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Endoplasmic reticulum (ER) stress mediates Cd-caused germ cell apoptosis in testis. The effects of 4-phenylbutyric acid (PBA), a classical chaperone, were investigated on Cd-induced apoptosis in mouse GC-1 spermatogonia cells. The cells were pretreated with PBA before Cd exposure. TUNEL and flow cytometry assays were applied to determine apoptosis. Some key biomarkers of ER stress were analyzed using RT-PCR and western blot. as expected, the apoptotic cells exposed to Cd apparently increased. The mRNA and protein expression levels of GRP78 and ATF6α, were elevated in the Cd groups. Additional experiments displayed that Cd notably increased IRE1α and JNK phosphorylation, and upregulated XBP-1 mRNA and protein expression. Moreover, p-eIF2α and CHOP expressions were clearly elevated in the Cd groups. Interestingly, PBA almost completely inhibited ER stress and protected spermatogonia against apoptosis induced by Cd. PBA alleviated Cd-induced ER stress and spermatogonia apoptosis, and may have the therapeutic role in Cd-induced male reproductive toxicity. • CdCl 2 induced spermatogonia apoptosis in vitro. • CdCl 2 triggered spermatogonia ER stress. • 4-PBA alleviated CdCl 2 -induced spermatogonia apoptosis. [ABSTRACT FROM AUTHOR]

Objectives: MicroRNAs possess essential effects on gastric cancer (GC), whereas the underlying mechanisms have not been fully uncovered. The present work focused on investigating the role of miR-381-3p in GC cellular processes and the possible mechanisms. Materials and Methods: miR-381-3p levels within GC tissues and cells were measured through quantitative real-time polymerase chain reaction (qRT-PCR). This study measured cell proliferation, apoptosis, and metastasis through EdU, colony formation, flow cytometry, and Transwell assays separately. TargetScan was adopted to predict the miR-381-3p targets, whereas luciferase reporter assay was adopted for confirmation. Results: miR-381-3p levels were decreased, whereas fibroblast growth factor receptor-2 (FGFR2) expression was increased in GC. miR-381-3p upregulation inhibited proliferation, migration, and invasion and it promoted the apoptosis of GC cells. Further, FGFR2 overexpression partly reversed the miR-381-3p-mediated impacts on GC cellular processes. Conclusions: This study provides an experimental basis, suggesting the potential of using miR-381-3p as the novel marker for GC. Clinical Trial Registration number: 2020-05. [ABSTRACT FROM AUTHOR]

Cerebral ischemia-reperfusion (I/R) injury is a major problem worldwide. The Notch signaling pathway plays an important role in neural progenitor cell differentiation and in the inflammatory response after central nervous system injury. This study evaluated whether the neuroprotective effect of cerebral ischemic preconditioning (cIPC) is mediated by the preactivation of the Notch signaling pathway. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and glucose deprivation/reoxygenation (OGD/R) cell model were constructed to detect the neuroprotective effects of cIPC. In in vivo experiments, cIPC reduces the neurological functional deficit, cerebral infarction, and cellular apoptosis in the hippocampus induced by middle cerebral artery occlusion/reperfusion (MCAO/R), thus indicating that cIPC can improve neurologic function. Moreover, cIPC can reveal the expression peak of Jagged1, Notch1, NICD, and Hes1 protein, thereby indicating that cIPC can preactivate Notch signaling. However, cIPC-induced improvements in neurologic function are compromised by the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl ester (DAPT). In in vitro experiments, OGD preconditioning (OGDPC) can clearly upregulate Notch1 expression in the OGD/R-treated neuron and neural stem cell. Notch1 pre-overexpression can decrease neuron death and apoptosis under OGD/R treatment. Notch1 pre-overexpression can decrease the percentage of G1 stage cells and increase the percentage of S stage cells in OGD/R-treated neural stem cell. Furthermore, Notch1 pre-knockdown has the opposite effect on cell survival, apoptosis, and cycle in both OGD/R-treated neuron and neural stem cell. In conclusion, our results demonstrate that the neuroprotective effects of cIPC in a rat MCAO/R model are mediated by the preactivation of the Notch signaling pathway. [ABSTRACT FROM AUTHOR]

