OBJECTIVE: To elucidate the molecular mechanisms underlying sepsis-induced injury in mouse intestinal organoids and investigate the possible mechanisms or potential drug targets of myeloid differentiation factor 88 inhibitor [TJ-M2010-5 (TJ5)] on this condition. METHODS: Small intestinal organoids from C57BL/6 mice aged 6-8 weeks were established and characterized using immunofluorescence for cell growth and proliferation marker nuclear antigen Ki-67, goblet cell marker mucin-2 (MUC-2), epithelial cell marker E-cadherin, and Paneth cell marker lysozyme (Lyz). Small intestinal organoids after 3 days of passaging were divided into different groups: a normal control group treated with culture medium containing 0.2% dimethyl sulfoxide (DMSO) for 10 hours, a lipopolysaccharide (LPS) group treated with culture medium containing 200 mg/L LPS and 0.2% DMSO for 10 hours, and a TJ5 group pre-treated with 10 mmol/L TJ5 for 2 hours followed by treatment with culture medium containing 200 mg/L LPS for 10 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to measure the expression levels of interleukin-6 (IL-6) and zonula occludens-1 (ZO-1) in the small intestinal organoids. RNA transcriptome sequencing was performed on the small intestinal organoids from each group to analyze differentially expressed genes between groups, and significant enrichment was analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: By the 7th day of primary culture, mature organoids had formed, and their growth rate increased after passaging. Immunofluorescence identification showed expressions of Ki-67, MUC-2, E-cadherin, and Lyz, indicating that the mouse small intestinal organoids maintained their cellular composition and functional characteristics under in vitro culture conditions. RT-qPCR results showed that compared with the normal control group, the mRNA expression of IL-6 in the small intestinal organoids of the LPS group was significantly increased (2^-ΔΔCT: 1.83±0.16 vs. 1.02±0.28, P < 0.05), while the mRNA expression of ZO-1 was significantly decreased (2^-ΔΔCT: 0.53±0.11 vs. 1.01±0.18, P < 0.05). In contrast, the mRNA expression trends of both IL-6 and ZO-1 were reversed in the TJ5 group, showing statistically significant differences as compared with the LPS group (2^-ΔΔCT: IL-6 mRNA was 1.24±0.01 vs. 1.83±0.16, ZO-1 mRNA was 1.97±0.29 vs. 0.53±0.11, both P < 0.05). RNA transcriptome sequencing showed 49 differentially expressed genes in the LPS group compared to the normal control group, with 42 upregulated and 7 downregulated. Compared to the LPS group, the TJ5 group showed 84 differentially expressed genes, with 47 upregulated and 37 downregulated. GO enrichment analysis of these differentially expressed genes showed that the significantly enriched biological processes of the differentially expressed genes between the normal control group and the LPS group included responses to LPS, responses to molecule of bacterial origin and responses to bacterium. The significantly enriched biological processes of the differentially expressed genes between the LPS group and the TJ5 group included glutathione metabolic processes, responses to stress cellular and responses to chemical stimulus. In molecular function groups, glutathione binding and oligopeptide binding were significantly enriched by the differentially expressed genes. In cellular component classifications, the enrichment of the differentially expressed genes was mainly observed in the cytoplasm, endoplasmic reticulum, and microsomes. KEGG pathway enrichment analysis indicated that the differentially expressed genes between the normal control group and LPS group were enriched in IL-17 signaling pathways, tumor necrosis factor (TNF) signaling pathways, viral protein interactions with cytokines and cytokine receptors signaling pathways, and cytokine-cytokine receptor interaction signaling pathways. In contrast, the differentially expressed genes between the LPS and TJ5 groups were mainly enriched in atherosclerosis signaling pathways, ferroptosis signaling pathways, glutathione metabolism signaling pathways, and cytochrome P450-mediated drug metabolism signaling pathways. CONCLUSIONS Mouse small intestinal organoids were successfully extracted and cultured. TJ5 may exert its protective effects by regulating gene expression and related signaling pathways (fluid shear stress and atherosclerosis, ferroptosis, glutathione metabolism, cytochrome P450 drug metabolism, etc.) in sepsis-injured mouse small intestinal organoids. These genes and signaling pathways may be key targets for treating sepsis-induced intestinal injury.
Animals ; Mice ; Sepsis/genetics* ; Organoids/drug effects* ; Mice, Inbred C57BL ; Intestine, Small/metabolism* ; Gene Expression Profiling ; Transcriptome ; Lipopolysaccharides

