DKK1 Ameliorates Myofibroblast Differentiation in Urethral Fibrosis in Vivo and in Vitro by Regulating the Canonical Wnt Pathway.

Background: Urethral stricture is a common disorder of the lower urinary tract in men. A complex network of pathways and interactions are involved in the pathogenesis of urethral fibrosis. However, the mechanisms underlying urethral fibrosis remain poorly understood. Objectives: To investigate the critical role of the canonical Wnt pathway in development of urethral fibrosis and explore DKK1, the endogenous inhibitor of Wnt pathway, as a potential target to prevent urethral fibrosis in vitro and in vivo. Methods: Urethral fibrosis tissue derived from patients and rat models were harvested to assess the activation of the canonical Wnt pathway by using western blot, qRT-PCR and immunohistochemistryWe performed histological staining, western blot, qRT-PCR and immunohistochemistry to examine the effects of DKK1 treatment on in vivo rat urethral fibrosis models. In vitro, human urethral fibroblasts (HUFs) were cultured to examine the effects of DKK1 in TGFß1-induced HUFs by CCK-8 assay, hydroxyproline assay, flow cytometry, cell migration assay, western blot, qRT-PCR and immunofluorescence. Results: The key components of Wnt signaling were upregulated in urethral fibrosis tissue derived from patients and rat models while DKK 1 was downregulated. DKK1 ameliorated TGFß1-induced urethral fibrosis in rats. TGFß1 induced myofibroblast differentiation by upregulating collagen I, collagen III, α-SMA, ß-catenin and p-GSK-3ß, while DKK1 was decreased. DKK1 significantly inhibited cell proliferation, collagen content, cell migration and promoted cell apoptosis in TGFß1-induced HUFs. DKK1 significantly suppressed myofibroblast differentiation of TGFß1-induced HUFs by downregulating collagen I, collagen III, α-SMA, ß-catenin and p-GSK-3ß with a mechanism independent of Smad2/3. Conclusions: Our study demonstrated that canonical Wnt pathway may be an essential mechanism underlying the pathogenesis of urethral fibrosis and explored the potential role of DKK1 participation in the development of urethral fibrosis.

MMP9 and TYROBP affect the survival of circulating tumor cells in clear cell renal cell carcinoma by adapting to tumor immune microenvironment.

Circulating tumor cells (CTCs) play a key role in tumor metastasis. CTCs have altered gene expression and can survive in the bloodstream. Finding the key genes whose expression are altered in CTCs could help explain the mechanism of tumor metastasis. We searched for genes differentially expressed in CTCs by analyzing four CTCs and primary tumor gene expression datasets in the GEO database. Key genes of clear cell renal cell carcinoma (ccRCC) CTCs were identified. The correlation between key genes and the immune microenvironment of ccRCC was explored. Finally, the CTCs cell model of ccRCC was constructed by in vivo screening method, and the expression of key genes was detected at the cell and tissue levels. A total of 771 DEGs were obtained. Gene enrichment analysis showed that DEGs of CTCs were mainly involved in the regulation of the tumor immune process and tumor cell apoptosis. Finally, we found 2 key genes, MMP9 and TYROBP in ccRCC CTCs. The high expression of these 2 genes predicted a poor prognosis of ccRCC, and the expression levels of these 2 genes were significantly increased in CTCs and ccRCC tissues. Our study suggested that genetic alterations in CTCs contribute to the ability of CTCs to survive in the blood by adapting to the tumor microenvironment. MMP9 and TYROBP are potential therapeutic and prognostic targets for ccRCC.

SERPINE1 and its co-expressed genes are associated with the progression of clear cell renal cell carcinoma.

