The Identification of Stemness-Related Genes in the Risk of Head and Neck Squamous Cell Carcinoma

ObjectivesThis study aimed to identify genes regulating cancer stemness of head and neck squamous cell carcinoma (HNSCC) and evaluate the ability of these genes to predict clinical outcomes.Materials and MethodsThe stemness index (mRNAsi) was obtained using a one-class logistic regression machine learning algorithm based on sequencing data of HNSCC patients. Stemness-related genes were identified by weighted gene co-expression network analysis and least absolute shrinkage and selection operator analysis (LASSO). The coefficient of LASSO was applied to construct a diagnostic risk score model. The Cancer Genome Atlas database, the Gene Expression Omnibus database, Oncomine database and the Human Protein Atlas database were used to validate the expression of key genes. Interaction network analysis was performed using String database and DisNor database. The Connectivity Map database was used to screen potential compounds. The expressions of stemness-related genes were validated using quantitative real‐time polymerase chain reaction (qRT‐PCR).ResultsTTK, KIF14, KIF18A and DLGAP5 were identified. Stemness-related genes were upregulated in HNSCC samples. The risk score model had a significant predictive ability. CDK inhibitor was the top hit of potential compounds.ConclusionStemness-related gene expression profiles may be a potential biomarker for HNSCC

A teosinte-derived allele of ZmSC improves salt tolerance in maize

Maize, a salt-sensitive crop, frequently suffers severe yield losses due to soil salinization. Enhancing salt tolerance in maize is crucial for maintaining yield stability. To address this, we developed an introgression line (IL76) through introgressive hybridization between maize wild relatives Zea perennis, Tripsacum dactyloides, and inbred Zheng58, utilizing the tri-species hybrid MTP as a genetic bridge. Previously, genetic variation analysis identified a polymorphic marker on Zm00001eb244520 (designated as ZmSC), which encodes a vesicle-sorting protein described as a salt-tolerant protein in the NCBI database. To characterize the identified polymorphic marker, we employed gene cloning and homologous cloning techniques. Gene cloning analysis revealed a non-synonymous mutation at the 1847th base of ZmSCIL76, where a guanine-to-cytosine substitution resulted in the mutation of serine to threonine at the 119th amino acid sequence (using ZmSCZ58 as the reference sequence). Moreover, homologous cloning demonstrated that the variation site derived from Z. perennis. Functional analyses showed that transgenic Arabidopsis lines overexpressing ZmSCZ58 exhibited significant reductions in leaf number, root length, and pod number, alongside suppression of the expression of genes in the SOS and CDPK pathways associated with Ca2+ signaling. Similarly, fission yeast strains expressing ZmSCZ58 displayed inhibited growth. In contrast, the ZmSCIL76 allele from Z. perennis alleviated these negative effects in both Arabidopsis and yeast, with the lines overexpressing ZmSCIL76 exhibiting significantly higher abscisic acid (ABA) content compared to those overexpressing ZmSCZ58. Our findings suggest that ZmSC negatively regulates salt tolerance in maize by suppressing downstream gene expression associated with Ca2+ signaling in the CDPK and SOS pathways. The ZmSCIL76 allele from Z. perennis, however, can mitigate this negative regulatory effect. These results provide valuable insights and genetic resources for future maize salt tolerance breeding programs

Melatonin Promotes Ubiquitination of Phosphorylated Pro-Apoptotic Protein Bcl-2-Interacting Mediator of Cell Death-Extra Long (BimEL) in Porcine Granulosa Cells

Melatonin (N-acetyl-5-methoxytryptamine) is found in ovarian follicular fluid, and its concentration is closely related to follicular health status. Nevertheless, the molecular mechanisms underlying melatonin function in follicles are uncertain. In this study, melatonin concentration was measured in porcine follicular fluid at different stages of health. The melatonin concentration decreased as the follicles underwent atresia, suggesting that melatonin may participate in the maintenance of follicular health. The molecular pathway through which melatonin may regulate follicular development was further investigated. The pro-apoptotic protein BimEL (Bcl-2-interacting mediator of cell death-Extra Long), a key protein controlling granulosa cell apoptosis during follicular atresia, was selected as the target molecule. BimEL was downregulated when porcine granulosa cells were cultured in medium containing 10−9 M melatonin and isolated cumulus oocyte complexes (COCs) or follicle stimulating hormone (FSH). Interestingly, ERK-mediated phosphorylation was a prerequisite for the melatonin-induced decline in BimEL, and melatonin only promoted the ubiquitination of phosphorylated BimEL, and did not affect the activities of the lysosome or the proteasome. Moreover, the melatonin-induced downregulation of BimEL was independent of its receptor and its antioxidant properties. In conclusion, melatonin may maintain follicular health by inducing BimEL ubiquitination to inhibit the apoptosis of granulosa cells

