Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway†.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug and immunosuppressant in the clinic, and the hypoandrogenism caused by CP is receiving more attention. Some studies found that ferroptosis is a new mechanism of cell death closely related to chemotherapeutic drugs and plays a key role in regulating reproductive injuries. The purpose of this study is to explore ferroptosis' role in testicular Leydig cell dysfunction and molecular mechanisms relating to it. In this study, the level of ferroptosis in the mouse model of testicular Leydig cell dysfunction induced by CP was significantly increased and further affected testosterone synthesis. The ferroptosis inhibitors ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) can improve injury induced by CP. The results of immunohistochemistry showed that Fer-1 and DFO could improve the structural disorder of seminiferous tubules and the decrease of the number of Leydig cells in testicular tissue induced by CP. Immunofluorescence and western blot confirmed that Fer-1 and DFO could improve the expression of key enzymes in testosterone synthesis. The activation of SMAD family member 2 (Smad2)/cyclin-dependent kinase inhibitor 1A (Cdkn1a) pathway can improve the ferroptosis of Leydig cells induced by CP and protect the function of Leydig cells. By inhibiting the Smad2/Cdkn1a signal pathway, CP can regulate ferroptosis, resulting in testicular Leydig cell dysfunction. In this study, CP-induced hypoandrogenism is explained theoretically and a potential therapeutic strategy is provided.

Artificial intelligence-based model for lymph node metastases detection on whole slide images in bladder cancer: a retrospective, multicentre, diagnostic study.

BACKGROUND: Accurate lymph node staging is important for the diagnosis and treatment of patients with bladder cancer. We aimed to develop a lymph node metastases diagnostic model (LNMDM) on whole slide images and to assess the clinical effect of an artificial intelligence-assisted (AI) workflow. METHODS: In this retrospective, multicentre, diagnostic study in China, we included consecutive patients with bladder cancer who had radical cystectomy and pelvic lymph node dissection, and from whom whole slide images of lymph node sections were available, for model development. We excluded patients with non-bladder cancer and concurrent surgery, or low-quality images. Patients from two hospitals (Sun Yat-sen Memorial Hospital of Sun Yat-sen University and Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong, China) were assigned before a cutoff date to a training set and after the date to internal validation sets for each hospital. Patients from three other hospitals (the Third Affiliated Hospital of Sun Yat-sen University, Nanfang Hospital of Southern Medical University, and the Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong, China) were included as external validation sets. A validation subset of challenging cases from the five validation sets was used to compare performance between the LNMDM and pathologists, and two other datasets (breast cancer from the CAMELYON16 dataset and prostate cancer from the Sun Yat-sen Memorial Hospital of Sun Yat-sen University) were collected for a multi-cancer test. The primary endpoint was diagnostic sensitivity in the four prespecified groups (ie, the five validation sets, a single-lymph-node test set, the multi-cancer test set, and the subset for a performance comparison between the LNMDM and pathologists). FINDINGS: Between Jan 1, 2013 and Dec 31, 2021, 1012 patients with bladder cancer had radical cystectomy and pelvic lymph node dissection and were included (8177 images and 20 954 lymph nodes). We excluded 14 patients (165 images) with concurrent non-bladder cancer and also excluded 21 low-quality images. We included 998 patients and 7991 images (881 [88%] men; 117 [12%] women; median age 64 years [IQR 56-72]; ethnicity data not available; 268 [27%] with lymph node metastases) to develop the LNMDM. The area under the curve (AUC) for accurate diagnosis of the LNMDM ranged from 0·978 (95% CI 0·960-0·996) to 0·998 (0·996-1·000) in the five validation sets. Performance comparisons between the LNMDM and pathologists showed that the diagnostic sensitivity of the model (0·983 [95% CI 0·941-0·998]) substantially exceeded that of both junior pathologists (0·906 [0·871-0·934]) and senior pathologists (0·947 [0·919-0·968]), and that AI assistance improved sensitivity for both junior (from 0·906 without AI to 0·953 with AI) and senior (from 0·947 to 0·986) pathologists. In the multi-cancer test, the LNMDM maintained an AUC of 0·943 (95% CI 0·918-0·969) in breast cancer images and 0·922 (0·884-0·960) in prostate cancer images. In 13 patients, the LNMDM detected tumour micrometastases that had been missed by pathologists who had previously classified these patients' results as negative. Receiver operating characteristic curves showed that the LNMDM would enable pathologists to exclude 80-92% of negative slides while maintaining 100% sensitivity in clinical application. INTERPRETATION: We developed an AI-based diagnostic model that did well in detecting lymph node metastases, particularly micrometastases. The LNMDM showed substantial potential for clinical applications in improving the accuracy and efficiency of pathologists' work. FUNDING: National Natural Science Foundation of China, the Science and Technology Planning Project of Guangdong Province, the National Key Research and Development Programme of China, and the Guangdong Provincial Clinical Research Centre for Urological Diseases.