BACKGROUND: Clear cell renal cell carcinoma(ccRCC) is a frequently occurring malignant tumor of the urinary system. Despite extensive research, the regulatory mechanisms underlying the pathogenesis and progression of ccRCC remain largely unknown. METHODS: We downloaded 5 ccRCC expression profiles from the Gene Expression Omnibus (GEO) database and obtained the list of differentially expressed genes (DEGs). Using String and Cytoscape tools, we determined the hub genes of ccRCC, and then analyzed their relationship with ccRCC patient survival. Ultimately, we identified SERPINE1 as a prognostic factor in ccRCC. Meanwhile, we confirmed the role of SERPINE1 in 786-O cells by cell transfection and in vitro experiments. RESULTS: Our analysis yielded a total of 258 differentially expressed genes, comprising 105 down-regulated genes and 153 up-regulated genes. Survival analysis of SERPINE1 expression in The Cancer Genome Atlas (TCGA) confirmed its association with the increase of tumor grade, lymph node metastasis, and tumor stage, as well as with shorter survival. Furthermore, we found that SERPINE1 expression levels were associated with CD8 + T cells, CD4 + T cells, B cells, macrophages, neutrophils, and dendritic cells. Cell experiments showed that knockdown SERPINE1 expression could inhibit the proliferation, migration and invasion of ccRCC cells. Among the co-expressed genes with the highest correlation, ITGA5, SLC2A3, SLC2A14, SHC1, CEBPB, and ADA were overexpressed and associated with shorter overall survival (OS) in ccRCC. CONCLUSIONS: In this study, we identified hub genes that are strongly related to ccRCC, and highlights the potential utility of overexpressed SERPINE1 and its co-expressed genes could be used as prognostic and diagnostic biomarkers in ccRCC.

Injectable adhesive self-healing biocompatible hydrogel for haemostasis, wound healing, and postoperative tissue adhesion prevention in nephron-sparing surgery.

Nephron-sparing surgery is a well-established treatment in patients with T1a renal cell carcinoma; however, the complex suturing process prolongs warm ischaemia time, affects the preservation of normal renal parenchymal function, and causes avoidable postoperative tissue adhesion complications, including chronic abdominal pain, intestinal obstruction, and female infertility. Hence, the design of multifunctional biomaterials with haemostasis, postoperative wound management, and postoperative tissue adhesion prevention properties for nephron-sparing surgeries is urgently needed. In this study, a series of injectable adhesive multifunctional biocompatible hydrogels were designed based on the free-radical polymerisation of monomers acryloyl-6-aminocaproic acid (AA) and N-acryloyl 2-glycine (NAG), and the ionic coordination between Ca2+ and the abundant carboxyl groups in AA and NAG. AA/NAG/Ca (AA, NAG, and Ca refer to acryloyl-6-aminocaproic acid, N-acryloyl 2-glycine and calcium chloride, respectively) hydrogel exhibited good mechanical properties, swelling and adhesion properties, flexibility, in vitro blood-clotting ability, and cytocompatibility. In vivo experiments on liver injury models and rat/rabbit nephron-sparing surgery models elucidated that the AA/NAG/Ca hydrogel had haemostasis performance and wound healing properties that led to short bleeding time, reduced bleeding volume, and well-organised nephron structures. An abdomen-caecum adhesion model indicated that the AA/NAG/Ca hydrogel showed excellent anti-adhesion properties. In summary, this multifunctional hydrogel exhibited potential for improving haemostasis and wound management in nephron-sparing surgeries, showing potential for clinical application. STATEMENT OF SIGNIFICANCE: Extended warm ischemia time during nephron sparing surgery negatively affected postoperative renal function due to the need for hemostasis at the wound with abundant blood supply, and postoperative wound healing and additional adhesions caused by the surgical procedure deserve attention. Based on the efficient and stable adhesion properties of hydrogels and the ability to promote wound healing. Herein, a series of adhesive self-healing biocompatible hydrogels were prepared based on free-radical polymerization of acryloyl-6-aminocaproic acid (AA) and N-acryloyl 2-glycine (NAG) and the ionic coordination between Ca2+ with the abundant carboxyl groups in AA and NAG. AA/NAG/Ca hydrogel showed hemostasis property in nephron sparing surgery model, promote kidney wound healing, and could perform anti-postoperative adhesion efficacy in an abdomen-caecum adhesion model.

Comprehensive analysis of the collagen family members as prognostic markers in clear cell renal cell carcinoma.

Background: Clear cell renal cell carcinoma (ccRCC) is one of the common malignant tumors worldwide. There is still a lack of effective diagnostic and therapeutic targets for the recurrence and metastasis of ccRCC. In this study, we sought to identify effective diagnostic and therapeutic targets for ccRCC recurrence and metastasis. Methods: Gene Expression Omnibus (GEO) dataset was used to obtain differentially expressed genes (DEGs) between primary and metastasis ccRCC. We used The Cancer Genome Atlas (TCGA), GeneMANIA, cBioPortal, MethSurv, and TIMER to analyze the expression differences, mutation status, prognostic value, molecular function, and immune infiltration of hub genes in renal cell carcinoma (RCC). Results: We obtained a total of 35 different gene lists. Six collagen family members were identified as hub genes. The expression level of collagen family members was closely related to ccRCC. Moreover, differences in the expression levels of collagen family members were closely related to the stage and prognosis of ccRCC. Members of the collagen family were responsible for more than 15% of the genetic alterations in ccRCC and are involved in multiple signaling pathways. The expression level of collagen family members was closely related to the infiltration of tumor-associated immune cells. Univariate and multivariate Cox regression identified the prognosis-related genes: COL5A1. Conclusions: Our study implied that members of the collagen family may serve as a biomarker for ccRCC metastasis and prognosis.

