CDX2 alleviates hypoxia-induced apoptosis and oxidative stress in spermatogenic cells through suppression of reactive oxygen species-mediated Wnt/ß-catenin pathway.

Hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are considered to be important factors leading to male infertility. It was reported that CDX2 expression was downregulated in hypoxia-stimulated spermatogenic cells. However, the effects of CDX2 on hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are still unknown. This study aimed to explore the roles of CDX2 in hypoxia-induced injury of spermatogenic cells, as well as its mechanism of action. Spermatogenic cells were cultured under 1% oxygen for 48 h to established hypoxia damage model. Reactive oxygen species (ROS) generation was determined using 2',7'-dichlorofluorescein diacetate assay. Apoptosis was assessed using flow cytometry. Enzyme-linked immunosorbent assay was used to evaluate oxidative stress markers, including malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidases (GSH-Px). Protein levels were detected using western blotting. Hypoxia exposure induced increase in ROS generation, apoptosis rate, and oxidative stress in spermatogenic cells. ROS scavenger inhibited hypoxia-induced apoptosis, oxidative stress, and Wnt/ß-catenin pathway activation. Hypoxia exposure induced CDX2 downregulation. CDX2 overexpression suppressed hypoxia-induced ROS generation, apoptosis rate, oxidative stress, and Wnt/ß-catenin pathway activation. Moreover, CDX2 knockdown restores the inhibitory effects of si-ß-catenin or NAC on hypoxia-induced activation of the Wnt/ß-catenin pathway, apoptosis, and oxidative stress. In conclusion, our study suggests that CDX2 overexpression alleviates hypoxia-induced apoptosis and oxidative stress by suppression of ROS-mediated Wnt/ß-catenin pathway in spermatogenic cells.

Kurarinone attenuates hydrogen peroxide-induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells.

Dysregulated follicular development may lead to follicular atresia, and this is associated with oxidative stress in granulosa cells. Kurarinone is a natural compound possessing multiple activities, including antioxidative ability. However, the role of kurarinone in granulosa cell damage during follicular atresia remains unknown. Human ovarian granulosa KGN cells were treated with hydrogen peroxide (H2 O2 ) to induce cellular damage. Cytotoxicity was investigated by lactate dehydrogenase (LDH) release assay. Oxidative stress was evaluated by detection of reactive oxygen species (ROS) generation and oxidative biomarker levels. Cell apoptosis was evaluated by flow cytometry, a Cell Death Detection ELISA Kit, and a Caspase-3 Assay Kit. The downstream target and related signaling pathway were analyzed by western blotting. Kurarinone attenuated H2 O2 -induced LDH release in KGN cells. Kurarinone relieved H2 O2 -induced increase in ROS generation and malondialdehyde level as well as decrease in superoxide dismutase-1 activity and heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 mRNA levels. Kurarinone inhibited H2 O2 -induced apoptosis in KGN cells. Kurarinone targeted insulin-like growth factor 1 (IGF1) and upregulated IGF1 expression to activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling. IGF1 silencing attenuated the suppressive effects of kurarinone on H2 O2 -induced oxidative stress and apoptosis in KGN cells. In conclusion, kurarinone attenuates H2 O2 -induced oxidative stress and apoptosis in KGN cells through activating the PI3K/Akt signaling by upregulating IGF1 expression, indicating the therapeutic potential of kurarinone in follicular atresia.

Identification of an Immune Gene-Based Cisplatin Response Model and CD27 as a Therapeutic Target against Cisplatin Resistance for Ovarian Cancer.

Objective: Evidence demonstrates that the immune microenvironment is extensively associated with chemotherapy response of ovarian cancer (OV). Herein, this study is aimed at establishing a cisplatin response prediction model for OV on the basis of immune genes. Methods: The expression profiles of cisplatin-sensitive and cisplatin-resistant OV specimens were integrated from multiple public datasets. The abundance scores of 22 immune cells were estimated with CIBERSORT algorithm. Differentially expressed immune genes (DEGs) were determined between cisplatin-sensitive and cisplatin-resistant groups. Thereafter, a cisplatin response model was constructed based on prognostic DEGs with logistic regression analysis. The prediction performance was validated in independent cohorts. The possible relationships between the model and immunotherapy were then assessed. Results: Treg scores were significantly decreased in cisplatin-resistant than cisplatin-sensitive OV specimens, with the opposite results for naïve B cells and activated dendritic cells. Fourteen prognostic DEGs were identified and used to develop a cisplatin-response model. The response scores, estimated by the model, showed favorable performance in discriminating cisplatin-response and nonresponse samples. The response scores also presented significantly negative correlations with three well-known cisplatin-resistant pathways and a positive correlation with the expression of CD274 (PD-L1). Moreover, the decreased CD27 expression was observed in cisplatin-resistant groups, and OV specimens with higher CD27 expressions were more sensitive to cisplatin treatment. Conclusion: Altogether, our findings proposed a cisplatin response prediction model and identified CD27 that might be involved in cisplatin resistance. Further investigations suggested that CD27 could be a promising immunotherapeutic target for cisplatin-resistant subset of OV.

