Prognostic significance and immune landscape of a cell cycle progression-related risk model in bladder cancer.

BACKGROUND: A greater emphasis has been placed on the part of cell cycle progression (CCP) in cancer in recent years. Nevertheless, the precise connection between CCP-related genes and bladder cancer (BCa) has remained elusive. This study endeavors to establish and validate a reliable risk model incorporating CCP-related factors, aiming to predict both the prognosis and immune landscape of BCa. METHODS: Clinical information and RNA sequencing data were collected from the GEO and TCGA databases. Univariate and multivariate Cox regression analyses were conducted to construct a risk model associated with CCP. The performance of the model was assessed using ROC and Kaplan-Meier survival analyses. Functional enrichment analysis was employed to investigate potential cellular functions and signaling pathways. The immune landscape was characterized using CIBERSORT algorithms. Integration of the risk model with various clinical variables led to the development of a nomogram. RESULTS: To build the risk model, three CCP-related genes (RAD54B, KPNA2, and TPM1) were carefully chosen. ROC and Kaplan-Meier survival analysis confirm that our model has good performance. About immunological infiltration, the high-risk group showed decreased levels of regulatory T cells and dendritic cells coupled with increased levels of activated CD4 + memory T cells, M2 macrophages, and neutrophils. Furthermore, the nomogram showed impressive predictive power for OS at 1, 3, and 5 years. CONCLUSION: This study provides new insights into the association between the CCP-related risk model and the prognosis of BCa, as well as its impact on the immune landscape.

Autophagy-related 7 proteindependent autophagy mediates resveratrol-caused upregulation of mitochondrial biogenesis and steroidogenesis in aged Leydig cell.

BACKGROUND: Mitochondrial dysfunction may contribute to decreased testosterone synthesis in aged Leydig cells. Resveratrol (RSV) as an antioxidant has been shown to exhibit multiple positive effects on mitochondrion, where steroidogenesis takes place. Whether RSV can improve steroidogenesis in aged testis is still unknown. This study investigates the effect of RSV on testosterone production during aging and corresponding changes in mitochondrial biogenesis and autophagy activity, which are closely associated with steroidogenesis. Whether ATG7, an important autophagy-related protein, functions in RSV-treated aged Leydig cells will also be explored. METHODS AND RESULTS: Two-month-old male C57BL/6 mice were fed for 16 months by customized regular diet with or without RSV as diet supplement. Leydig cell line TM3 cells were treated with D-galactose to induce senescence, followed with or without RSV treatment. Results found that RSV supplement increased testosterone production in both aged mice and D-galactose-induced senescent Leydig cells. Western blot results revealed that RSV treatment elevated levels of steroidogenic rate-limiting enzymes StAR and 3ß-HSD, as well as autophagy-related proteins LC3II, Beclin1, ATG5 and ATG7 and mitochondrial function-related proteins mtTFA and COXIV. However, after Atg7 was knocked down in senescent Leydig cells, even though RSV was added, levels of these proteins declined significantly, accompanied by decreased levels of mitochondrial transcript factors PGC-1α, mtTFA and NRF-1 and more fragmented mitochondria, demonstrating that Atg7 knockdown wrecked the protective effects of RSV on steroidogenesis in senescent Leydig cells. CONCLUSION: ATG7-dependent autophagy plays a key role in RSV-brought testosterone production increase through regulating mitochondrial biogenesis in senescent Leydig cells.

ORC6, a novel prognostic biomarker, correlates with T regulatory cell infiltration in prostate adenocarcinoma: a pan-cancer analysis.

BACKGROUND: The origin recognition complex (ORC), a six-subunit DNA-binding complex, participates in DNA replication in cancer cells. Specifically in prostate cancers, ORC participates the androgen receptor (AR) regulated genomic amplification and tumor proliferation throughout the entire cell cycle. Of note, ORC6, the smallest subunit of ORC, has been reported to be dysregulated in some types of cancers (including prostate cancer), however, its prognostic and immunological significances remain yet to be elucidated. METHODS: In the current study, we comprehensively investigated the potential prognostic and immunological role of ORC6 in 33 human tumors using multiple databases, such as TCGA, Genotype-Tissue Expression, CCLE, UCSC Xena, cBioPortal, Human Protein Atlas, GeneCards, STRING, MSigDB, TISIDB, and TIMER2 databases. RESULTS: ORC6 expression was significantly upregulated in 29 types of cancers compared to the corresponding normal adjacent tissues. ORC6 overexpression correlated with higher stage and worse prognostic outcomes in most cancer types analyzed. Additionally, ORC6 was involved in the cell cycle pathway, DNA replication, and mismatch repair pathways in most tumor types. A negative correlation was observed between the tumor endothelial cell infiltration and ORC6 expression in almost all tumors, whereas the immune infiltration of T regulatory cell was noted to be statistically positively correlated with the expression of ORC6 in prostate cancer tissues. Furthermore, in most tumor types, immunosuppression-related genes, especially TGFBR1 and PD-L1 (CD274), exhibited a specific correlation with the expression of ORC6. CONCLUSIONS: This comprehensive pan-cancer analysis revealed that ORC6 expression serves as a prognostic biomarker and that ORC6 is involved in the regulation of various biological pathways, the tumor microenvironment, and the immunosuppression status in several human cancers, suggesting its potential diagnostic, prognostic, and therapeutic value in pan-cancer, especially in prostate adenocarcinoma.

