TFAP2C Knockdown Sensitizes Bladder Cancer Cells to Cisplatin Treatment via Regulation of EGFR and NF-κB.

Cisplatin is the first-line chemotherapy for advanced or metastatic bladder cancer. Nevertheless, approximately half of patients with BCa are insensitive to cisplatin therapy or develop cisplatin resistance during the treatment process. Therefore, it is especially crucial to investigate ways to enhance the sensitivity of tumor cells to cisplatin. Transcription factor AP-2 gamma (TFAP2C) is involved in cancer development and chemotherapy sensitivity. However, its relationship with chemotherapy has not been studied in BCa. In this study, we aimed to investigate the therapeutic potential of TFAP2C in human BCa. Results based on TCGA (The Cancer Genome Atlas), GTEx (The Genotype-Tissue Expression) and GEO (Gene Expression Omnibus) data showed that TFAP2C expression was upregulated in BCa tissues and that its high expression was associated with poor prognosis. Meanwhile, we demonstrated the overexpression of TFAP2C in BCa clinical specimens. Subsequently, in vitro, we knocked down TFAP2C in BCa cells and found that TFAP2C knockdown further increased cell cycle arrest and apoptosis caused by cisplatin. In addition, the inhibitory effect of cisplatin on BCa cell migration and invasion was enhanced by TFAP2C knockdown. Our data indicated that cisplatin increased epidermal growth factor receptor (EGFR) and nuclear factor-kappaB (NF-κB) activation levels, but TFAP2C knockdown suppressed this effect. Finally, in vivo data further validated these findings. Our study showed that TFAP2C knockdown affected the activation levels of EGFR and NF-κB and enhanced the anti-tumor effects of cisplatin in vivo and in vitro. This provides a new direction to improve the efficacy of traditional cisplatin chemotherapy.

Phototherapy with Cancer-Specific Nanoporphyrin Potentiates Immunotherapy in Bladder Cancer.

PURPOSE: Immune checkpoint inhibitors (ICI) in general have shown poor efficacy in bladder cancer. The purpose of this project was to determine whether photodynamic therapy (PDT) with bladder cancer-specific porphyrin-based PLZ4-nanoparticles (PNP) potentiated ICI. EXPERIMENTAL DESIGN: SV40 T/Ras double-transgenic mice bearing spontaneous bladder cancer and C57BL/6 mice carrying syngeneic bladder cancer models were used to determine the efficacy and conduct molecular correlative studies. RESULTS: PDT with PNP generated reactive oxygen species, and induced protein carbonylation and dendritic cell maturation. In SV40 T/Ras double-transgenic mice carrying spontaneous bladder cancer, the median survival was 33.7 days in the control, compared with 44.8 (P = 0.0123), 52.6 (P = 0.0054), and over 75 (P = 0.0001) days in the anti-programmed cell death-1 antibody (anti-PD-1), PNP PDT, and combination groups, respectively. At Day 75 when all mice in other groups died, only 1 in 7 mice in the combination group died. For the direct anti-tumor activity, compared with the control, the anti-PD-1, PNP PDT, and combination groups induced a 40.25% (P = 0.0003), 80.72% (P < 0.0001), and 93.03% (P < 0.0001) tumor reduction, respectively. For the abscopal anticancer immunity, the anti-PD-1, PNP PDT, and combination groups induced tumor reduction of 45.73% (P = 0.0001), 54.92% (P < 0.0001), and 75.96% (P < 0.0001), respectively. The combination treatment also diminished spontaneous and induced lung metastasis. Potential of immunotherapy by PNP PDT is multifactorial. CONCLUSIONS: In addition to its potential for photodynamic diagnosis and therapy, PNP PDT can synergize immunotherapy in treating locally advanced and metastatic bladder cancer. Clinical trials are warranted to determine the efficacy and toxicity of this combination.

Corrigendum: Comprehensive Analysis of N6-Methylandenosine-Related Long Non-Coding RNAs Signature in Prognosis and Tumor Microenvironment of Bladder Cancer.

[This corrects the article DOI: 10.3389/fonc.2022.774307.].

Comprehensive Analysis of N6-Methylandenosine-Related Long Non-Coding RNAs Signature in Prognosis and Tumor Microenvironment of Bladder Cancer.