Pristimerin, which is one of the compounds present in Celastraceae and Hippocrateaceae, has antitumor effects. However, its mechanism of action in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to investigate the efficacy and mechanism of pristimerin on ESCC in vitro and in vivo. The inhibitory effect of pristimerin on cell growth was assessed using trypan blue exclusion and colony formation assays. Cell apoptosis was evaluated by flow cytometry. Gene and protein expressions were analyzed through quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry. RNA sequencing (RNA-Seq) was employed to identify significantly differentially expressed genes (DEGs). Cell transfection and RNA interference assays were utilized to examine the role of key proteins in pristimerin?s effect. Xenograft models were established to evaluate the antitumor efficiency of pristimerin in vivo. Pristimerin inhibited cell growth and induced apoptosis in ESCC cells. Upregulation of Noxa was crucial for pristimerin-induced apoptosis. Pristimerin activated the Forkhead box O3a (FoxO3a) signaling pathway and triggered FoxO3a recruitment to the Noxa promoter, leading to Noxa transcription. Blocking FoxO3a reversed pristimerin-induced Noxa upregulation and cell apoptosis. Pristimerin treatment suppressed xenograft tumors in nude mice, but these effects were largely negated in Noxa-KO tumors. Furthermore, the chemosensitization effects of pristimerin in vitro and in vivo were mediated by Noxa. This study demonstrates that pristimerin exerts an antitumor effect on ESCC by inducing AKT/FoxO3a-mediated Noxa upregulation. These findings suggest that pristimerin may serve as a potent anticancer agent for ESCC treatment.
Forkhead Box Protein O3/genetics* ; Humans ; Apoptosis/drug effects* ; Esophageal Squamous Cell Carcinoma/physiopathology* ; Esophageal Neoplasms/physiopathology* ; Pentacyclic Triterpenes ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Mice ; Signal Transduction/drug effects* ; Mice, Nude ; Cell Proliferation/drug effects* ; Triterpenes/pharmacology* ; Xenograft Model Antitumor Assays ; Mice, Inbred BALB C ; Male ; Gene Expression Regulation, Neoplastic/drug effects*

OBJECTIVE: miR-34c-3p is down-regulated in nasopharyngeal carcinoma (NPC). The biological role of miR-34c-3p in NPC and its underlying mechanisms are unknown and were explored in this study. METHODS: Flow cytometry and immunohistochemical staining were employed to detect cluster of differentiation 86 (CD86) and cluster of differentiation 206 (CD206) expression; quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were employed to examine mRNA expression and protein levels; cell counting kit-8 (CCK8) and transwell assays were employed to assess cell proliferation, migration, and invasion; and hematoxylin-eosin (HE) staining was employed to assess pathological changes in tumor tissues. RESULTS: Our results revealed that the miR-34c-3p mimic markedly inhibited M2 polarization of macrophages by targeting SLC7A11, and M2 macrophages transfected with the miR-34c-3p mimic inhibited the proliferation, migration, and invasion of NPC cells. The in vivo experiments further confirmed that miR-34c-3p mimics blocked tumor growth and reduced inflammatory infiltration in tumor tissues. CONCLUSION This study provides novel insights into the pathogenesis of NPC and a new treatment strategy.
MicroRNAs/metabolism* ; Nasopharyngeal Carcinoma/genetics* ; Humans ; Animals ; Nasopharyngeal Neoplasms/genetics* ; Macrophages/physiology* ; Cell Line, Tumor ; Mice ; Cell Proliferation ; Mice, Inbred BALB C ; Cell Movement ; Male ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; Female