Corrigendum: A New Prognostic Risk Score: Based on the Analysis of Autophagy-Related Genes and Renal Cell Carcinoma.

[This corrects the article DOI: 10.3389/fgene.2021.820154.].

Abnormal expression and the significant prognostic value of aquaporins in clear cell renal cell carcinoma.

Aquaporins (AQPs) are a kind of transmembrane proteins that exist in various organs of the human body. AQPs play an important role in regulating water transport, lipid metabolism and glycolysis of cells. Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor of the kidney, and the prognosis is worse than other types of renal cell cancer (RCC). The impact of AQPs on the prognosis of ccRCC and the potential relationship between AQPs and the occurrence and development of ccRCC are demanded to be investigated. In this study, we first explored the expression pattern of AQPs by using Oncomine, UALCAN, and HPA databases. Secondly, we constructed protein-protein interaction (PPI) network and performed function enrichment analysis through STRING, GeneMANIA, and Metascape. Then a comprehensive analysis of the genetic mutant frequency of AQPs in ccRCC was carried out using the cBioPortal database. In addition, we also analyzed the main enriched biological functions of AQPs and the correlation with seven main immune cells. Finally, we confirmed the prognostic value of AQPs throughGEPIA and Cox regression analysis. We found that the mRNA expression levels of AQP0/8/9/10 were up-regulated in patients with ccRCC, while those of AQP1/2/3/4/5/6/7/11 showed the opposite. Among them, the expression differences of AQP1/2/3/4/5/6/7/8/9/11 were statistically significant. The differences in protein expression levels of AQP1/2/3/4/5/6 in ccRCC and normal renal tissues were consistent with the change trends of mRNA. The biological functions of AQPs were mainly concentrated in water transport, homeostasis maintenance, glycerol transport, and intracellular movement of sugar transporters. The high mRNA expression levels of AQP0/8/9 were significantly correlated with worse overall survival (OS), while those of AQP1/4/7 were correlated with better OS. AQP0/1/4/9 were prognostic-related factors, and AQP1/9 were independent prognostic factors. In general, this research has investigated the values of AQPs in ccRCC, which could become new survival markers for ccRCC targeted therapy.

Prognostic value of circulating tumor cells and immune-inflammatory cells in patients with renal cell carcinoma.

PURPOSE: The relationships among circulating tumor cells (CTCs), inflammatory cells, and platelets in patients with renal cell carcinoma (RCC) are not transparent. We evaluated the correlations among CTCs, blood inflammatory cells, and platelets in patients with RCC and their prognostic value for metastasis-free survival. METHODS: CTC and typical tumor cell chip data were collected and analyzed by the GEO database. The baseline data, survival data, CTCs data, and blood test results were statistically analyzed. RESULTS: Bioinformatics analysis showed that the function of the differentially expressed genes between CTCs and normal tumor cells mainly involved platelets and immune inflammation. A total of 82 patients whose follow-up time was 3 to 68 months were included in the analysis. Clinical data of the patients confirmed that there is a correlation between platelets and mesenchymal CTCs. Simultaneously, there was a correlation between immune inflammatory cells and platelets. The univariate Cox proportional hazards model indicated that staging, mesenchymal CTCs, and the monocyte-to-neutrophil ratio (MNR) had prognostic value. The multivariate Cox proportional hazards model indicated that staging and the MNR had prognostic value and high accuracy. CONCLUSIONS: Bioinformatics analysis showed that CTCs were related to platelets and immune-inflammatory cells. Furthermore, the clinical data confirmed that platelets were correlated with mesenchymal CTCs and immune-inflammatory cells in the blood. By using mesenchymal CTCs, the MNR, or staging respectively, it is possible to predict the risk of postoperative metastasis in RCC patients. As a compound prognostic factor, staging, and the MNR can provide more convenient and accurate condition monitoring.

