OBJECTIVE: To explore the underlying molecular mechanism of Notch1/cadherin 5 (CDH5) pathway in modulating in cell malignant behaviors of gastric cancer (GC).
METHODS: We performed bioinformatic analyses to screen the potential target genes of Notch1 from cadherins in GC. Western blot and RT-PCR were conducted to detect CDH5 expression in GC tissues and cells. We utilized chromatin immunoprecipitation (CHIP) assays to assess the interaction of Notch1 with CDH5 gene. The effects of Notch1/CDH5 axis on the proliferation, invasion, migration and vasculogenic mimicry in GC cells were evaluated by EdU, wound healing, transwell, and tubule formation assays.
RESULTS: Significantly increased CDH5 expression was found in GC tissues compared with paracancerous tissues and associated to clinical stage and poor overall survival (OS) in patients with GC. Notch1 positively regulate the expression of CDH5 in GC cells. CHIP assays validated that CDH5 was a direct target of Notch1. In addition, Notch1 upregulation enhanced the proliferation, migration, invasion and vasculogenic mimicry capacity of GC cells, which could be attenuated by CDH5 silencing.
CONCLUSIONS: These results indicated Notch1 upregulation enhanced GC malignant behaviors by triggering CDH5, suggesting that targeting Notch1/CDH5 axis could be a potential therapeutic strategy for GC progression.

In chronic lymphocytic leukemia (CLL), TP53 mutations or deletions on chromosome 17p lead to adverse prognosis and reduced levels of miR-34a, which targets NOTCH1. Also, hyperactivated NOTCH1 signaling is crucial for CLL progression. Here we explored the interaction between p53, miR-34a, and NOTCH1 in CLL. We investigated the effect of p53 and miR-34a on NOTCH1 signaling and expression in CLL cells with altered TP53. Our results indicate that miR-34a reduces NOTCH1 3\' UTR activity but might not be a mediator between p53 signaling and NOTCH1. p53 activation increases miR-34a expression and NOTCH1 protein levels, correlating with decreased NOTCH1 and miR-34a levels in primary CLL cells with TP53 alterations. Some samples with high NOTCH1 levels presented increased BCL-2, suggesting an anti-apoptotic mechanism of a potentially direct p53-NOTCH1 relation in CLL. This study deepens the understanding of the p53-miR-34a-NOTCH1 signaling network, providing insights that could guide future therapeutic strategies for CLL.

Breast cancer ranks as the most prevalent cancer globally, surpassing lung cancer, with recurrence/metastasis to be its main account for the cancer-related mortality. MicroRNAs (miRNAs) participate critically in various physiological and pathological processes through posttranscriptional regulation of downstream genes. Our preliminary findings identified miR-338-5p, potentially linked to metastasis in breast cancer, a previously unexplored area. Analysis of the GSE38867 dataset revealed the decreased miR-338-5p expression in metastatic breast cancer compared to normal tissues. Cellular function experiments and a xenograft tumor model demonstrated the inhibitory function of miR-338-5p on the progression of breast cancer in vitro and in vivo. Furthermore, it downregulated the expression of mesenchymal biomarkers and NOTCH1 significantly. With the predicting targets of miR-338-5p and transcription factors of the NOTCH1 gene, coupled with dual luciferase reporter assays, it is identified ETS1 as the interactor between miR-338-5p and NOTCH1. In breast cancer tissues, as well as in our xenograft tumor model, expression of ETS1 and NOTCH1 was positively correlated using immunohistochemical staining. This study reports, for the first time, on the miR-338-5p/ETS1/NOTCH1 axis and its pivotal role in breast cancer proliferation and metastasis. These findings propose a novel therapeutic strategy for breast cancer patients and lays a foundation for its clinical detection and treatment evaluation.

