O and ACHN cells after culturing with PB28. But, the outcomes of sigma-2 receptor antagonist 1 presented the opposite tendency. PB28 significantly inhibited the proliferative and invasive ability of OS-RC-2 cells in vivo. Treatment resulted in decreased phosphorylation of constituents of the PI3K-AKT-mTOR pathway. The combination of PB28 and cisplatin showed enhanced efficacy in the inhibition of renal cancer cell proliferation. Taken together, PB28 inhibited the tumorigenic behaviours of renal cancer cells by regulating the PI3K-AKT-mTOR signalling pathway and was expected to be a sensitizer of cisplatin.

Non-metabolic function of MTHFD2 activates CDK2 in bladder cancer.

Bladder cancer is a common tumor with a high recurrence rate and high fatality rate, and its mechanism of occurrence and development remains unclear. Many proteins and metabolites reprogram at different stages of tumor development to support tumor cell growth. The moonlighting effect happens when a protein performs multiple functions simultaneously in a cell. In this study, we identified a metabolic protein, MTHFD2, which participates in the cell cycle by binding to CDK2 in bladder cancer. MTHFD2 has been shown to affect bladder cancer cell growth, which is independent of its metabolic function. We found that MTHFD2 was involved in cell cycle regulation and could encourage cell cycle progression by activating CDK2 and sequentially affecting E2F1 activation. In addition, moonlighting MTHFD2 might be regulated by the dynamics of the mitochondria. In conclusion, MTHFD2 localizes in the nucleus to perform a distinct function of catalyzing metabolic reactions. Moreover, the nuclear MTHFD2 activates CDK2 and promotes bladder cancer cell growth by modulating the cell cycle.

A novel immune-related gene pair prognostic signature for predicting overall survival in bladder cancer.

BACKGROUND: Bladder cancer (BC) is the ninth most common malignant tumor. We constructed a risk signature using immune-related gene pairs (IRGPs) to predict the prognosis of BC patients. METHODS: The mRNA transcriptome, simple nucleotide variation and clinical data of BC patients were downloaded from The Cancer Genome Atlas (TCGA) database (TCGA-BLCA). The mRNA transcriptome and clinical data were also extracted from Gene Expression Omnibus (GEO) datasets (GSE31684). A risk signature was built based on the IRGPs. The ability of the signature to predict prognosis was analyzed with survival curves and Cox regression. The relationships between immunological parameters [immune cell infiltration, immune checkpoints, tumor microenvironment (TME) and tumor mutation burden (TMB)] and the risk score were investigated. Finally, gene set enrichment analysis (GSEA) was used to explore molecular mechanisms underlying the risk score. RESULTS: The risk signature utilized 30 selected IRGPs. The prognosis of the high-risk group was significantly worse than that of the low-risk group. We used the GSE31684 dataset to validate the signature. Close relationships were found between the risk score and immunological parameters. Finally, GSEA showed that gene sets related to the extracellular matrix (ECM), stromal cells and epithelial-mesenchymal transition (EMT) were enriched in the high-risk group. In the low-risk group, we found a number of immune-related pathways in the enriched pathways and biofunctions. CONCLUSIONS: We used a new tool, IRGPs, to build a risk signature to predict the prognosis of BC. By evaluating immune parameters and molecular mechanisms, we gained a better understanding of the mechanisms underlying the risk signature. This signature can also be used as a tool to predict the effect of immunotherapy in patients with BC.

A multicenter retrospective study on evaluation of predicative factors for positive biopsy of prostate cancer in real-world setting.

OBJECTIVE: This study aimed to evaluate the predictors for positive biopsy in prostate cancer (PCa) patients and develop a risk-stratification score model for positive biopsy rate in patients with prostate specific antigen (PSA) in the gray zone. METHODS: In this retrospective, multicenter, real-world study, Chinese patients receiving prostate biopsy for the first time were included. The study evaluated the positive biopsy rate, predictors for positive biopsy and a risk prediction model for PSA 4-10 ng/mL PCa was developed. The univariate and multivariate logistic regression analyses were used to identify the risk factors. RESULTS: A total of 2426 patients were included in the study. The biopsy positive rate was 47.57%, 25.77%, and 60.57% among overall patients, total PSA (t-PSA) 4-10 ng/mL patients, and PSA > 10 ng/mL patients respectively. Elderly age 60-74, ≥75, multi parametric magnetic resonance imaging (MP-MRI), pre-operative PSA > 10 and PSA density (PSAD) significantly increased the positive rate in overall population, and elderly age, MP-MRI, positive digital rectal examination and f-PSA were significant predictors for positive biopsy in PSA 4-10 ng/mL population. A risk prediction model for positive biopsy rate in patients with PSA in the gray zone was developed. Area under curve (AUC) was associated with low accuracy for all the variables used such as tPSA (0.53), PSAD (0.57), frequency of puncture (0.53) and MP-MRI (0.64) in prediction of biopsy positive rate. CONCLUSION: Our study evaluated the significant predicative factors for positive biopsy and the PCa risk prediction model developed might help Clinicians to avoid unnecessary biopsy in patients with PSA in gray zone.

Construction and analysis of a ceRNA network and patterns of immune infiltration in bladder cancer.

BACKGROUND: Bladder cancer (BC) is the ninth most common malignant tumor, accounting for an estimate of 549,000 new BC cases and 200,000 BC-related deaths worldwide in 2018. The prognosis of BC has not substantially improved despite significant advances in the diagnosis and treatment of the disease. METHODS: The RNA sequencing (RNA-seq) data and clinical information of BC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm was used to assess immune infiltration. The survival analyses were performed using the selected components of a ceRNA network and selected immune cell types by least absolute shrinkage and selection operator (LASSO) Cox regression to calculate the risk score. The accuracy of prognosis prediction was determined by receiver operating characteristic (ROC) curves, survival curves, and nomograms. Finally, the correlation analysis was performed to investigate the relationships between the signature components of the ceRNA network and the immune cell signature. RESULTS: Two completed survival analyses included selected components of the ceRNA network (ELN, SREBF1, DSC2, TTLL7, DIP2C, SATB1, hsa-miR-20a-5p, and hsa-miR-29c-3p) and selected immune cell types (M0 macrophages, M2 macrophages, resting mast cells, and neutrophils). ROC curves, survival curves (all P values <0.05), nomograms, and calibration curves indicated that the accuracy of the two survival analyses was acceptable. Moreover, the correlations between TTLL7 and resting mast cells (R=0.24, P<0.001), DSC2 and resting mast cells (R=-0.23, P<0.001), ELN and resting mast cells (R=0.44, P<0.001), and hsa-miR-29c-3p and M0 macrophages (R=-0.29, P<0.001) were significant, indicating that interactions of these factors may play significant roles in the prognosis of BC. CONCLUSIONS: TTLL7, DSC2, ELN, hsa-miR-29c-3p, resting mast cells, and M0 macrophages may play an important role in the development of BC. However, additional studies are needed to confirm this hypothesis.