STEAP4 knockdown inhibits the proliferation of prostate cancer cells by activating the cGMP-PKG pathway under lipopolysaccharide-induced inflammatory microenvironment.

Six-transmembrane epithelial antigen of prostate 4 (STEAP4) is involved in the development of human cancers. However, the role of STEAP4 in prostate cancer remains largely unknown. The purpose of this research is to explore the role and action mechanism of STEAP4 in prostate cancer development under lipopolysaccharide (LPS)-induced inflammatory microenvironment. STEAP4 expression was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN and Cancer Cell Line Encyclopedia (CCLE), and its prognostic value was analyzed by LinkedOmics. STEAP4-correlated genes were analyzed by LinkedOmics and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. STEAP4 level was detected by Western blotting or qRT-PCR. Proliferation was investigated by CCK-8 and EdU staining. Inflammatory cytokine levels were detected by ELISA. The cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway was detected by ELISA and Western blotting. STEAP4 level was increased in prostate cancer tissues, and high expression of STEAP4 was associated with the poor overall survival. LPS promoted cell viability and STEAP4 expression. STEAP4 knockdown attenuated LPS-induced inflammation in prostate cancer cells. STEAP4 downregulation mitigated LPS-induced tumorigenesis by decreasing cell proliferation. STEAP4 silencing reversed LPS-induced inactivation of the cGMP-PKG pathway. Inhibition of the cGMP-PKG pathway using inhibitor KT5823 relieved STEAP4 silencing-mediated suppression of cell proliferation and inflammation in LPS-stimulated cells. In conclusion, STEAP4 silencing inhibits LPS-induced proliferation of prostate cancer cells by activating the cGMP-PKG pathway.

Inhibition of NCAPG expression inactivates the Wnt/ß-catenin signal to suppresses endometrial cancer cell growth in vitro.

Endometrial cancer (EC) ranks as the most prevalent malignancy occurring in the female genital tract. Non-SMC condensin I complex subunit G (NCAPG), a mitotic associated chromosomal condensing protein, is reported to be frequently abnormally expressed in several tumors and plays a vital role in carcinogenesis. Our study aimed to explore the effect of NCAPG on cell proliferation and apoptosis in EC cells and to determine the underlying mechanism. Expression and survival data of NCAPG in EC tissues were analyzed by bioinformatics methods. Cell proliferation was evaluated by EdU and CCK-8 assays. Apoptosis was assessed by flow cytometry analysis. Expression of NCAPG, proliferating cell nuclear antigen (PCNA), Ki67, Bcl-2, Bax, active caspase-3, active ß-catenin, and c-Myc were determined by western blotting. NCAPG was highly expressed in EC tissues and cells and predicted poor survival for EC patients. NCAPG knockdown inhibited cell proliferation and induced apoptosis in EC cells. Additionally, NCAPG knockdown inactivated the Wnt/ß-catenin pathway in EC cells. Mechanistically, ß-catenin overexpression blocked the tumorigenic effects of NCAPG in EC cells. In conclusion, NCAPG silencing inhibited cell proliferation and induced apoptosis in EC cells via inhibiting the Wnt/ß-catenin pathway.

SMC4 knockdown inhibits malignant biological behaviors of endometrial cancer cells by regulation of FoxO1 activity.

Structural maintenance of chromosomes 4 (SMC4) has an important role in chromosome condensation and segregation, which is involved in regulating multiple tumor development. However, the role of SMC4 in endometrial cancer is uncertain. The expression and prognostic value of SMC4 were predicted by UALCAN, Gene Expression Omnibus (GEO), The Human Protein Atlas and Kaplan Meier plotter tools. SMC4-related genes were analyzed by LinkedOmics, Gene Ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Forkhead box protein O1 (FoxO1) activity was suppressed by AS1842856 (AS). SMC4, Ki67, B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein (Bax), FoxO1, phosphorylated FoxO1 (p-FoxO1), and p27 protein levels were detected by Western blotting. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) analyses. Cell apoptosis was measured using TUNEL analysis. SMC4 abundance was increased in endometrial cancer, and predicted a worse overall survival. SMC4 knockdown repressed proliferative ability of endometrial cancer cells and promoted cell apoptosis. SMC4 knockdown promoted FoxO1 transactivation by decreasing its phosphorylated level. Addition of AS inhibited FoxO1 activity by increasing the phosphorylated level of FoxO1. The inhibition of FoxO1 activity reversed the effect of SMC4 silencing on cell proliferation and apoptosis. In conclusion, SMC4 silencing restrained cell proliferation and facilitated apoptosis in endometrial cancer via regulating FoxO1 activity.