Central and systemic inflammation play pivotal roles in epileptogenesis and proepileptogenesis in temporal lobe epilepsy (TLE). The interplay between peripheral CD4+ T cells and central microglia orchestrates the \"systemic-central\" immune response in TLE. However, the precise molecular mechanisms linking central and systemic inflammation in TLE remain unknown. This preliminary findings revealed an imbalance in Th1/Th2 subsets in the periphery，accompanied by related cytokines release in TLE patients. they proposed that this peripheral Th1/Th2 imbalance may influence central inflammation by mediating microglial state dynamics within epileptic foci and distant brain regions. In Li-pilocarpine-induced TLE rats, a peripheral Th1/Th2 imbalance and observed corresponding central and systemic responses is confirmed. Notably, CD4+ T cells infiltrated through the compromised blood-brain barrierand are spatially close to microglia around epileptic foci. Intravenous depletion and reinfusion of CD4+ T cells modulated microglia state dynamics and altered neuroinflammatory cytokines secretion. Moreover, mRNA sequencing of the human hippocampus identified Notch1 as a key regulator of Th1/Th2 differentiation, CD4+ T cell recruitment to brain infiltration sites, and the regulation of microglial responses, seizure frequency, and cognition. This study underscores the significance of Th1/Th2 imbalance in modulating the \"systemic-central\" response in TLE, highlighting Notch1 as a potential therapeutic target.

Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region (ccRING3) drive MIB1 dimerization and that MIB1 ubiquitin transfer activity relies solely on ccRING3. We report X-ray crystal structures of a UbcH5B-ccRING3 complex and the ANK domain. Directly tethering the MIB1 N-terminal region to ccRING3 forms a minimal MIB1 protein sufficient to induce a Notch response in receiver cells and rescue mib knockout phenotypes in flies. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.

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BACKGROUND: Hepatic ischemia-reperfusion injury (HIRI) is commonly observed in cases of extensive hepatic resection and involves complex mechanisms. Cell senescence has been recognized as a factor in liver injury including HIRI, where it presents as a pro-inflammatory phenotype called senescence-associated secretory phenotype (SASP). Radix Rehmanniae Praeparata (RRP) is a commonly utilized traditional Chinese medicine known for its hepatoprotective, anti-aging and antioxidant qualities. Despite its recognized benefits, the specific mechanisms by which RRP may impede the progression of HIRI through the regulation of cell senescence and the identification of the most potent anti-aging extracts from RRP remain unclear.
METHODS: Here, we first applied different chemical analysis methods to identify the RRP aqueous extract (RRPAE) and active fractions of RRP. Next, we constructed a surgically established mouse model and a hypoxia-reoxygenation (HR)-stimulated liver sinusoidal endothelial cells (LSECs) model to explore the underlying mechanism of RRP against HIRI through transcriptomics and multiple molecular biology experiments.
RESULTS: After identifying active ingredients in RRP, we observed that RRP and its factions effectively restored LSECs fenestration and improved inflammation, cellular swelling and vascular continuity in the hepatic sinusoidal region during HIRI. Transcriptomic results revealed that RRP might reverse HIRI-induced senescence through the NOTCH signaling pathway and cell categorization further showed that the senescent cell population in HIRI liver was primarily LSECs rather than other cell types. Different RRPAE, especially RRP glucoside (RRPGLY), improved LSECs senescence and suppressed the expression of pro-inflammatory SASP genes either induced by HR insult or NOTCH1 activator, which was accompanied with the inhibition of LRP1-NOTCH1-C/EBPβ pathways. Additionally, the specific inhibition of NOTCH1 by siRNA synergistically enhanced the hepatoprotective effect of RRPGLY. The ChIP-qPCR results further showed that C/EBPβ was enriched at the promoter of a representative SASP, Il-1β, in hypoxic LSECs but was significantly inhibited by RRPGLY.
CONCLUSIONS: Our study not only clarified the potential mechanism of RRP active extractions in alleviating HIRI, but also highlighted RRPGLY was the main component of RRP that exerted anti-aging and anti-HIRI effects, providing a fresh perspective on the use of RRP to improve HIRI.