Melatonin ameliorates diabetic hyperglycaemia-induced impairment of Leydig cell steroidogenic function through activation of SIRT1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related complications are important health problems worldwide. The underlying mechanisms for diabetic male subfertility/infertility are considerably complicated and need to be unveiled for therapeutic intervention. Melatonin treatment was investigated to assess the beneficial effects on injured steroidogenic function in DM due to its regulatory roles in mitochondria and autophagy. METHODS: Diabetic hyperglycaemia was induced in rats injected with streptozotocin (STZ, 55 mg/kg/d) or simulated in TM3 Leydig cell line cultured with medium containing 30 mM D-glucose. Then, diabetic rats or the TM3 cells under high glucose were treated with melatonin. The diabetic rats were randomly divided into diabetes mellitus group (DM group), insulin treatment group (DM + INS group) and melatonin treatment group (DM + MT group). The TM3 Leydig cells were divided into a normal glucose control group (NG group), a high glucose treatment group (HG group), and a melatonin treatment group (HG + MT group). Then, Sirt1 (silent mating type information regulation 2 homologue) 1 expression was knocked down by siRNA. RESULTS: The results showed that hyperglycaemia induced a decline in steroidogenesis, accompanied by autophagy defects, mitochondrial dysfunction and oxidative stress, in rats in the DM group or TM3 Leydig cells in the HG group. Furthermore, reduced SIRT1 expression levels and hyperacetylation were found in Leydig cells of DM group. Melatonin treatment ameliorated hyperglycaemia-induced impairment of Leydig cell function with simultaneous stimulation of 5'-adenosine monophosphate activated protein kinase (AMPK)/SIRT1 activity and the expression of autophagy-related genes. With regards to mitochondrial function, it promoted mitochondrial biogenesis with elevated PGC-1α, NRF1 and mtTFA, improved mitochondrial morphology, increased BNIP3L-related mitophagy and alleviated oxidative stress. Further results revealed that knockdown of Sirt1 in Leydig cells prevented the protective effects provided by melatonin against high glucose treatment, and interestingly, neutralization of reactive oxygen species (ROS) by N-acetyl-L-cysteine pretreatment abolished the stimulatory effect of melatonin on AMPK/SIRT1 activity in Leydig cells and prevented the induction of autophagy and mitochondrial biogenesis in the context of high glucose, indicating that modulation of SIRT1 pathway by melatonin was closely linked to ROS levels and oxidative stress. CONCLUSIONS: These findings suggest that SIRT1 pathway plays essential roles in the pleiotropic actions of melatonin on Leydig cells and in the prevention of hyperglycaemia-induced steroidogenic dysfunction. The stimulatory action of melatonin on SIRT1 pathway is related to oxidative stress and its antioxidant property. Our data provide new evidence for the relationship of melatonin and SIRT1 pathway in the context of hyperglycaemia, and melatonin as a combination therapy may be useful to combat DM-related complications, especially male reproductive system injury.

Autophagy modulation in resveratrol protective effects on steroidogenesis in high-fat diet-fed mice and H2O2-challenged TM3 cells.