To investigate the role of N6-methyladenosine (m6A)- related long non-coding RNAs (lncRNAs) in bladder cancer (BC). 50 m6A-related lncRNAs were screened out and were correlated with prognosis from BC samples in The Cancer Genome Atlas (TCGA). The lncRNAs were subdivided into cluster 1 and cluster 2 with consensus cluster analysis, and it was found that lncRNAs in cluster 2 were associated with poor prognosis and increased PD-L1 expression. Gene set enrichment analysis (GSEA) revealed tumor-related pathways in cluster 2. Through least absolute shrinkage and selection operator (LASSO) Cox regression analysis, univariate and multivariate Cox regression, and ROC analyses, 14 prognostic lncRNAs were selected and used to construct the m6A-related lncRNA prognostic signature (m6A-LPS), furthermore, that m6A-LPS was as a valuable independent prognostic factor. Interestingly, the m6A-LPS risk score was positively correlated with the immune score, PD-L1 expression, and the infiltration of immune cell subtypes in BC. SNHG16, a member of the high-risk group based on m6A-LPS, was highly expressed in BC tissues and cell lines and interfered with siRNA resulted in suppressed proliferation, migration, and invasion in vitro. Our study illustrates the role of m6A-related lncRNAs in BC. The m6A-LPS may be an important regulatory target of the tumor microenvironment (TME) in BC.

Synergistic antitumor activity of pan-PI3K inhibition and immune checkpoint blockade in bladder cancer.

BACKGROUND: Immune checkpoint blockade (ICB) induces durable response in approximately 20% of patients with advanced bladder urothelial cancer (aUC). Over 50% of aUCs harbor genomic alterations along the phosphoinositide 3-kinase (PI3K) pathway. The goal of this project was to determine the synergistic effects and mechanisms of action of PI3K inhibition and ICB combination in aUC. METHODS: Alterations affecting the PI3K pathway were examined in The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map databases. Human and mouse cells with Pten deletion were used for in vitro studies. C57BL/6 mice carrying syngeneic tumors were used to determine in vivo activity, mechanisms of action and secondary resistance of pan-PI3K inhibition, ICB and combination. RESULTS: Alterations along the PI3K pathway occurred in 57% of aUCs in TCGA. CRISPR (clustered regularly interspaced short palindromic repeats) knockout of PIK3CA induced pronounced inhibition of cell proliferation (p=0.0046). PI3K inhibition suppressed cancer cell growth, migration and colony formation in vitro. Pan-PI3K inhibition, antiprogrammed death 1 (aPD1) therapy and combination improved the overall survival (OS) of syngeneic mice with PTEN-deleted tumors from 27 days of the control to 48, 37, and 65 days, respectively. In mice with tumors not containing a PI3K pathway alteration, OS was prolonged by the combination but not single treatments. Pan-PI3K inhibition significantly upregulated CD80, CD86, MHC-I, and MHC-II in dendritic cells, and downregulated the transforming growth factor beta pathway with a false discovery rate-adjusted q value of 0.001. Interferon alpha response was significantly upregulated with aPD1 therapy (q value: <0.001) and combination (q value: 0.027). Compared with the control, combination treatment increased CD8+ T-cell infiltration (p=0.005), decreased Treg-cell infiltration (p=0.036), and upregulated the expression of multiple immunostimulatory cytokines and granzyme B (p<0.01). Secondary resistance was associated with upregulation of the mammalian target of rapamycin (mTOR) pathway and multiple Sprr family genes. CONCLUSIONS: The combination Pan-PI3K inhibition and ICB has significant antitumor effects in aUC with or without activated PI3K pathway and warrants further clinical investigation. This combination creates an immunostimulatory tumor milieu. Secondary resistance is associated with upregulation of the mTOR pathway and Sprr family genes.

SC66 inhibits the proliferation and induces apoptosis of human bladder cancer cells by targeting the AKT/ß-catenin pathway.