Effects of a gamma-secretase inhibitor of notch signalling on transforming growth factor ß1-induced urethral fibrosis.

Urethral stricture (US) is a common disorder of the lower urinary tract in men caused by fibrosis. The recurrence rate of US is high; however, there are no effective therapies to prevent or treat urethral fibrosis. The pathogenesis of urethral fibrosis involves myofibroblast activation and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying this pathological activation are not completely understood. It has been demonstrated that Notch signalling contributes to the development of fibrosis and inflammation. However, whether this contributes to urethral fibrosis remains unclear. In this study, activation of Notch signalling was observed in patients with US. Additionally, it was noted that activation of Notch signalling promoted ECM production and myofibroblast activation in human urethral scar fibroblasts (HUSFs) treated with transforming growth factor (TGF) ß1. However, the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) suppressed activation of Notch signalling as well as proliferation and migration of the TGFß1-treated HUSFs. Additionally, DAPT ameliorated TGFß1-induced urethral fibrosis in Sprague Dawley rats by suppressing ECM production, myofibroblast activation and the TGFß signalling pathway. These findings demonstrate that Notch signalling may be a promising and potential target in the prevention or treatment of urethral fibrosis.

The prognostic value of circulating tumour cells (CTCs) and CTC white blood cell clusters in patients with renal cell carcinoma.

PURPOSE: Circulating tumour cell (CTC) and CTC-white blood cell (CTC-WBC) clusters are related to the prognosis of tumour patients. However, the relationship between CTC-WBC clusters and prognosis in renal cell carcinoma (RCC) patients is not clear. We evaluated the prognostic value of CTC-WBC clusters using metastasis-free survival (MFS) and overall survival (OS) in patients with RCC. MATERIALS AND METHODS: The baseline, survival, and CTC data of patients with RCC were statistically analysed by R. RESULTS: The Cox risk proportional regression model suggests that the total CTCs, pathology type, and CTC-WBC clusters can be used as prognostic indicators for the MFS of RCC patients. Total CTCs and solid tumour diameter can be used as prognostic indicators for the OS of RCC patients. Using Kaplan-Meier survival analysis, we found that patients with total CTCs, pathology, and CTC-WBC clusters greater than the cut-off value had a worse MFS, and patients with total CTCs greater than the cut-off value had a worse OS. CONCLUSION: The analysis of the clinical sample data in patients with RCC shows that CTC-WBC clusters play an important role in monitoring the prognosis of RCC. Among them, total CTCs, pathology, and CTC-WBC clusters were combined as prognostic factors for the MFS of RCC patients. Total CTCs and solid tumour diameter can be combined as prognostic factors for the OS of RCC patients. These prognostic factors provide more convenient and accurate condition monitoring for renal cancer patients and can be used to actively improve the prognosis of patients.

A New Prognostic Risk Score: Based on the Analysis of Autophagy-Related Genes and Renal Cell Carcinoma.

Introduction: Clear cell renal cell carcinoma (ccRCC) patients suffer from its high recurrence and metastasis rate, and a new prognostic risk score to predict individuals with high possibility of recurrence or metastasis is in urgent need. Autophagy has been found to have a dual influence on tumorigenesis. In this study we aim to analyze autophagy related genes (ATGs) and ccRCC patients and find a new prognostic risk score. Method: Analyzing differential expression genes (DEGs) in TCGA-KIRC dataset, and took intersection with ATGs. Through lasso, univariate, and multivariate cox regression, DEGs were chosen, and the coefficients and expression levels of them were components constructing the formula of risk score. We analyzed mRNA expression of DEGs in tumor and normal tissue in ONCOMINE database and TCGA-KIRC dataset. The Human Protein Atlas (HPA) was used to analyze protein levels of DEGs. The protein-protein interaction (PPI) network was examined in STRING and visualized in cytoscape. Functional enrichment analysis was performed in RStudio. To prove the ability and practicibility of risk score, we analyzed univariate and multivariate cox regression, Kaplan-Meier curve (K-M curve), risk factor association diagram, receiver operating characteristic curve (ROC curve) of survival and nomogram, and the performance of nomogram was evaluated by calibration curve. Then we further explored functional enrichment related to risk groups through Gene Set Enrichment Analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and Metascape database. At last, we investigated immune cell infiltration of DEGs and two risk groups through TIMER database and "Cibersort" algorithm. Result: We identified 7 DEGs (BIRC5, CAPS, CLDN7, CLVS1, GMIP, IFI16, and TCIRG1) as components of construction of risk score. All 7 DEGs were differently expressed in ccRCC and normal tissue according to ONCOMINE database and TCGA-KIRC dataset. Functional enrichment analysis indicated DEGs, and their most associated genes were shown to be abundant in autophagy-related pathways and played roles in tumorigenesis and progression processes. A serious analysis proved that this risk score is independent from the risk signature of ccRCC patients. Conclusion: The risk score constructed by 7 DEGs had the ability of predicting prognosis of ccRCC patients and was conducive to the identification of novel prognostic molecular markers. However, further experiment is still needed to verify its ability and practicability.