Purpose: Ferroptosis is a form of regulated cell death that has the potential to be targeted as a cancer therapeutic strategy. But cancer cells have a wide range of sensitivities to ferroptosis, which limits its therapeutic potential. Accumulation of lipid peroxides determines the occurrence of ferroptosis. However, the type of lipid involved in peroxidation and the mechanism of lipid peroxide accumulation are less studied. Methods: The effects of fatty acids (10 μM) with different carbon chain length and unsaturation on ferroptosis were evaluated by MTT and LDH release assay in cell lines derived from prostate cancer (PC3, 22RV1, DU145 and LNCaP), colorectal cancer (HT-29), cervical cancer (HeLa) and liver cancer (HepG2). Inhibitors of apoptosis, necroptosis, autophagy and ferroptosis were used to determine the type of cell death. Then the regulation of reactive oxygen species (ROS) and lipid peroxidation by docosahexaenoic acid (DHA) was measured by HPLC–MS and flow cytometry. The avtive form of DHA was determined by siRNA mediated gene silencing. The role of lipoxygenases was checked by inhibitors and gene silencing. Finally, the effect of DHA on ferroptosis-mediated tumor killing was verified in xenografts. Results: The sensitivity of ferroptosis was positively correlated with the unsaturation of exogenously added fatty acid. DHA (22:6 n-3) sensitized cancer cells to ferroptosis-inducing reagents (FINs) at the highest level in vitro and in vivo. In this process, DHA increased ROS accumulation, lipid peroxidation and protein oxidation independent of its membrane receptor, GPR120. Inhibition of long chain fatty acid-CoA ligases and lysophosphatidylcholine acyltransferases didn't affect the role of DHA. DHA-involved ferroptosis can be induced in both arachidonate lipoxygenase 5 (ALOX5) negative and positive cells. Down regulation of ALOX5 inhibited ferroptosis, while overexpression of ALOX5 promoted ferroptosis. Conclusion: DHA can effectively promote ferroptosis-mediated tumor killing by increasing intracellular lipid peroxidation. Both ALOX5 dependent and independent pathways are involved in DHA-FIN induced ferroptosis. And during this process, free DHA plays an important role. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-associated mortality. Cisplatin is one of the most effective chemotherapeutic drugs used in EOC; however, its use can lead to relapse due to cisplatin resistance. MYCN sensitizes neuroblastoma to undergo cisplatin-induced apoptosis. However, to the best of our knowledge, there have been no studies to date on the association between MYCN and cisplatin resistance in EOC. Therefore, the present study assessed this association. Datasets from The Cancer Genome Atlas database were used. The overall survival (OS) of patients receiving platin-based therapy was analyzed using Kaplan-Meier Plotter software. RNA sequencing data of 300 patients with EOC were downloaded from cBioportal. The co-expressed genes were subjected to 'Kyoto Encyclopedia of Genes and Genomes' analysis using DAVID software. For gene set enrichment analysis, the expression matrix was separated according to the median expression of MYCN, which was selected for hallmark gene set enrichment. Immunohistochemistry was used to assess MYCN expression in EOC tissue. Western blotting was used to evaluate MYCN, p53, Bax and Bcl-2 protein expression levels in EOC cells. Cell viability and apoptosis were assessed using Cell Counting Kit-8 and flow cytometry, respectively. The results demonstrated that MYCN upregulation was associated with increased cisplatin sensitivity and prolonged OS of patients with EOC and patients receiving platin-based therapy. Cisplatin downregulated MYCN expression in cisplatin-sensitive, but not resistant, EOC cells. The genes co-expressed with MYCN were primarily involved in pathways involved in 'chemotherapeutic resistance' and 'apoptosis'. MYCN enriched the apoptosis and p53 signaling pathways in hallmark gene sets. Cells in which MYCN was knocked down demonstrated significantly increased cisplatin resistance; however, MYCN overexpression in cisplatin-resistant cells restored cisplatin sensitivity. Collectively, the present study demonstrated that MYCN downregulation promoted cisplatin resistance by suppressing cisplatin-induced apoptosis in EOC. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction (AMI) is the main cause of death in the whole world. This study aimed to investigate whether forkhead box O4 (FoxO4) could negatively modulate ubiquitin specific peptidase 10 (USP10) transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation (H/R)-induced cardiomyocytes through Hippo/YAP pathway. mRNA expression as well as protein expressions of USP10 and FoxO4 in H9C2 cells after H/R induction or transfection were respectively detected by Reverse transcription-quantitative (RT-q) PCR analysis and Western blot. The viability and apoptosis of H9C2 cells after H/R induction or transfection were respectively detected by CCK-8 and TUNEL assays. The expressions of lactate dehydrogenase (LDH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in H9C2 cells after H/R induction or transfection were analyzed using appropriate kits and intracellular reactive oxygen species (ROS) levels were detected using a ROS Assay Kit. Dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP) have adopted to confirm the combination of USP10 and FoxO4. Western blot was also used to analyze the expression of apoptosis-related proteins and Hippo/YAP pathway-related proteins. As a result, USP10 expression was decreased in H/R-induced H9C2 cells in a time-dependent manner. USP10 overexpression increased the viability and suppressed the apoptosis and oxidative stress of H/R-induced H9C2 cells. In addition, FoxO4 modulated USP10 transcription. FoxO4 expression was increased in H9C2 cells induced by H/R. FoxO4 overexpression could reverse the protective effects of USP10 overexpression on H/R-induced H9C2 cells by regulating the Hippo/YAP signaling pathway. In conclusion, FoxO4 negatively modulated USP10 transcription to aggravate the apoptosis and oxidative stress of H/R-induced H9C2 cells via blocking Hippo/YAP pathway. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3'-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma. [ABSTRACT FROM AUTHOR]

Astrocytomas are common malignant intracranial tumors that comprise the majority of adult primary central nervous system tumors. MicroRNAs (miRNAs) are small, non-coding RNAs (20–24 nucleotides) that post-transcriptionally modulate gene expression by negatively regulating the stability or translational efficiency of their target mRNAs. In our previous studies, we found that the downregulation of miR-106a-5p in astrocytomas is associated with poor prognosis. However, its specific gene target(s) and underlying functional mechanism(s) in astrocytomas remain unclear. In this study, we used mRNA microarray experiments to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels. We then performed bioinformatics analysis based on multiple target prediction algorithms to obtain candidate target genes that were further validated by computational predictions, western blot analysis, quantitative real-time PCR, and the luciferase reporter assay. Fas-activated serine/threonine kinase (FASTK) was identified as a direct target of miR-106a-5p. In human astrocytomas, miR-106a-5p is downregulated and negatively associated with clinical staging, whereas FASTK is upregulated and positively associated with advanced clinical stages, at both the protein and mRNA levels. Furthermore, Kaplan-Meier analysis revealed that the reduced expression of miR-106a-5p or the increased expression of FASTK is significantly associated with poor survival outcome. These results further supported the finding that FASTK is a direct target gene of miR-106a-5p. Next, we explored the function of miR-106a-5p and FASTK during astrocytoma progression. Through gain-of-function and loss-of-function studies, we demonstrated that miR-106a-5p can significantly inhibit cell proliferation and migration and can promote cell apoptosis in vitro. The knockdown of FASTK induced similar effects on astrocytoma cells as those induced by the overexpression of miR-106a-5p. These observations suggest that miR-106a-5p functions as a tumor suppressor during the development of astrocytomas by targeting FASTK. [ABSTRACT FROM AUTHOR]