Expression of miR-135a-5p and its target gene JAK2 in spermatozoa of patients with asthenozoospermia.

This study aimed to identify the molecular mechanism by which JAK2 mRNA and microRNA-135a-5p (miR-135a-5p) affect asthenozoospermia. The expression levels of JAK2 mRNA in the spermatozoa of 30 asthenozoospermia patients and 30 normal controls were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). MiR-135a-5p that targeted JAK2 mRNA was predicted by bioinformatics. A dual luciferase reporter vector containing miR-135a-5p 3'UTR was constructed. The binding of miR-135a-5p to JAK2 mRNA was verified by luciferase reporter assay. The protein expression levels of JAK2 and STAT3 in spermatozoa were examined by Western bolt. The relative expression levels of JAK2 mRNA and protein in the asthenozoospermia group were significantly lower than those of the normal group. MiR-135a-5p overexpression inhibited JAK2 mRNA and protein expression by targeting JAK2 mRNA 3'UTR. Correlation analysis showed that miR-135a-5p level was significantly negatively correlated with progressive sperm motility, while JAK2 mRNA level was significantly positively correlated with progressive sperm activity. Low expression of JAK2 mRNA and high expression of miR-135a-5p were related to asthenozoospermia and male infertility.

The Effects of Follistatin on the Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Neurons-Like Cells.

OBJECTIVE: The present study was designed to evaluate the effects of Follistatin (FST) on the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into neuron-like cells. MATERIALS AND METHODS: hBMSCs were isolated and characterized by cell surface markers including CD29, CD44, CD166, CD34, CD14, and CD45. Subsequently, 0.3, 3, and 10 nmol/L recombinant human FST (rhFST) were used to stimulate hBMSCs, respectively. Neuron-like cell differentiation and Nissl's body within the cytoplasm of hBMSCs were investigated by a transmission electron microscope (TEM). Meanwhile, nestin and NSE were determined by immunofluorescence. The expression level of Activin A, BMP4, Moysatin, and Smad3 were detected by Western blotting. RESULTS: The isolated hBMSCs were positive for CD29, CD44, and CD166, but negative for CD34, CD14, and CD45. The level of nestin and NSE mRNAs were significantly higher than those before induction (both P<0.05). Additionally, immunofluorescence revealed that nestin and NSE positive cells significantly increased as the rhFST concentration increased. With the increase of rhFST concentration, the expression level of Activin A gradually decreased accordingly, but the expression levels of BMP4 and Moysatin did not change significantly. Furthermore, the expression level of Smad3 gradually decreased with the increase of rhFST concentration. CONCLUSIONS: Our study indicates that FST could effectively induce hBMSCs to differentiate into neuron-like cells in vitro. This differentiation mechanism may be related to the Activin A signalling pathway, partially through binding to Activin receptors and inhibiting expression of Smad3.

MicroRNA-15a Inhibits Glucose Transporter 4 Translocation and Impairs Glucose Metabolism in L6 Skeletal Muscle Via Targeting of Vesicle-Associated Membrane Protein-Associated Protein A.

OBJECTIVES: MicroRNAs have been reported to participate in various important cell biological processes, such as glucose metabolism. The aim of this study was to explore the roles of microRNA-15a (miR-15a) in regulating insulin sensitivity. METHODS: In L6 rat skeletal muscle cells, we observed the effect of miR-15a on glucose metabolism and glucose transporter 4 (GLUT4) translocation by targeting vesicle-associated membrane protein-associated protein A (VAP-A) after insulin treatment. Luciferase reporter assays were performed to demonstrate a direct interaction between miR-15a and the 3'-untranslated region of VAP-A microRNA. RESULTS: We identified miR-15a as an extremely important regulator of GLUT4 translocation via targeting of VAP-A. Additionally, knockdown of endogenous miR-15a or overexpression of VAP-A could increase extracellular glucose by inhibiting the translocation of GLUT4 to the cell membrane after insulin treatment. However, overexpression of miR-15a or knockdown of VAP-A had no significant effect on glucose metabolism. CONCLUSIONS: These findings reveal the following: 1) VAP-A is a marker of skeletal muscle glucose disposal and 2) a novel mechanism for GLUT4 translocation by miR-15a.