Bladder cancer (BC) is a major disease of the genitourinary tract, and chemotherapy is one of the main treatments commonly used at present. SC66 is a new type of allosteric AKT inhibitor that is reported to play an effective inhibitory role in the progression of many other types of tumours, but there is no reported research on its role in BC. In this study, we found that SC66 significantly inhibited the proliferation and EMT-mediated migration and invasion of T24 and 5637 cells. In addition, experiments confirmed that SC66 achieved its antitumour effect by inducing cell apoptosis and affecting the cell cycle. Luciferase assays confirmed that SC66 exerted an antitumour effect through the AKT/ß-catenin signalling pathway, and this inhibitory effect was reversed after the addition of the ß-catenin signalling pathway activator, CHIR-99021. In addition, animal studies have shown that, compared with the control group, the experimental group with SC66 intraperitoneal injection showed significantly reduced the tumour weight and volume in nude mice with T24 tumours and that SC66 combined with cisplatin achieved better inhibition on tumours. Western blot analysis and immunohistochemistry staining confirmed that SC66 inhibited the EMT process in vivo and induced apoptosis through the AKT/ß-catenin signalling pathway. In conclusion, our study demonstrated that SC66 exerts a significant antitumour effect through the AKT/ß-catenin signalling pathway, thereby providing a new potential treatment for BC.

Combination strategies to maximize the benefits of cancer immunotherapy.

Immunotherapies such as immune checkpoint blockade (ICB) and adoptive cell therapy (ACT) have revolutionized cancer treatment, especially in patients whose disease was otherwise considered incurable. However, primary and secondary resistance to single agent immunotherapy often results in treatment failure, and only a minority of patients experience long-term benefits. This review article will discuss the relationship between cancer immune response and mechanisms of resistance to immunotherapy. It will also provide a comprehensive review on the latest clinical status of combination therapies (e.g., immunotherapy with chemotherapy, radiation therapy and targeted therapy), and discuss combination therapies approved by the US Food and Drug Administration. It will provide an overview of therapies targeting cytokines and other soluble immunoregulatory factors, ACT, virotherapy, innate immune modifiers and cancer vaccines, as well as combination therapies that exploit alternative immune targets and other therapeutic modalities. Finally, this review will include the stimulating insights from the 2020 China Immuno-Oncology Workshop co-organized by the Chinese American Hematologist and Oncologist Network (CAHON), the China National Medical Product Administration (NMPA) and Tsinghua University School of Medicine.

Preclinical Models for Bladder Cancer Research.

At diagnosis, more than 70% of bladder cancers (BCs) are at the non-muscle-invasive bladder cancer (NMIBC) stages, which are usually treated with transurethral resection followed by intravesical instillation. For the remaining advanced cancers, systemic therapy is the standard of care, with addition of radical cystectomy in cases of locally advanced cancer. Because of the difference in treatment modalities, different models are needed to advance the care of NMIBC and advanced BC. This article gives a comprehensive review of both in vitro and in vivo BC models and compares the advantages and drawbacks of these preclinical systems in BC research.

Upregulation of NFKBIZ affects bladder cancer progression via the PTEN/PI3K/Akt signaling pathway.

NF­κB inhibitor ζ (NFKBIZ), a member of the IκB family that interacts with NF­κB, has been reported to be an important regulator of inflammation, cell proliferation and survival. However, the role of NFKBIZ in bladder cancer (BC) remains unknown. The present study aimed to investigate the functions of NFKBIZ in BC. First, the expression levels of NFKBIZ and the associations between NFKBIZ expression and the clinical survival of patients were determined using BC tissue samples, BC cell lines and datasets from different databases. Two BC cell lines (T24 and 5637) were selected to overexpress NFKBIZ, and the proliferative, migratory and invasive abilities of cells were determined; additionally, tumor growth following transplantation in in vivo mouse models was analyzed using T24 cells overexpressing NFKBIZ. Subsequently, the association between NFKBIZ and PTEN was determined using data from databases and immunohistochemistry analysis of clinical and nude mice tumor tissues. Finally, the interactions between NFKBIZ, PTEN and the downstream PI3K/AKT/mTOR signaling pathway were evaluated using western blotting. In conclusion, the present results indicated that NFKBIZ expression was low in BC, and NFKBIZ inhibited the proliferation of BC cells through the PTEN/PI3K/Akt signaling pathway, suggesting that NFKBIZ may represent a novel prognostic biomarker in BC and may provide a potential therapeutic tumor­associated antigen for BC.

Volunteering, Charitable Donation, and Psychological Well-Being of College Students in China.