Construction and experimental validation of a macrophage cell senescence-related gene signature to evaluate the prognosis, immunotherapeutic sensitivity, and chemotherapy response in bladder cancer.

Tumor-associated macrophages (TAMs) are pivotal components of tumor microenvironment (TME), and senescent TAMs contribute to the alternation of the profiles of TME. However, the potential biological mechanisms and the prognosis value of senescent macrophages are largely unknown, especially in bladder cancer (BLCA). Based on the single-cell RNA sequencing of a primary BLCA sample, 23 macrophage-related genes were identified. Genomic difference analysis, LASSO, and Cox regression were used to develop the risk model. TCGA-BLCA cohort (n = 406) was utilized as the training cohort, and then, three independent cohorts (n = 90, n = 221, n = 165) from Gene Expression Omnibus, clinical samples from the local hospital (n = 27), and in vitro cell experiments were used for external validation. Aldo-keto reductase family 1 member B (AKR1B1), inhibitor of DNA binding 1 (ID1), and transforming growth factor beta 1 (TGFB1I1) were determined and included in the predictive model. The model serves as a promising tool to evaluate the prognosis in BLCA (pooled hazard ratio = 2.51, 95% confidence interval = [1.43; 4.39]). The model was also effective for the prediction of immunotherapeutic sensitivity and chemotherapy treatment outcomes, which were further confirmed by IMvigor210 cohort (P < 0.01) and GDSC dataset, respectively. Twenty-seven BLCA samples from the local hospital proved that the risk model was associated with the malignant degree (P < 0.05). At last, the human macrophage THP-1 and U937 cells were treated with H2O2 to mimic the senescent process in macrophage, and the expressions of these molecules in the model were detected (all P < 0.05).Overall, a macrophage cell senescence-related gene signature was constructed to predict the prognosis, immunotherapeutic response, and chemotherapy sensitivity in BLCA, which provides novel insights to uncover the underlying mechanisms of macrophage senescence.

Ergothioneine attenuates varicocele-induced testicular damage by upregulating HSP90AA1 in rats.

This study investigates the therapeutic effect and the underlying mechanisms of ergothioneine (EGT) on the testicular damage caused by varicocele (VC) in vivo, in vitro, and in silico. This preclinical study combines a series of biological experiments and network pharmacology analyses. A total of 18 Sprague Dawley (SD) male rats were randomly and averagely divided into three groups: the sham-operated, VC model, and VC model with EGT treatment (VC + EGT) groups. The left renal vein of the VC model and the VC + EGT groups were half-ligated for 4 weeks. Meanwhile, the VC + EGT group was intragastrically administrated with EGT (10 mg/kg). GC1 and GC2 cells were exposed to H2 O2 with or without EGT treatment to re-verify the conclusion. The structure disorder of seminiferous tubules ameliorated the apoptosis decrease in the VC rats receiving EGT. EGT can also increase the sperm quality of the VC model rats (p < 0.05). The exposure to H2 O2 decreased proliferation and increased apoptosis of GC1 and GC2 cells, which was revisable by adding EGT to the plates (p < 0.05). The network pharmacology and molecular docking were conducted to explore the potential targets of EGT in VC, and HSP90AA1 was identified as the pivotal gene, which was validated by western blot, immunohistochemistry, and RT-qPCR both in vivo and in vitro (p < 0.05). Overall, EGT attenuates the testicular injury in the VC model both in vivo and in vitro by potentially potentiating the expression of HSP90AA1.

[Bone marrow mesenchymal stem cell-derived exosomes protect TM3 Leydig cells from cyclophosphamide-induced injury].