Female Reproductive Toxicity of Harmful Algal Blooms-Produced Toxin Microcystin

Harmful algal blooms (HABs) have become a global emerging environmental concern; however, its impact on women’s reproductive health is poorly understood. Microcystin-LR (MC-LR) is the most common and hazardous type of HAB toxin. Using both a chronic oral exposure mouse model and an acute exposure mouse model, I found that human-relevant exposure to MC-LR could compromise follicle ovulation via affecting follicle maturation. Both in vivo mouse exposure model and encapsulated in vitro follicle growth (eIVFG) model revealed that exposure to MC-LR did not affect follicle development, but significantly reduced the expression of some follicle maturation-related genes, such as Lhcgr and Pappa. Mechanistic studies demonstrated that MC-LR could inhibit the protein phosphatase 1 (PP1) and further block the activation of PI3K/AKT/FOXO1 signaling pathway. In addition, OATP inhibitor Ritonavir could inhibit the accumulation of MC-LR in the follicles and rescue the MC-LR-disrupted follicle ovulation. Taken together, these results demonstrate that human-relevant exposure to MCLR compromises ovulation via disrupting follicle maturation by inhibiting PP1/PI3K/AKT/FOXO1 signaling pathway in the granulosa cells. More than 279 MC congeners have been identified and a broad spectrum of environmental chemicals and pharmaceutical compounds cause female ovarian toxicity (ovotoxicity). The eIVFG is a robust in vitro model for ovotoxicity testing. However, the follicle preparation process is complex and highly dependent on technical skills. Here, I aimed to use vitrification method to cryopreserve murine immature follicles for a high-content eIVFG, chemical exposure, and ovotoxicity screening. Results indicated that a closed vitrification system combined with optimized vitrification protocols preserved mouse follicle viability and functionality and vitrified follicles exhibited comparable follicle and oocyte reproductive outcomes to freshly harvested follicles during eIVFG, including follicle survival and development, ovarian steroidogenesis, and oocyte maturation and ovulation. Moreover, vitrified follicles consistently responded to ovotoxic chemical, doxorubicin (DOX). I further used vitrified follicles to test the ovotoxicity of 4 MC congeners, including MC-LA, LF, LR, and LY, and found that different congeners of MCs exhibited differential ovotoxicities. In summary, my research study demonstrates a novel toxic mechanism of HAB toxin MC-LR in compromising follicle ovulation and introduces a follicle vitrification strategy to build a long-term-storage and ready-to-use ovarian follicle bank for high-throughput ovotoxicity screening, which identifies endocrine disrupting effects of MCs

Melatonin Promotes Ubiquitination of Phosphorylated Pro-Apoptotic Protein Bcl-2-Interacting Mediator of Cell Death-Extra Long (Bim_EL) in Porcine Granulosa Cells

Melatonin (N-acetyl-5-methoxytryptamine) is found in ovarian follicular fluid, and its concentration is closely related to follicular health status. Nevertheless, the molecular mechanisms underlying melatonin function in follicles are uncertain. In this study, melatonin concentration was measured in porcine follicular fluid at different stages of health. The melatonin concentration decreased as the follicles underwent atresia, suggesting that melatonin may participate in the maintenance of follicular health. The molecular pathway through which melatonin may regulate follicular development was further investigated. The pro-apoptotic protein BimEL (Bcl-2-interacting mediator of cell death-Extra Long), a key protein controlling granulosa cell apoptosis during follicular atresia, was selected as the target molecule. BimEL was downregulated when porcine granulosa cells were cultured in medium containing 10−9 M melatonin and isolated cumulus oocyte complexes (COCs) or follicle stimulating hormone (FSH). Interestingly, ERK-mediated phosphorylation was a prerequisite for the melatonin-induced decline in BimEL, and melatonin only promoted the ubiquitination of phosphorylated BimEL, and did not affect the activities of the lysosome or the proteasome. Moreover, the melatonin-induced downregulation of BimEL was independent of its receptor and its antioxidant properties. In conclusion, melatonin may maintain follicular health by inducing BimEL ubiquitination to inhibit the apoptosis of granulosa cells

IGF-1 Inhibits Apoptosis of Porcine Primary Granulosa Cell by Targeting Degradation of BimEL

Insulin-like growth factor-1 (IGF-1) is an intra-ovarian growth factor that plays important endocrine or paracrine roles during ovarian development. IGF-1 affects ovarian function and female fertility through reducing apoptosis of granulosa cells, yet the underlying mechanism remains poorly characterized. Here, we aimed to address these knowledge gaps using porcine primary granulosa cells and examining the anti-apoptotic mechanisms of IGF-1. IGF-1 prevented the granulosa cell from apoptosis, as shown by TUNEL and Annexin V/PI detection, and gained the anti-apoptotic index, the ratio of Bcl-2/Bax. This process was partly mediated by reducing the pro-apoptotic BimEL (Bcl-2 Interacting Mediator of Cell Death-Extra Long) protein level. Western blotting showed that IGF-1 promoted BimEL phosphorylation through activating p-ERK1/2, and that the proteasome system was responsible for degradation of phosphorylated BimEL. Meanwhile, IGF-1 enhanced the Beclin1 level and the rate of LC3 II/LC3 I, indicating that autophagy was induced by IGF-1. By blocking the proteolysis processes of both proteasome and autophagy flux with MG132 and chloroquine, respectively, the BimEL did not reduce and the phosphorylated BimEL protein accumulated, thereby indicating that both proteasome and autophagy pathways were involved in the degradation of BimEL stimulated by IGF-1. In conclusion, IGF-1 inhibited porcine primary granulosa cell apoptosis via degradation of pro-apoptotic BimEL. This study is critical for us to further understand the mechanisms of follicular survival and atresia regulated by IGF-1. Moreover, it provides a direction for the treatment of infertility caused by ovarian dysplasia, such as polycystic ovary syndrome and the improvement of assisted reproductive technology