Psychological well-being indicates individuals' positive psychological functioning and well-being. A growing body of literature, largely based on adults and old people, suggests that volunteering and charitable donations are related to individuals' psychological well-being. As emerging adulthood is a vital time for lifespan development, the aim of this study is to examine the effects of volunteering and charitable donation on individuals' psychological well-being on college students. Relying on theories of altruism and the warm-glow theory, this study estimates the relationships among hours of volunteering, amount of charitable donations, and psychological well-being from 1,871 Chinese college students. The linear regression results indicate that students' hours of volunteering and the amount of charitable donations in the past year were positively associated with their psychological well-being. Volunteering, compared to charitable donations, had a slightly stronger association with psychological well-being. This study provides a rationale for academic institutions to integrate social service activities into the curriculum as a potential tool to promote students' psychological well-being.

Job Demands and Resources and Employee Well-Being in the Chinese Nonprofit Sector.

Although the nonprofit sector in China has grown substantially in past decades, its future is threatened by high turnover and burnout. It is thus necessary to investigate the factors that contribute to employee well-being (EWB) among nonprofit employees in China. This study used 233 foundation employees in China to examine the effects of job demands and resources (JD-R) on EWB. Estimates produced by regression analyses indicated that job resources (JR) have a strong effect on EWB (Beta = 0.53), as well as on the three EWB subscales (workplace, psychological, and life well-being). While job demands (JD) had no effects on overall well-being, they were negatively associated with workplace well-being (WWB) (Beta = -0.12). Robustness tests were conducted to further examine how JD and JR dimensions affect EWB and its subscales. Based on the findings, we underscore the importance of JR for EWB among foundation employees in China as well as that of implementing interventions that may alleviate the cost of emotional workload as a JD.

Single Small Molecule-Assembled Mitochondria Targeting Nanofibers for Enhanced Photodynamic Cancer Therapy in Vivo.

Photodynamic therapy (PDT) has emerged as an attractive alternative in cancer therapy, but its therapeutic effects are limited by the nonselective subcellular localization and poor intratumoral retention of small-molecule photosensitizes. Here a fiber-forming nanophotosensitizer (PQC NF) that is composed of mitochondria targeting small molecules of amphiphilicity is reported. Harnessing the specific mitochondria targeting, the light-activated PQC NFs produce approximately 110-fold higher amount of reactive oxygen species (ROS) in cells than free photosensitizers and can dramatically induce mitochondrial disruption to trigger intense apoptosis, showing 20-50 times better in vitro anticancer potency than traditional photosensitizers. As fiber-shaped nanomaterials, PQC NFs also demonstrated a long-term retention in tumor sites, solving the challenge of rapid clearance of small-molecule photosensitizers from tumors. With these advantages, PQC NFs achieve a 100% complete cure rate in both subcutaneous and orthotopic oral cancer models with the administration of only a single dose. This type of single small molecule-assembled mitochondria targeting nanofibers offer an advantageous strategy to improve the in vivo therapeutic effects of conventional PDT.

[Effect of testicular autophagy on spermatogenic cells in varicocele rats].

OBJECTIVE: To study the effect of different levels of autophagy in the testis on the apoptosis of spermatogenic cells in the rat model of varicocele (VC). METHODS: We randomly divided 54 SD male rats into six groups, blank control (n = 6), rapamycin control (n = 6), chloroquine control (n = 6), VC model control (n = 12), VC + rapamycin (n = 12), and VC + chloroquine (n = 12). We observed the histomorphological changes of the testis and epididymis by HE staining, obtained the scores on spermatogenesis in the testis and epididymis, calculated the apoptosis index (AI) of the testicular spermatogenic cells by TUNEL, and determined the expressions of LC3-Ⅱ, LC3-Ⅰ, p62, Bax and Bcl-2 proteins in the testis tissue by Western blot. RESULTS: There were no significant morphological changes in the testis and epididymis of the rats in the blank control, rapamycin control and chloroquine control groups, or significant differences in the scores on testicular and epididymal spermatogenesis and AI of the testicular spermatogenic cells (P>0.05). The animals in the VC model control group exhibited significant pathological damage in the testicular and epididymal tissues, with remarkably decreased scores on spermatogenesis (P<0.01) and increased AI (P<0.01), which were markedly improved in the VC + rapamycin group and slightly aggravated in the VC + chloroquine group compared with the VC model controls. In comparison with the rats in the blank control group, those in the VC model control group showed significantly up-regulated expressions of the autophagy-related protein LC3 (including the LC3-Ⅱ/LC3-Ⅰ ratio) and the pro-apoptotic protein Bax in testicular tissue (P<0.01) but down-regulated expression of the anti-apoptotic protein Bcl-2 (P<0.01). The expressions of LC3 and Bcl-2 in the testis tissue were significantly higher in the VC + rapamycin (P<0.01) but lower in the VC + chloroquine group (P<0.01), while those of p62 and Bax remarkably lower in the VC + rapamycin (P<0.01) but higher in the VC + chloroquine group than in the VC model controls (P<0.01). CONCLUSIONS: Varicocele induces autophagy in the testis and apoptosis of spermatogenic cells in rats. Up-regulating autophagy can inhibit while blocking autophagy can promote the apoptosis of spermatogenic cells.