OBJECTIVE: To investigate the effect of exosomes derived from mouse bone marrow mesenchymal stem cells (BMSC) on the injury of TM3 Leydig cells induced by cyclophosphamide (CP). METHODS: The exosomes from BMSCs were extracted by ultrahigh speed centrifugation, and their particle size and morphology observed under the electron microscope, and their typical marker proteins examined by Western blot. The uptake of exosomes by TM3 Leydig cells was observed by co-culturing the exosomes with the TM3 cells. The viability and apoptosis rate of the TM3 cells in the normal control, CP-induction and CP+exosomes groups were detected using the CCK-8 method and flow cytometry respectively. ELISA was used to measure the testosterone (T) level in the cell supernatant, and Western blot adopted to determine the expression level of the steroidogenic acute regulatory (StAR) protein, a key enzyme related to T synthesis. RESULTS: The viability of the TM3 Leydig cells was markedly decreased and the apoptosis rate of the cells remarkably increased in the CP-induction group compared with that in the normal control, but both significantly restored after co-culture with exosomes (P < 0.01 and P < 0.05). The T level in the supernatant and the expression of the StAR protein in the cells were lower in the CP-induction than in the normal control group, but both dramatically increased in the CP+exosomes group (P < 0.01). CONCLUSION: Exosomes from BMSCs and protect TM3 Leydig cells from cyclophosphamide-induced injury and restore the level of testosterone secreted by the TM3 cells to a certain extent.

Activating transcription factor 5 (ATF5) promotes tumorigenic capability and activates the Wnt/b-catenin pathway in bladder cancer.

BACKGROUND: In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. METHODS: Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. RESULTS: ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/ß-catenin pathway. CONCLUSIONS: This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

Down regulating PHGDH affects the lactate production of sertoli cells in varicocele.

BACKGROUND: Although varicocele is considered to be one of the leading causes of male infertility, the precise mechanism underlying how varicocele leads to male infertility is not completely understood. We found the lactate concentration on the varicocele side of the patients was decreased compare with peripheral venous blood. In the testicles, the lactate produced by the sertoli cells through the glycolysis pathway provides most of the energy needed for spermatogenesis, the reduction of lactate will affect spermatogenesis. The objective of this study was to investigate the mechanism of this abnormal energy metabolism phenomenon in varicocele. METHODS: In this study, we collected the testicular tissue from patients with varicocele, the glycolysis related proteins PHGDH was identified by iTRAQ proteomics technology. Experimental rat varicocele model was constructed according to our new clip technique, the mRNA and protein expression levels of PHGDH were examined with qRT-PCR and Western blotting. We constructed a sertoli cell of PHGDH down-regulation model, and then detected the glucose consumption, LDH activities and lactate production in the sertoli cells. Western blot was conducted to investigate the effects of PHGDH on the expression of phosphoserine phosphatase (PSPH) and Pyruvate kinase M2 (PKM2). Flow cytometry was used to detect the cell apoptosis and cell cycle in sertoli cells. RESULTS: The results showed that testicular protein PHGDH was down-regulated in patients with varicocele and in experimental rat varicocele model. Down-regulation of PHGDH in sertoli cells significantly decreased the glucose consumption, LDH activities and lactate production in the sertoli cells, indicating that the low expression of PHGDH ultimately led to a decrease in lactate production by affecting the glycolysis. The Western blot results showed that the down-regulation of PHGDH significantly reduced the expression of pathway protein PSPH and PKM2, leading to the reduction of lactate production. Moreover, PHGDH knockdown can promote apoptosis and inhibit cell cycle to affect cell growth. CONCLUSIONS: Overall, we conformed that varicocele lead to the decreasing of testis lactate production. Down-regulation of PHGDH in sertoli cells may mediate the process of abnormal glucose metabolism. Our study provide new insight into the mechanisms underlying metabolism-associated male infertility and suggests a novel therapeutic target for male infertility.

Predictive value of cadherin-11 for subsequent recurrence and progression in non-muscle invasive bladder cancer.