BACKGROUND: There is evidence suggesting that Notch1 signaling pathway contributes to the development of hand, foot, and mouth disease (HFMD); however, the role of Notch1 gene polymorphisms in the severity of coxsackievirus A6 (CVA6)-related HFMD remains unclear. This study aimed to investigate the correlation between Notch1 gene polymorphisms and the severity of CVA6-related HFMD.
METHODS: A total of 196 patients (Chinese Han population) diagnosed with CVA6-related HFMD through nucleic acid testing were included in this study. Among them, 97 patients were classified as severe cases, while 99 cases were categorized as mild. The mRNA levels of Notch1 in the peripheral blood leukocytes of HFMD patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was utilized for genotyping of rs3124599, rs3124603, and rs3124591.
RESULTS: The frequencies of rs3124599 alleles were G (39.0%) and A (61.0%), while the frequencies of rs3124599 genotypes were GG (12.2%), GA (53.6%), and AA (34.2%), respectively. In the recessive model, the frequency of rs3124599 AA genotypes significantly increased in severe patients, compared to mild patients (P < 0.05). Due to the low frequency of alleles for rs3124591 and rs3124603 in patients, as well as the absence of any difference in their distribution between the two groups (P > 0.05), no additional statistical analysis was performed. After adjusting for age and sex, patients with rs3124599 AA genotype had a significantly higher risk of severe HFMD in comparison to G allele carriers (GA/GG), with an odds ratio (95% confidence interval) of 2.010 (1.094, 3.691). Meanwhile, the mRNA levels of Notch1 were found to be significantly higher in severe patients compared to mild patients (P < 0.05), and a positive correlation was observed between Notch1 mRNA levels and the peripheral blood monocyte count (r = 0.42, P < 0.001). Additionally, there were significant differences observed in Notch1 mRNA levels and peripheral blood monocyte counts between patients with the AA genotype of rs3124599 and those with the GA genotype or G allele carriers (P < 0.05).
CONCLUSIONS: In the Chinese Han population, there is a strong correlation between the Notch1 rs3124599 allele and the severity of CVA6-related HFMD. This correlation may be attributed to genetic polymorphism of rs3124599 regulating Notch1 transcription levels. These findings reveal the important role of Notch1 gene polymorphism in CVA6 infection, establishing a scientific foundation for the precise control of severe HFMD.

Cutaneous squamous cell carcinoma (cSCC) is a common and increasingly prevalent form of skin cancer, posing significant health challenges. Understanding the molecular mechanisms involved in cSCC progression is crucial for developing effective treatments. The primary aim of this research was to evaluate the activation of NOTCH1 and FGFR2 oncogenes in inducing skin cancer in FVB/N mice through a stepwise chemical process. Forty female FVB/N mice, aged four weeks, were randomly divided into a control group (n = 8) and two experimental groups (group A: n = 16, group B: n = 16). This study involved subjecting the groups to a two-stage carcinogenesis procedure. This included an initial application of 97.4 nmol DMBA on shaved skin on their backs, followed by applications of 32.4 nmol TPA after thirteen weeks for group A and after twenty weeks for group B. The control group did not receive any treatment. Their skin conditions were monitored weekly to detect tumor development. After the experiment, the animals were euthanized for further tissue sampling. The examination of skin lesions in the experimental groups showed a correlation with tumor progression, ranging from dysplasia to carcinoma. Tumor samples were assessed both histologically and immunohistochemically. Notably, FGFR2 expression was higher in benign, precancerous, and malignant tumors compared to normal tissue. NOTCH1 expression was only elevated in benign tumors compared to normal tissue. This study demonstrates a clear correlation of FGFR2 expression and the progression of cutaneous neoplasms, while NOTCH 1 expression is inversely correlated in FVB/N mice. This suggests an early involvement of these oncogenes in the development of skin tumors.

Genome-wide association studies and experimental mouse models implicate the MIB1 and GATA6 genes in congenital heart disease (CHD). Their close physical proximity and conserved synteny suggest that these two genes might be involved in analogous cardiac developmental processes. Heterozygous Gata6 loss-of-function mutations alone or humanized Mib1 mutations in a NOTCH1-sensitized genetic background cause bicuspid aortic valve (BAV) and a membranous ventricular septal defect (VSD), consistent with MIB1 and NOTCH1 functioning in the same pathway. To determine if MIB1-NOTCH and GATA6 interact in valvular and septal development, we generated compound heterozygote mice carrying different Mib1 missense (Mib1K735R and Mib1V943F) or nonsense (Mib1R530X) mutations with the Gata6STOP/+ heterozygous null mutation. Combining Mib1R530X/+ or Mib1K735R/+ with Gata6STOP/+ does not affect Gata6STOP/+ single mutant phenotypes. In contrast, combining Mib1V943F/+ with Gata6STOP/+ decreases the incidence of BAV and VSD by 50%, suggesting a suppressive effect of Mib1V943F/+ on Gata6STOP/+. Transcriptomic and functional analyses revealed that while the EMT pathway term is depleted in the Gata6STOP/+ mutant, introducing the Mib1V943F variant robustly enriches this term, consistent with the Mib1V943F/+ phenotypic suppression of Gata6STOP/+. Interestingly, combined Notch1 and Gata6 insufficiency led to a nearly fully penetrant VSD but did not affect the BAV phenotype, underscoring the complex functional relationship between MIB1, NOTCH, and GATA6 in valvular and septal development.