Ultrasound for Measuring the Cross-Sectional Area of Biceps Brachii Muscle in Sarcopenia.

Background: Ultrasound is emerging as an effective method for measuring muscle mass in elderly people. It has been applied in numerous studies to obtain measurement of lower limbs. The study aims to explore the relationship between sarcopenia and ultrasound measurements of biceps brachii. Methods: Participants (n=179) aged over 60 years were enrolled from the first affiliated hospital of Zhejiang University. The muscle thickness (MT), cross-sectional area (CSA) and fat thickness (FT) of these participants were recorded. Spearman test and partial correlation test was used to determine the correlation between indicators. Mann-Whitney U test was performed to compare ultrasonic parameters between sarcopenia group and non-sarcopenia group. The binary logistic regression analysis was employed to detect the potential indicators and prediction equation of sarcopenia. Receiver operating characteristic (ROC) curve analysis was performed for the accuracy of equation. Results: The prevalence of sarcopenia were 16.3% and 10.8% respectively in men and women. CSA was significantly lower in sarcopenia group than non-sarcopenia group in women (P<0.05). CSA was positively correlated with skeletal muscle mass index (SMI) and grip strength (men: r=0.460, 0.433; women: r=0.267, 0.392). After controlling of age and BMI, these correlations disappeared. Binary logistic regression analysis showed that age (OR=1.149, 95%CI: 1.060-1.246; P=0.001) and CSA (OR=0.465, 95%CI: 0.225-0.963; P=0.039) was significant indicators associated with sarcopenia. Area Under Curve was 0.822 (95%CI: 0.725-0.919, P<0.001) for the prediction equation composed of age, gender and CSA for sarcopenia. Conclusion: CSA of the biceps brachii measured with ultrasound is an important indicator associated with sarcopenia.

Traditional Classification and Novel Subtyping Systems for Bladder Cancer.

Bladder cancer is the most common tumor in the urinary system, with approximately 420,000 new cases and 160,000 deaths per year. The European Organization for Research and Treatment of Cancer (EOTRC) classifies non-muscular invasive bladder cancer (NMIBC) into low-risk, medium-risk and high-risk groups based on a comprehensive analysis of NMIBC pathological parameters and the risk of recurrence and progression to muscular invasive bladder cancer (MIBC). Traditional classification systems are based on pathologic grading, staging systems, and clinical prognosis. However, the pathological parameters of the tumor cannot fully reflect the "intrinsic characteristics" of bladder cancer, and tumors with a similar pathology exhibit different biological behaviors. Furthermore, although the traditional classification system cannot accurately predict the risk of recurrence or the progression of bladder cancer patients (BCs) individually, this method is widely used in clinical practice because of its convenient operation. With the development of sequencing and other technologies, the genetics-based molecular subtyping of bladder cancer has become increasingly studied. Compared with the traditional classification system, it provides more abundant tumor biological information and is expected to assist or even replace the traditional typing system in the future.

Overexpression of the transcription factor HAC1 improves nerolidol production in engineered yeast.

Increasing the metabolic flux of the mevalonate pathway, reducing the metabolic flux of competing pathway and utilizing the diauxie-inducible system constructed by GAL promoters are strategies commonly used in yeast metabolic engineering for the production of terpenoids. Using these strategies, we constructed a series of yeast strains with a strengthened mevalonate pathway and finally produced 336.5 mg/L nerolidol in a shake flask. The spliced HAC1 mRNA assay indicated that the unfolded protein response (UPR) occurred in the strains that we constructed. UPR strains exhibited the low transcriptional activities of GAL1 promoter. HAC1-overexpressing strain exhibited dramatically enhanced transcriptional activity of GAL1 promoter at 72 h of fermentation in flasks. HAC1 overexpression also increased the nerolidol titer by 47.7 %, reaching 497.0 mg/L and increased cell vitality. RNA-seq showed that the genes whose transcription responded to HAC1-overexpression were involved in the regulation of monocarboxylic acid metabolic processes and cellular amino acid biosynthetic process, indicating that the metabolic regulation may be part of the reason of the improved nerolidol synthesis. Our findings enrich the knowledge of the relationship between the construction of sesquiterpene-producing cell factories and UPR regulation. This study provides an effective strategy for sesquiterpene production in yeast.