BACKGROUND: Autophagy dysregulation and oxidative stress play critical pathophysiological roles in developing obesity-related metabolic health disorders. This study aims to investigate how autophagy modulation is related to resveratrol (RSV) antioxidant activities and preventive effects on steroidogenesis decline associated with a high-fat diet (HFD) and oxidative damage. METHODS AND RESULTS: Eight-week-old C57BL/6 J male mice were fed with HFD with or without supplement RSV (400 mg/kg/day) by gavage for 16 weeks. The control group was fed with a standard diet with no RSV or the same amount of RSV. Mouse Leydig cell line TM3 cell was used for in vitro studies. Oxidative stress was induced in TM3 cells with H2O2, followed by RSV treatment plus autophagy activator rapamycin or autophagy inhibitor 3-methyladenine, respectively. RSV supplement could upregulate proteins level of StAR and mitochondrial proteins COX4 and mtTFA, indicating the amelioration of steroidogenesis decline and mitochondrial dysfunction caused by HFD. Antioxidants such as GPx4 and SOD2 were improved by RSV as well. The observation of autophagosomes and the changes in expressions of LC3II/I, Beclin1, and Atg7 indicated that RSV could reverse the autophagy defect associated with HFD. 3-methyladenine inhibition of autophagy partially abolished RSV protection on mitochondrial function and steroidogenesis in H2O2-challenged TM3 cells. However, the combination use of rapamycin and RSV did not improve protection on Leydig cells against oxidative damage. CONCLUSIONS: The stimulation of autophagy by RSV is closely linked to its antioxidant actions and positive impact on steroidogenesis in HFD mice. The findings suggest RSV is protective against obesity-related Leydig cell impairment.

Insulin on changes in expressions of aquaporin-1, aquaporin-5, and aquaporin-8 in submandibular salivary glands of rats with Streptozotocin-induced diabetes.

OBJECTIVE: This study aimed to explore the relationship between diabetic xerostomia and changes in aquaporin-1 (AQP1), aquaporin-5 (AQP5), and aquaporin-8 (AQP8) expression in the submandibular glands (SMGs), to further study the pathogenesis of diabetic xerostomia and to observe the therapeutic effect of insulin (INS). METHODS: Thirty SD rats were randomized equally into 3 groups: control group, diabetic model (DM) group and insulin (INS) group (n=10, respectively). The control group received no treatment. DM group and INS group were induced by a high-fat diet and streptozotocin intraperitoneal injection. After establishment of a diabetic rat model, the rats in INS group were treated with insulin. Then all rats were fed continuously with ordinary diet for 2 months. H&E staining was used to describe morphologic changes in the SMGs of rats. Immunohistochemistry was used to analyze the expressions and localization of AQP1, AQP5, and AQP8 in the SMGs. Computer image analysis was used to detect the mean optical density (MOD) values of AQP1, AQP5, and AQP8 expression, and changes in the diameters of acini and ducts. RESULTS: The acini were mildly atrophied and the acinar cells were rearranged in an irregular way. The morphology of insulin-administered diabetic SMGs was similar to that of the control group. The acinar average circumference and GCT average diameter in DM group were significantly reduced (P<0.05). The acinar average circumference and GCT average diameter of INS group were significantly increased (P<0.05). The expressions of AQP1, AQP5, and AQP8 were significantly reduced in DM group (P<0.05). The expressions of AQP1, AQP5, and AQP8 in INS group were significantly increased (P<0.05). CONCLUSION: The decreased expressions of AQP1, AQP5, and AQP8 led to decreased salivary secretion of SMGs in diabetic rats, which may be involved in the pathogenesis of diabetic xerostomia. Insulin could up-regulate the expressions of AQP1, AQP5 and AQP8, and play a protective role in the secretory function of diabetic SMGs.

Impact of diabetic hyperglycaemia and insulin therapy on autophagy and impairment in rat epididymis.

Blood glucose dysregulation and hyperglycaemia caused by diabetes mellitus are intimately associated with male infertility. Two-month-old Sprague-Dawley rats were given a single dose of streptozotocin (55 mg/kg) by intraperitoneal injection to induce type I diabetes mellitus (DM group). The treatment group was given 1 unit/day of insulin for 16 weeks (INS group). The normal control group (NC group) was given food ad libitum. In the DM group, the histological analysis of caput and cauda epididymal ducts showed broken stereocilia and more lipid vacuolisations in the principal cells. The interstitial hyperplasia and inflammatory cell infiltration were observed in epididymal tissues. Transmission electron microscopy observation showed that the principal cells in the DM group contained more vacuoles, partly lost stereocilia, and swollen mitochondria. The autophagosomes were observed as well. Western blotting results of LC3II/I and P62 protein expression indicated that autophagy was downregulated in the DM group. The total antioxidant activity and GPx5 expression of epididymal tissues were also decreased. In the INS group, significant improvements were observed in epididymal tissues. Our study suggests that diabetic hyperglycaemia causes autophagy dysregulation in epididymal tissues, which may play a role in diabetes-induced rat epididymal injury. Insulin treatment is beneficial for diabetic-associated epididymal dysfunction.

Salmonella enteritidis Hcp distribute in the cytoplasm and regulate TNF signaling pathway in BHK-21 cells.