Polydatin enhances glomerular podocyte autophagy homeostasis by improving Nrf2-dependent antioxidant capacity in fructose-fed rats.

High fructose is considered a causative factor for oxidative stress and autophagy imbalance that cause kidney pathogenesis. Antioxidant polydatin isolated from Polygonum cuspidatum has been reported to protect against kidney injury. In this study, polydatin was found to ameliorate fructose-induced podocyte injury. It activated mammalian target of rapamycin complex 1 (mTORC1) and suppressed autophagy in glomeruli of fructose-fed rats and in fructose-exposed conditionally immortalized human podocytes (HPCs). Polydatin also enhanced nuclear factor-E2-related factor 2 (Nrf2)-dependent antioxidant capacity to suppress fructose-induced autophagy activation in vivo and in vitro, with the attenuation of fructose-induced up-regulation of cellular light chain 3 (LC3) II/I protein levels. This effect was abolished by Raptor siRNA in fructose-exposed HPCs. These results demonstrated that polydatin ameliorated fructose-induced autophagy imbalance in an mTORC1-dependent manner via improving Nrf2-dependent antioxidant capacity during podocyte injury. In conclusion, polydatin with anti-oxidation activity suppressed autophagy to protect against fructose-induced podocyte injury.

Identification of key genes and functions of circulating tumor cells in multiple cancers through bioinformatic analysis.

BACKGROUND: Circulating tumor cells (CTCs) play a key role in cancer progression, especially metastasis, due to the rarity and heterogeneity of CTCs, fewer researches have been conducted on them at the molecular level. However, through the Gene Expression Omnibus (GEO) database, this kind of minority researches can be well integrated, the gene expression differences between CTCs and primary tumors can be identified, and molecular targets for CTCs can be found. METHODS: We analyzed 7 sets of gene chips (GSE82198, GSE99394, GSE31023, GSE65505, GSE67982, GSE76250, GSE50746) obtained by GEO. Analysis of differentially expressed genes (DEGs) between CTCs and corresponding primary tumors by NetworkAnalyst. Metascape tool for Gene Ontology (GO) / Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differential genes and visual display. Cytoscape performs protein-protein interaction (PPI) analysis and obtains the hub genes. Renal cancer patients' clinical specimens to verify the correctness of enrichment results. Prognostic analysis of hub genes in kidney cancer patients using the Kaplan-Meier plotter survival analysis tool. RESULTS: We obtained a total of 589 DEGs. The GO / KEGG enrichment results indicate that the DEGs are mainly concentrated in cell adhesion, epithelial-mesenchymal transition (EMT), and apoptosis. Renal cancer clinical specimens suggest that CTCs have epithelial and mesenchymal types. At the same time, PSMC2 can be used as a poor prognostic indicator for renal cancer patients. CONCLUSIONS: In summary, our study suggests that compared with primary tumors, CTCs mainly change cell adhesion, EMT, and apoptosis. PSMC2 can be used as a poor prognostic factor.

Pathology of circulating tumor cells and the available capture tools (Review).

Circulating tumor cells (CTCs), are tumor cells that diffuse into the circulating blood and serve an important role in the progress of cancer. During the early stages of cancer, CTCs undergo an epithelial­mesenchymal transition and obtain a more invasive phenotype. Subsequently, the tumor cells enter the circulating blood with the aid of immune cells, and enter a dormant state upon reaching distal organs. As the tumor progresses, metastasis may occur under certain conditions. The capture technologies available for CTCs are based on antibody­based capture, or capture based on the physical properties of CTCs, as well as modern technologies that integrate both these methods. Emerging modern technologies have increased the accuracy and efficiency of tumor cell capture, and have thus improved our understanding of tumor cells, and the molecular mechanisms underlying their properties. CTCs serve an important role in disease progression, prediction of patient prognosis and individualized treatment.