TSPAN7 Exerts Anti-Tumor Effects in Bladder Cancer Through the PTEN/PI3K/AKT Pathway.

The tetraspanin protein superfamily participate in the dynamic regulation of cellular membrane compartments expressed in a variety of tumor types, which may alter the biological properties of cancer cells such as cell development, activation, growth and motility. The role of tetraspanin 7 (TSPAN7) has never been investigated in bladder cancer (BCa). In this study, we aimed to investigate the biological function of TSPAN7 and its therapeutic potential in human BCa. First, via reverse transcription and quantitative real-time PCR (qRT-PCR), we observed downregulation of TSPAN7 in BCa tissues samples and cell lines and found that this downregulation was associated with a relatively high tumor stage and tumor grade. Low expression of TSPAN7 was significantly correlated with a much poorer prognosis for BCa patients than was high expression. Immunohistochemistry (IHC) showed that low TSPAN7 expression was a high-risk predictor of BCa patient overall survival. Furthermore, the inhibitory effects of TSPAN7 on the proliferation and migration of BCa cell lines were detected by CCK-8, wound-healing, colony formation and transwell assays in vitro. Flow cytometry analysis revealed that TSPAN7 induced BCa cell lines apoptosis and cell cycle arrest. In vivo, tumor growth in nude mice bearing tumor xenografts could be obviously affected by overexpression of TSPAN7. Western blotting showed that overexpression of TSPAN7 activated Bax, cleaved caspase-3 and PTEN but inactivated Bcl-2, p-PI3K, and p-AKT to inhibit BCa cell growth via the PTEN/PI3K/AKT pathway. Taken together, our study will help identify a potential marker for BCa diagnosis and supply a target molecule for BCa treatment.

Identification of differentially expressed genes and signaling pathways in ovarian cancer by integrated bioinformatics analysis.

BACKGROUND: The mortality rate associated with ovarian cancer ranks the highest among gynecological malignancies. However, the cause and underlying molecular events of ovarian cancer are not clear. Here, we applied integrated bioinformatics to identify key pathogenic genes involved in ovarian cancer and reveal potential molecular mechanisms. RESULTS: The expression profiles of GDS3592, GSE54388, and GSE66957 were downloaded from the Gene Expression Omnibus (GEO) database, which contained 115 samples, including 85 cases of ovarian cancer samples and 30 cases of normal ovarian samples. The three microarray datasets were integrated to obtain differentially expressed genes (DEGs) and were deeply analyzed by bioinformatics methods. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments of DEGs were performed by DAVID and KOBAS online analyses, respectively. The protein-protein interaction (PPI) networks of the DEGs were constructed from the STRING database. A total of 190 DEGs were identified in the three GEO datasets, of which 99 genes were upregulated and 91 genes were downregulated. GO analysis showed that the biological functions of DEGs focused primarily on regulating cell proliferation, adhesion, and differentiation and intracellular signal cascades. The main cellular components include cell membranes, exosomes, the cytoskeleton, and the extracellular matrix. The molecular functions include growth factor activity, protein kinase regulation, DNA binding, and oxygen transport activity. KEGG pathway analysis showed that these DEGs were mainly involved in the Wnt signaling pathway, amino acid metabolism, and the tumor signaling pathway. The 17 most closely related genes among DEGs were identified from the PPI network. CONCLUSION: This study indicates that screening for DEGs and pathways in ovarian cancer using integrated bioinformatics analyses could help us understand the molecular mechanism underlying the development of ovarian cancer, be of clinical significance for the early diagnosis and prevention of ovarian cancer, and provide effective targets for the treatment of ovarian cancer.

Effects of autophagy on the spermatogonia of chronic hypoxic mice.