BACKGROUND: Cadherin-11 (CDH11) is a type II cadherin and reported to function as an oncogene in various cancers. Our present study aims to investigate the role of CDH11 in bladder cancer (BCA). METHODS: Bioinformatics analysis was performed in four independent microarray data including 56 non-muscle-invasive bladder cancer (NMIBC) and 132 muscle-invasive bladder cancer (MIBC) tissues from Gene Expression Omnibus to screen out differentially expressed genes. Next, we detected CDH11 expression in BCA specimens and cell lines by qPCR and western blotting assays. Immunohistochemical analyses were performed in 209 paraffin-embedded BCA samples and 30 adjacent normal bladder tissues. RESULTS: Bioinformatics analysis revealed that CDH11 had a higher expression level in MIBC tissues than in NMIBC, which was consistent with our clinical BCA specimens and cell lines at both mRNA and protein levels. Immunohistochemical analysis demonstrated that over-expression of CDH11 was closely related to the histological grade, pT status, tumour size and poor outcomes of BCA patients. What's more, CDH11 (area under curve (AUC) = 0.673 and 0.735) had a better predictive value than E-cadherin (AUC = 0.629 and 0.629) and a similar discrimination with the European Organization for Research and Treatment of Cancer (EORTC) score system (AUC = 0.719 and 0.667) in evaluating potential recurrence and progression of NMIBC. Moreover, combination of CDH11 and EORTC score system was the best predictive model in predicting recurrence of NMIBC (AUC = 0.779) among the three models. CONCLUSIONS: CDH11 was a reliable therapeutic target in BCA and a useful index to predict the possibilities of recurrence and progression in NMIBC patients.

Optimal Starting Age and Baseline Level for Repeat Tests: Economic Concerns of PSA Screening for Chinese Men - 10-Year Experience of a Single Center.

OBJECTIVE: To investigate the optimal age for the baseline serum prostate-specific antigen (PSA) test and for repeat screening and its economic burden in a single center in China. MATERIALS AND METHODS: 35,533 men with PSA screening were retrospectively enrolled in this study. Follow-ups were conducted in 1,586 men with PSA >4 ng/mL, and receiver-operating characteristic (ROC) curves were employed to investigate the optimal cutoffs. RESULTS: ROC analysis indicated that the optimal age for initial PSA screening was 57.5 years (AUC = 0.84), 62.5 years (AUC = 0.902), 60.5 years (AUC = 0.909), and 61.5 years (AUC = 0.890) for individuals with PSA >4 and >10 ng/mL, a diagnosis of prostate cancer (PCa), and clinically significant PCa defined as the focus events, respectively. For Chinese men aged 50-59, 60-69, and >70 years, the initial PSA levels of 1.305 ng/mL (AUC = 0.699), 1.975 ng/mL (AUC = 0.711), and 2.740 ng/mL (AUC = 0.720) might have a PSA velocity >0.75 ng/mL per year during the follow-up. In addition, the total cost amounts to CNY 13,609,260 in these cases, but only 60 of the 35,533 (0.17%) men gained benefit from PSA screening. CONCLUSION: In our opinion, the optimal starting age for initial PSA testing was 57.5 years. The necessity for repeat screening should be based on the first PSA level depending on age. A cost--benefit analysis should be included in population-based screening.

[Effects of silencing the SEMG1 protein on the cycle and apoptosis of GC-1 spg cells].

OBJECTIVE: To investigate the effects of silencing the semenogelin 1 (SEMG1) protein on the cycle and apoptosis of the spermatogonia germ cell line (GC-1 spg). METHODS: SEMG1-specific siRNA was transfected into GC-1 spg cells by lipofectamine 2000 (the siRNA-SEMG1 group), the relative expression levels of the SEMG1 protein in the GC-1 spg cells of the siRNA-SEMG1, blank control and negative control groups were detected by Western blot, and the apoptosis and cycle of the cells in different groups were determined by flow cytometry. RESULTS: The expression of the SEMG1 protein in the GC-1 spg cells was dramatically decreased in the siRNA-SEMG1 group compared with those in the blank and negative control groups (1.80±0.05 vs 2.51±0.13 and 2.50±0.12， P < 0.01), but the apoptosis rate was remarkably higher in the former than in the latter two groups (ï¼»6.77 ± 0.15ï¼½% vs ï¼»0.70 ± 0.06ï¼½% and ï¼»0.8 ± 0.06ï¼½%, P < 0.01). No statistically significant difference was observed in the cell cycles among the three groups (P > 0.05). In addition, Western blot showed that the expression of the caspase-3 protein was significantly higher and that of the BCL2 protein markedly lower in the siRNA-SEMG1 than in the blank and negative control groups (P < 0.05). CONCLUSIONS: SEMG1-specific siRNA can effectively silence the expression of the SEMG1 protein in GC-1 spg cells and promote their apoptosis.