Hemolysin-coregulated protein (Hcp) of Salmonella enteritidis is known to be a structural and effector protein of the T6SS, but little is known about the role of Hcp in host cells. In this study, Hcp was expressed by plasmid pEGFP-N1-hcp in BHK-21 cells and the results showed that the subcellular localization of Hcp was predominantly in the cytoplasm of BHK-21 cells. When Hcp was over-expressed by transfecting plasmid pCI-neo-hcp in BHK-21 cells and mRNA sequencing was performed to analyze differentially expressed genes, the results showed a change in the expression levels of 307 mRNAs (fold change > 2, and p < 0.01). Amongst these, 125 mRNAs were up-regulated and 182 mRNAs were down-regulated. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in tumor necrosis factor (TNF) signaling pathway, IL-17 signaling pathway, and cytokine-cytokine receptor interaction. Subsequently, we selected differentially expressed genes of TNF signaling pathway and verified the changes by real-time PCR. The results were consistent with the trend observed for the sequencing results. In conclusion, we demonstrated that Hcp of Salmonella enteritidis caused the change of mRNAs expression of TNF signaling pathway in the cytoplasm of BHK-21 cells.

Comparative proteomics reveals protective effect of resveratrol on a high-fat diet-induced damage to mice testis.

In recent years, resveratrol has been shown to protect against metabolic damage, including obesity-associated subfertility/infertility. In the present study, proteomic alterations in testicular tissues were investigated by tandem mass tag (TMT) in mice fed with a high-fat diet (HFD) without or with resveratrol supplementation (HFD+RSV). Serum testosterone levels, spermatozoa parameters and testicular histological morphology were assessed. Resveratrol treatment was shown to significantly reduce serum cholesterol, prevent the HFD-induced reductions in serum testosterone and spermatozoa parameters, and decrease the ultrastructural degeneration of testicular tissues. The comparative proteomics analysis revealed 58 differentially expressed proteins between the HFD and control groups and 38 differentially expressed proteins between the HFD and HFD+RSV groups. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the most highly enriched differential proteins were correlated to spermatozoa function and cholesterol metabolism. The real-time RT-PCR and western blotting results confirmed the differential expression of the corresponding proteins related to spermatozoa function that were identified by proteomics. The present study provides new insight into the mechanisms of the beneficial effects of resveratrol, and may present it as a potential therapeutic strategy for obesity-associated male subfertility/infertility.Abbreviations:TMT: Tandem mass tag; HFD: High-fat diet; RSV: Resveratrol; GO: Gene ontology; Protein-proteinKEGG: Kyoto Encyclopedia of Genes and Genomes; RT-PCR: Reverse transcription-polymerase chain reaction; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF: Polyvinylidene fluoride; ECL: Enhanced chemiluminescence; RIPA: Radio-immunoprecipitation assay; CTRL: Control; PPI: interaction; RIA: Radioimmunoassay; T: Testosterone; TG: Triglycerides; TC: Total cholesterol; LDL-c: Low-density lipoprotein cholesterol; HDL-c: High-density lipoprotein cholesterol; Crisp1: Cysteine-rich secretory protein 1; SIRT1: Sirtuin 1; GPx5: Glutathione peroxidase 5; Svs4: Seminal vesicle secretory protein 4; Tssk3: Testis-specific serine kinase 3; Pate4: Prostate and testis expressed 4; Sva: Seminal vesicle antigen; Lcn5: Lipocalin 5; Spinkl: Serine protease inhibitor, Kazal type-like.

Vitamin D3 inhibits lipopolysaccharide-induced placental inflammation through reinforcing interaction between vitamin D receptor and nuclear factor kappa B p65 subunit.

It is increasingly recognized that vitamin D3 (VitD3) has an anti-inflammatory activity. The present study investigated the effects of maternal VitD3 supplementation during pregnancy on LPS-induced placental inflammation and fetal intrauterine growth restriction (IUGR). All pregnant mice except controls were intraperitoneally injected with LPS (100 µg/kg) daily from gestational day (GD)15-17. In VitD3 + LPS group, pregnant mice were orally administered with VitD3 (25 µg/kg) before LPS injection. As expected, maternal LPS exposure caused placental inflammation and fetal IUGR. Interestingly, pretreatment with VitD3 repressed placental inflammation and protected against LPS-induced fetal IUGR. Further analysis showed that pretreatment with VitD3, which activated placental vitamin D receptor (VDR) signaling, specifically suppressed LPS-induced activation of nuclear factor kappa B (NF-κB) and significantly blocked nuclear translocation of NF-κB p65 subunit in trophoblast gaint cells of the labyrinth layer. Conversely, LPS, which activated placental NF-κB signaling, suppressed placental VDR activation and its target gene expression. Moreover, VitD3 reinforced physical interaction between placental VDR and NF-κB p65 subunit. The further study demonstrates that VitD3 inhibits placental NF-κB signaling in VDR-dependent manner. These results provide a mechanistic explanation for VitD3-mediated anti-inflammatory activity. Overall, the present study provides evidence for roles of VDR as a key regulator of placental inflammation.