Varicocele (VC) is a common cause of male infertility. An increasing number of studies have confirmed that chronic hypoxia plays an important role in male infertility caused by VC. Our previous study suggested that autophagy may have a crucial function in VC. Whether autophagy plays an important role in hypoxic spermatogonia remains unknown. We established a new method to study VC by culturing spermatogonia under hypoxic conditions to simulate the chronic hypoxic environment of VC. Moreover, the effects of autophagy on hypoxic spermatogonia were observed by regulating autophagy. Spermatogonia from GC-1 spg mice were cultured in vitro in chronic hypoxic conditions (2% O2, 93% N2, 5% CO2) with a tri-gas incubator. Cells were separately cultured under normal oxygen and hypoxic conditions, and then, the autophagy blocker drug LY294002 was subsequently administered. Cytoactivity and proliferation were tested by CCK8 assays. Cells cultured in hypoxic conditions were divided into the hypoxia for 24 h, 48 h, and 72 h groups and the LY294002+hypoxia for 24 h, 48 h, and 72 h groups. Hoechst staining and flow cytometry/Annexin-V-FITC/PI staining were used to detect cell apoptosis, while transmission electron microscopy was used to observe the structure of the spermatogonia. Western blot analysis was used to observe the expression of Hif-1α, Beclin-1, LC3, SOD2, GPX4, and Cyt C. As the hypoxia time increased, CCK8 detection showed that the cytoactivity of the spermatogonia was suppressed. After administration of the inhibitor LY294002, the inhibition ratio of the spermatogonia increased. As the hypoxia time was prolonged, the apoptosis index of spermatogonia increased. After administration of LY294002, the apoptosis index increased significantly. Transmission electron microscopy showed that there were abundant structurally abnormal mitochondria and autophagosomes and autolysosomes in hypoxic spermatogonia. In addition, LY294002 reduced the expression of autophagosomes and autophagosomes, but the number of abnormal mitochondria and vacuoles in hypoxic cells increased significantly. As the hypoxia time was prolonged, the expression levels of Hif-1α, Beclin-1, LC3 II/LC3-I, and Cyt C increased gradually, but there were no significant changes in SOD2 and GPX4. After administration of LY294002, the expression levels of Beclin-1, LC3 II/LC3-I, and GPX4 significantly decreased, but Cyt C significantly increased. Thus, chronic hypoxia decreases cytoactivity and proliferation of spermatogonia. Moreover, hypoxia damages the mitochondria and other organelles. At the same time, hypoxia activates the autophagic system to clear these damaged organelles. The inhibition of autophagy in spermatogonia under chronic hypoxic conditions prevents the removal of damaged mitochondria and eventually results in a significant decrease in the antioxidant system molecule GPX4, rather than SOD2, and a significant increase in the apoptosis rate.

Effect of varicocelectomy treatment on spermatogenesis and apoptosis via the induction of heat shock protein 70 in varicocele­induced rats.

In the current study, the hypothesis that testicular varicocelectomy improves spermatogenesis and attenuates apoptosis via the induction of heat shock protein 70 (Hsp70) in a rat model of varicocele was investigated. Adult male Wistar rats (n=75) were randomly divided into 5 groups of 15 each: Control, sham, varicocele, varicocelectomy, and varicocelectomy plus Quercetin. A total of 6 weeks after the varicocelectomy, the left testis of all rats was removed for subsequent examination. Histological changes were compared between the groups. The expression of Hsp70 and apoptosis­associated indicators were evaluated based on immunohistochemical, western blot and mRNA expression analyses. Compared with the varicocele group, the varicocelectomy group exhibited a markedly reduced Bcl­2­associated X protein/B­cell lymphoma 2 (Bax/Bcl­2) ratio, and had a decreased expression of caspase­9, cytochrome c (cyt c) and caspase­3 through the intrinsic signal transduction pathways. Quercetin treatment inhibited the protective effects of varicocelectomy. The expression of Hsp70 was increased in the varicocele group which was further elevated by the varicocelectomy. These results indicated that varicocelectomy can reduce the Bax/Bcl­2 ratio, and decrease the levels of caspase­9, cyt c and caspase­3 via the mitochondrial signal transduction pathway. Such protective effects on left testis spermatogenesis and against apoptosis may be due to the induction of Hsp70. The findings of the present study suggested that varicocelectomy has a clear advantage in protecting testicular function and ameliorating spermatogenic cells apoptosis.