With a new clip technique surgically inducing varicocele in Sprague-Dawley rats.

BACKGROUND: We introduced and recreated a more consistent and effective experimental varicocele rat model by a new clip technique. METHODS: A total of 40 rats were numbered and randomly assigned to 5 groups of 8 each, including sham surgery (Group I), conventional (Group II) and clip groups with 0.7, 0.8, 0.9 mm gap widths, respectively (Group III, IV, V). All of the rats in each group were sacrificed at 8 weeks after initial surgery, and the rats forming out with less than 1 mm diameter of left spermatic vein or no presence of the pampiniform plexus dilation were excluded from the experimental groups. The left spermatic vein (LSV) diameter, testicular weight, left kidney weight to body weight coefficients, kidney and testicular histology were determined. RESULTS: The baseline mean diameter of the LSV in Group I, II and III was 0.22 ± 0.02, 0.23 ± 0.02 and 0.22 ± 0.03 mm, respectively (P = 0.7504). At 8 weeks after initial surgery, varicocele was successfully created in 6/8 (75%), 7/8 (87.5%), 3/8 (37.5%), 3/8 (37.5%) in GroupII-V, no varicocele was observed in Group I. In Group I, II and III, no pathological changes were observed and the left kidney weight to body weight coefficients showed no significant differences. The diameter of LSV was remarkably increased both in Group II and III compared to Group I (1.72 ± 0.13, 1.57 ± 0.19 and 0.25 ± 0.02, respectively), and Group II and III had a smaller testicular weight than the rats in Group I (1.67 ± 0.05, 1.62 ± 0.06, and 1.92 ± 0.12, respectively). CONCLUSIONS: With a new clip technique, surgically inducing varicocele rat model becomes convenient and safe. This appears to improve the effectiveness of the model and this innovation may allow us to further understand the pathophysiology of varicocele.

C14orf166 is a high-risk biomarker for bladder cancer and promotes bladder cancer cell proliferation.

BACKGROUND: C14orf166 (chromosome 14 open reading frame 166) plays a crucial role in some tumors, but its role in bladder cancer hasn't been explored. METHOD: We determined C14orf166 expression in uroepithelial cell, bladder cancer cells, normal bladder tissues and bladder cancer tissues using quantitative RT-PCR and western blot, we then analyzed the correlation between C14orf166 expression and clinicopathologic characteristics in a cohort of 149 patients with bladder cancer. Finally we downregulated C14orf166 and determined its role in the proliferation of bladder cancer cell lines using MTT assay, colony formation assay and cell cycle assay. RESULTS: We demonstrated C14orf166 was upregulated in bladder cancer cells and tissues, C14orf166 expression was significantly correlated with larger tumor size (P = 0.001), lymph node involvement (P < 0.001), histological differentiation (P < 0.001), survival time and vital states, and high C14orf166 expression correlated with poor survival, these results suggested C14orf166 served as a high-risk marker for bladder cancer. Knockdown of C14orf166 decreased the proliferation rate and colony formation ability of bladder cancer cells, and arrested cell cycle in G1/S transition. Further analysis showed that C14orf166 knockdown caused abnormal expression of key proteins for G1/S transition, such as Cyclin D1, P21, P27 and Rb phosphorylation. CONCLUSIONS: This study demonstrates that C14orf166 promotes bladder cancer cell proliferation and can be a novel prognostic biomarker for patients with bladder cancer.

[Changed percentage of myeloid-derived suppressor cells in the peripheral blood of prostate cancer patients and its clinical implication].