Resveratrol attenuates inflammation and oxidative stress in epididymal white adipose tissue: implications for its involvement in improving steroidogenesis in diet-induced obese mice.

Chronic, low-grade systemic inflammation has been shown to play an important role in the development of obesity-related complications. Epididymal white adipose tissue (WAT) can influence testicular function through its endocrine function. The purpose of this study was to assess the effects of resveratrol on the epididymal WAT inflammatory response and on testicular steroidogenesis in obese individuals. Seven-week-old male C57BL/6J mice were fed a high-calorie and high-cholesterol diet (HCD group) or HCD supplemented with resveratrol (HCD+Res group) for 18 weeks. As we previously showed that resveratrol protects against Leydig cell steroidogenesis in HCD-induced obese mice, this study assessed macrophage infiltration in fat depots by measuring crown-like structure (CLS) density. Histological analysis showed that adipocyte size was significantly smaller and CLSs were less numerous in the HCD+Res group than the HCD group (P < 0.01). Additionally, resveratrol supplementation decreased Nfkb1 expression (P < 0.01) and increased the IκB-α protein abundance (P < 0.01) in epididymal WAT. Consistent with this alteration in NF-κB signaling, the expression of two classic proinflammatory cytokines, TNF-α (Tnfa) and IL-1ß (Il1b), were significantly decreased in the HCD+Res group compared with the HCD group (P < 0.01). Significant differences were also found in the expression of sirtuin1 (Sirt1) (P < 0.01) and manganese superoxide dismutase (Sod2) (P < 0.01) between the HCD and HCD+Res groups. Our data suggest that resveratrol can attenuate obesity-induced inflammation and oxidative stress in epididymal WAT, which partly accounts for its beneficial effects in testicular steroidogenesis.

Antifertility characteristics of the N-terminal region of mouse equatorial segment protein.

To investigate antifertility characteristics of the equatorial segment protein (ESP) and its potential immunocontraceptive effect, three partially overlapping cDNA fragments P1/P2/P3, together covering the entire mouse ESP, were cloned, expressed, and purified. The roles of P1/P2/P3 in fertility were investigated through in vitro fertilization and mouse mating test. Antibodies against P1/P2 significantly reduced the rates of fertilization in vitro in the zona-intact experiments. Coincubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of antibodies against P1/P2 also inhibited sperm-oolemma binding and fusion, while anti-P3 antibody virtually had no effect on in vitro fertilization at the same concentration. Immunization of female BALB/c mice with N-terminal of mouse ESP (recombinant P1 and P2) resulted in a significant decrease in the fertility rate as well as the litter size. Double immunofluorescence staining showed that mouse ESP protein was localized to the equatorial segment of acrosome of mouse sperm, and was exposed and surface-accessible after acrosome reaction. Mouse ESP was also demonstrated to have complementary binding sites on the mouse egg plasma membrane by indirect immunofluorescence assay. These findings suggest that the N-terminal of mouse ESP could play an important role in fertility and might be a vaccine candidate for contraception.

Immunocontraceptive potential of the Ig-like domain of Izumo.

To investigate whether the Ig-like domain of sperm protein Izumo or the other part of the protein could be used as an immunocontraceptive antigen, three partially overlapping cDNA fragments (PA, PB, and PC), together covering entire mouse Izumo, were cloned, expressed, and purified. PB contains the whole Ig-like domain of mouse Izumo. The anti-PB antibody significantly inhibited the fusion of sperm with zona-free mouse eggs with no effect on sperm motility, while anti-PA and anti-PC antibodies virtually had no effect on sperm-egg fusion at the same concentration. Furthermore, in the presence of anti-PB antibody, the anti-sperm reactivity could be competitively inhibited by recombinant PB protein. The PB-specific antibody staining was restricted to the acrosome region in acrosome-reacted mouse spermatozoa by indirect immunofluorescence. Active immunization with the PB antigen sharply raised the antibody titers in mouse that were enough to cause a significant reduction in fertility compared to the PA and PC immunized groups. In conclusion, our data indicate that the Ig-like domain of Izumo plays an important role in the fertilization process, as verified by the dose-dependent reduction in fertilization rates in mouse IVF trials and mouse mating assay. These results indicate that the Ig-like domain of Izumo might be a new candidate for the development of a contraceptive vaccine.