OBJECTIVE: To investigate the changes in the percentage of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of prostate cancer (PCa) patients and explore the correlation of MDSCs and their subsets with the prognosis of PCa. METHODS: Using flow cytometry, we determined the percentage of MDSCs and the levels of Arg-1, iNOS and PD-L1 in the peripheral blood of 32 PCa patients and 25 healthy controls, detected the distribution of CD14+ Mo-MDSC and CD15+ PMN-MDSC subsets, and analyzed the correlation between the obtained parameters and the prognosis of PCa. RESULTS: Compared with the healthy controls, the PCa patients showed significant increases in the percentage of MDSCs (P<0.01) and levels of Arg-1, iNOS and PD-L1 in the peripheral blood. Statistically significant differences were observed in the distribution of the CD14+ Mo-MDSC and CD15+ PMN-MDSC subsets between the two groups（60.4% vs 72.2%, 29.5% vs 18.8%） (P<0.05). The percentages of MDSCs and Mo-MDSCs were remarkably correlated with the total survival rate of the PCa patients (P=0.025 and 0.017). CONCLUSIONS: The percentages of MDSCs and CD14+ Mo-MDSCs in the peripheral blood were correlated with the prognosis of PCa, which may provide a target or some evidence for the clinical treatment of PCa.

[Application value of real-time contrast-enhanced ultrasound in transrectal prostate biopsy].

OBJECTIVE: To explore the application value of real-time contrast-enhanced ultrasound (RTCEU) in improving the detection rate of transrectal ultrasound-guided prostate biopsy. METHODS: This prospective study included 91 male patients with abnormally high PSA (4－20 µg/L) or abnormalities in DRE or MRI, who underwent 12+X prostate biopsy following conventional transrectal ultrasonography (TRUS) and RTCEU examination. We compared the numbers of suspected prostatic nodules before and after RTCEU as well as the detection rates of prostate cancer between conventional TRUS-guided 12PBx and 12PBx plus lesion-targeted biopsy procedures. RESULTS: Totally, 57 of the 86 suspected lesions on TRUS (66.3%), and 108 of the 118 abnormal nodules on RTCEU (91.5%) were confirmed to be prostate cancer. RTCEU achieved a significantly higher detection rate than TRUS (P<0.01). A total of 39 cases of prostate cancer (42.8%) were detected by RTCEU, while only 28 (30.7%) by TRUS, with statistically significant difference in the detection rate between the two procedures (P=0.033). CONCLUSIONS: Real-time contrast-enhanced ultrasound can significantly improve the detection rate of prostate cancer and provide a valuable guide to targeted prostate biopsy.

The expression of cysteine-rich secretory protein 2 (CRISP2) and its specific regulator miR-27b in the spermatozoa of patients with asthenozoospermia.

Cysteine-rich secretory protein 2 (CRISP2) is an important sperm protein and plays roles in spermatogenesis, modulation of flagellar motility, acrosome reaction, and gamete fusion. Clinical evidence shows a reduced CRISP2 expression in spermatozoa from asthenozoospermic patients, but the molecular mechanism underlying its reduction remains unknown. Herein, we carried out a study focusing on the CRISP2 reduction and its roles in asthenozoospermia. Initially, through analyzing CRISP2 expression and methylation on CRISP2 promoter activity in sperm, we observed a decreased expression of CRISP2 protein rather than its mRNA in the ejaculated spermatozoa from asthenozoospermic patients and no methylation in the CRISP2 promoter, suggesting CRISP2 expression may be regulated in the sperm at the posttranscriptional level. Subsequently, we found that microRNA 27b (miR-27b), predicted as a candidate regulator of CRISP2 using bioinformatics, was highly expressed in the ejaculated spermatozoa from asthenozoospermic patients. Luciferase reporter assay and transfection experiments disclosed that this microRNA could target CRISP2 by specifically binding its 3' untranslated region, suppressing CRISP2 expression. Extended clinical observation further confirmed a highly expressed miR-27b and its obviously negative correlation with CRISP2 protein expression in ejaculated spermatozoa samples from asthenozoospermic patients. Finally, we conducted a retrospective follow-up study to support that either high miR-27b expression or low CRISP2 protein expression was significantly associated with low sperm progressive motility, abnormal morphology, and infertility. Thus, this study provides the first preliminary insight into the mechanism leading to the reduced CRISP2 expression in asthenozoospermia, offering a potential therapeutic target for treating male infertility or for male contraception.

Statin use and risk of gallstone disease: A meta-analysis.

AIM: There is emerging evidence from animal and human studies that current statins can decrease the formation of gallbladder cholesterol gallstones and subsequently decrease the risk of gallstone disease, but consistent results have not been reported. We performed a meta-analysis to provide an overview of the relevant studies. METHODS: Relevant studies published between January 1980 and February 2014 were identified by searching Medline, Embase and the Cochrane Library. Studies were selected using a priori defined criteria. The strength of the relationship between statin use and risk of gallstone disease was assessed by adjusted odds ratio (OR). RESULTS: A total of 622 868 participants from six studies (four case-control studies, one cohort study and one cross-sectional study) were identified in this meta-analysis. The studies provided adjusted overall OR estimates for current statin use versus non-use, leading to a pooled OR of 0.86 (95% confidence interval [CI], 0.77-0.97; P < 0.001). The overall OR of population-based case-control studies and cholecystectomy due to gallstone disease were 0.83 (95% CI, 0.73-0.95; P = 0.0131) and 0.78 (95% CI, 0.74-0.82; P = 0.615), respectively. CONCLUSION: There is evidence that current statin use lowers the risk of gallstone disease compared with non-use, especially for cholecystectomy due to gallstone disease. Low statin use (1-4 prescriptions) did not decrease the risk of gallstone disease, but moderate and high statin use significantly decreased the risk. Further multicenter and better controlled studies are needed to confirm these findings.

A 3-Gene Random Forest Model to Diagnose Non-obstructive Azoospermia Based on Transcription Factor-Related Henes.

Non-obstructive azoospermia (NOA) is one of the most severe forms of male infertility, but its diagnosis biomarkers with high sensitivity and specificity are largely unknown. Transcription factors (TFs) play essential roles in many pathological processes in different diseases. Herein, we aimed to identify the TFs showing high diagnosis ability for NOA through machine learning algorithms. The transcriptome data of the testicular tissue from 11 control and 47 NOA subjects were set as the training dataset; meanwhile, 1665 TFs were retrieved from the HumanTFDB. Through the feature extraction methods, including genomic difference analysis, Lasso, Boruta, SVM-RFE, and logistic regression, ETV2, TBX2, and ZNF689 were ultimately screened and then were included in the random forest (RF) diagnosis model. The RF model displayed high predictive power in the training (F-measure = 1) and two external validation (n = 31, F-measure = 0.902; n = 20, F-measure = 0.941) cohorts. The seminal plasma and testicular biopsy samples of 20 control and 20 NOA patients were collected from the local hospital, and the expression levels of ETV2, TBX2, and ZNF689 were measured via RT-qPCR and immunohistochemistry. The RF model could also distinguish the NOA samples in the local cohort (F-measure = 0.741). Single-cell RNA sequencing analysis, which was based on the 432 testicular cell samples from an NOA patient, showed that ETV2, TBX2, and ZNF689 were all significantly associated with spermatogenesis. In all, a 3-TF random forest diagnosis model was successfully established, providing novel insights into the latent mechanisms of NOA.

Hub gene associated with prognosis in bladder cancer is a novel therapeutic target.

Objective: Bladder cancer is a clinical and social conundrum due to its high incidence and recurrence rate. It is urgent to find new targets for the diagnosis and treatment of bladder cancer and improve the prognosis and survival rate of bladder cancer patients. We sought a prognosis-related gene, built related models of evaluated bladder cancer and identified the function of the hub gene in bladder cancer. Methods: We downloaded the data of bladder cancer patients from the TCGA database, and used differentially expressed genes (DEGs), copy number variation (CNV) and survival analysis to scan the hub genes associated with prognosis in bladder cancer. Then, multi-factor cox regression was used to obtain the bladder cancer prognosis correlation model. Then, we analyzed the relationship between the expression of hub gene and immune microenvironment of bladder cancer. The relationship between the expression of hub gene and prognosis in bladder cancer patients was verified by immunohistochemistry. Cell proliferation assay and drug sensitivity test in vivo were used to verify the inhibition of bladder cancer by targeted inhibitors. Results: In bladder cancer, we screened seven hub genes (ACLY, CNP, NKIRAS2, P3H4, PDIA6, VPS25 and XPO1) associated with survival. Moreover, the multifactor regression model constructed with hub gene can well distinguish the prognosis of bladder cancer. Hub gene is mostly associated with immune microenvironment. Immunohistochemical results basically confirmed the importance of XPO1 in bladder cancer. Selinexor (an inhibitor of XPO1) could effectively inhibit the proliferation of bladder cancer in the cell proliferation experiments by CCK-8 assays and it could suppress the growth of bladder cancer in mouse bladder cancer model. Conclusions: In this study, a prognostic model with seven hub genes has provided great help for the prognosis prediction of bladder cancer patients. And XPO1 is an important target affecting the prognosis of bladder cancer, and inhibition of XPO1 can effectively inhibit bladder cancer proliferation and growth.

Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway.

Cyclophosphamide-induced testosterone deficiency (CPTD) during the treatment of cancers and autoimmune disorders severely influences the quality of life of patients. Currently, several guidelines recommend patients suffering from CPTD receive testosterone replacement therapy (TRT). However, TRT has many disadvantages underscoring the requirement for alternative, nontoxic treatment strategies. We previously reported bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exos) could alleviate cyclophosphamide (CP)-induced spermatogenesis dysfunction, highlighting their role in the treatment of male reproductive disorders. Therefore, we further investigated whether BMSCs-exos affect autophagy and testosterone synthesis in Leydig cells (LCs). Here, we examined the effects and probed the molecular mechanisms of BMSCs-exos on CPTD in vivo and in vitro by detecting the expression levels of genes and proteins related to autophagy and testosterone synthesis. Furthermore, the testosterone concentration in serum and cell-conditioned medium, and the photophosphorylation protein levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were measured. Our results suggest that BMSCs-exos could be absorbed by LCs through the blood-testis barrier in mice, promoting autophagy in LCs and improving the CP-induced low serum testosterone levels. BMSCs-exos inhibited cell death in CP-exposed LCs, regulated the AMPK-mTOR signaling pathway to promote autophagy in LCs, and then improved the low testosterone synthesis ability of CP-induced LCs. Moreover, the autophagy inhibitor, 3-methyladenine (3-MA), significantly reversed the therapeutic effects of BMSCs-exos. These findings suggest that BMSCs-exos promote LC autophagy by regulating the AMPK-mTOR signaling pathway, thereby ameliorating CPTD. This study provides novel evidence for the clinical improvement of CPTD using BMSCs-exos.

Overexpression of SPHK1 associated with targeted therapy resistance in predicting poor prognosis in renal cell carcinoma.

Background: Sphingosine kinase 1 (SPHK1) is a key enzyme that catalyzes the phosphorylation of sphingosine. Recent studies reported SPHK1 to be associated with renal cell carcinoma (RCC) progression by inducing targeted therapy resistance. However, the expression and the clinical significance of SPHK1 on RCC in those having received targeted therapy have not been elucidated. The present study explored the expression of SPHK1 in RCC tissues from targeted therapy recipients, the correlation of SPHK1 with clinicopathological parameters, and the effect of SPHK1 on RCC patient prognosis. Methods: Differential gene expression analysis of RCC treated with and without targeted therapy was performed. The correlations of SPHK1 expression with clinical parameters of RCC were examined. Gene set enrichment analysis (GSEA) was performed to clarify the potential role of SPHK1 associated with targeted therapy resistance. The value of SPHK1 as a diagnostic marker for RCC was also evaluated. The Kaplan-Meier method was applied to analyze the correlation between SPHK1 expression and patient survival rate by using the clinical data from patients with RCC. Results: Significant overexpression of SPHK1 was detected in RCC treated with targeted therapy. SPHK1 expression was closely correlated with RCC progression-related clinicopathological parameters. Therefore, elevated SPHK1 could effectively diagnose RCC and distinguish RCC with an advanced clinical stage and a high pathological grade. SPHK1 was associated with the stemness of RCC cells via the activation of the Wnt, Hedgehog, or Notch signaling pathways in targeted drug-treated or untreated RCC. Survival analysis of a large cohort of RCC samples indicated overexpression of SPHK1 to be inversely correlated with the overall and disease-free survival of patients with RCC. Conclusions: Our study indicated that SPHK1 associated with targeted therapy resistance could serve as a potential prognostic marker and a valuable biomarker of response to angiogenic agents in RCC.