OBJECTIVE: To identify prognostic genes associated with lysosome-dependent cell death (LDCD) in patients with gastric cancer (GC). METHODS: Differentially expressed genes (DEGs) were identified using The Cancer Genome Atlas - Stomach Adenocarcinoma. Weighted gene co-expression network analysis was performed to identify the key module genes associated with LDCD score. Candidate genes were identified by DEGs and key module genes. Univariate Cox regression analysis, and least absolute shrinkage and selection operator regression and multivariate Cox regression analyses were performed for the selection of prognostic genes, and risk module was established. Subsequently, key cells were identified in the single-cell dataset (GSE183904), and prognostic gene expression was analyzed. Cell proliferation and migration were assessed using the Cell Counting Kit-8 assay and the wound healing assay. RESULTS: A total of 4,465 DEGs, 95 candidate genes, and 4 prognostic genes, including C19orf59, BATF2, TNFAIP2, and TNFSF18, were identified in the analysis. Receiver operating characteristic curves indicated the excellent predictive power of the risk model. Three key cell types (B cells, chief cells, and endothelial/pericyte cells) were identified in the GSE183904 dataset. C19orf59 and TNFAIP2 exhibited predominant expression in macrophage species, whereas TNFAIP2 evolved over time in endothelial/pericyte cells and chief cells. Functional experiments confirmed that interfering with C19orf59 inhibited proliferation and migration in GC cells. CONCLUSION C19orf59, BATF2, TNFAIP2, and TNFSF18 are prognostic genes associated with LDCD in GC. Furthermore, the risk model established in this study showed robust predictive power.
Stomach Neoplasms/pathology* ; Humans ; Prognosis ; Lysosomes/physiology* ; RNA-Seq ; Cell Death ; Single-Cell Analysis ; Gene Expression Regulation, Neoplastic ; Cell Proliferation ; Single-Cell Gene Expression Analysis

OBJECTIVES: This study aimed to investigate the impact of foam macrophages (FMs) on the intracellular survival of Mycobacterium tuberculosis (MTB) and identify the molecular mechanisms influencing MTB survival. METHODS: An in vitro FM model was established using oleic acid induction. Transcriptomic and metabolomic analyses were conducted to identify the key molecular pathways involved in FM-mediated MTB survival. RESULTS: Induced FMs effectively restricted MTB survival. Transcriptomic and metabolomic profiling revealed distinct changes in gene and metabolite expression in FMs during MTB infection compared with normal macrophages. Integrated analyses identified significant alterations in the cyclic adenosine monophosphate (cAMP) signaling pathway, indicating that its activation contributes to the FM-mediated restriction of MTB survival. CONCLUSIONS FMs inhibit MTB survival. The cAMP signaling pathway is a key contributor. These findings enhance the understanding of the role of FMs in tuberculosis progression, suggest potential targets for host-directed therapies, and offer new directions for developing diagnostic and therapeutic strategies against tuberculosis.
Mycobacterium tuberculosis/physiology* ; Transcriptome ; Metabolomics ; Foam Cells/microbiology* ; Humans ; Metabolome ; Tuberculosis/microbiology* ; Gene Expression Profiling

OBJECTIVE: To explore the correlation between chromosome 8 open reading frame 76 (C8orf76) and cyclin-dependent kinase 4 (CDK4) and the potential predictive effect of C8orf76 and CDK4 on the prognosis of colorectal cancer (CRC). METHODS: We constructed a protein-protein interaction network of C8orf76-related genes and analyzed the prognostic signatures of C8orf76 and CDK4. Clinicopathological features of C8orf76 and CDK4 were visualized using a nomogram. RESULTS: C8orf76 and CDK4 levels were positively correlated in two independent human CRC cohorts ( n = 83 and n = 597). A consistent positive correlation was observed between C8orf76 and CDK4 expression in the CRC cell lines. The nomogram included prognostic genes (C8orf76 and CDK4) and pathological N and M stages. The concordance index (C-index) in our cohort was 0.776, which suggests that the ability of the indicators to predict the overall survival of patients with CRC in our cohort was strong. CONCLUSION We found that C8orf76 was positively correlated with CDK4 in both the cohorts as well as in CRC cell lines. Therefore, C8orf76 and CDK4 can be used as potential biomarkers to predict the prognosis of CRC.
Humans ; Colorectal Neoplasms/diagnosis* ; Cyclin-Dependent Kinase 4/metabolism* ; Prognosis ; Male ; Female ; Middle Aged ; Biomarkers, Tumor/genetics* ; Aged ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic

OBJECTIVE: Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection frequently develop central nervous system damage, yet the mechanisms driving this pathology remain unclear. This study investigated the primary pathways and key factors underlying brain tissue damage induced by the SARS-CoV-2 beta variant (lineage B.1.351). METHODS: K18-hACE2 and C57BL/6 mice were intranasally infected with the SARS-CoV-2 beta variant. Viral replication, pathological phenotypes, and brain transcriptomes were analyzed. Gene Ontology (GO) analysis was performed to identify altered pathways. Expression changes of host genes were verified using reverse transcription-quantitative polymerase chain reaction and Western blot. RESULTS: Pathological alterations were observed in the lungs of both mouse strains. However, only K18-hACE2 mice exhibited elevated viral RNA loads and infectious titers in the brain at 3 days post-infection, accompanied by neuropathological injury and weight loss. GO analysis of infected K18-hACE2 brain tissue revealed significant dysregulation of genes associated with innate immunity and antiviral defense responses, including type I interferons, pro-inflammatory cytokines, Toll-like receptor signaling components, and interferon-stimulated genes. Neuroinflammation was evident, alongside activation of apoptotic and pyroptotic pathways. Furthermore, altered neural cell marker expression suggested viral-induced neuroglial activation, resulting in caspase 4 and lipocalin 2 release and disruption of neuronal molecular networks. CONCLUSION These findings elucidate mechanisms of neuropathogenicity associated with the SARS-CoV-2 beta variant and highlight therapeutic targets to mitigate COVID-19-related neurological dysfunction.
Animals ; COVID-19/genetics* ; Mice ; Brain/metabolism* ; Apoptosis ; Mice, Inbred C57BL ; SARS-CoV-2/physiology* ; Pyroptosis ; Gene Expression Profiling ; Transcriptome ; Male ; Female

OBJECTIVES: This study aimed to explore the biological functions and clinical value of mothers against decapentaplegic homolog (SMAD) 7 in head and neck squamous cell carcinoma (HNSCC) through bioinformatics analysis and basic experiments. METHODS: The expression of SMAD7 in HNSCC in public databases was studied. Western blot was used to detect the expression of SMAD7 in HNSCC cell lines and normal epithelial cells. The SMAD7 highly expressed HNSCC cell line HSC-4 was silenced, and CCK-8, Transwell assays, and cell scratch experiments were conducted to study the effect of SMAD7 on the biological functions of HSC-4 cells. HNSCC expression profile data were obtained from UCSC xena, and genes related to SMAD7 were selected for gene ontology and Kyoto encyclopedia of genes and genomes gene enrichment analysis, construction of a co-expression gene interaction network, and screening of related cell signaling pathways. Western blot was used to detect the expression changes of proteins in the related cell signaling pathways in HNSCC cells with silenced SMAD7. cBioPortal was utilized to analyze the mutation rate of the SMAD7 gene, and the MethSurv database was used to analyze the methylation level of the SMAD7 gene and its correlation with prognosis. The receiver operating characteristic curve was used to assess the diagnostic value of SMAD7 for HNSCC. TIMER2.0 was used to analyze the correlation between SMAD7 expression and immune cell infiltration. RESULTS: SMAD7 was highly expressed in HNSCC tumor tissues and some cell lines. Silencing the expression of SMAD7 can significantly inhibit the proliferation, migration, and invasion of cancer cells. Silencing SMAD7 can induce the downregulation of vascular cell adhesion molecule 1 (VCAM-1). The bioinformatics analysis showed that the mutation rate of the SMAD7 gene and the methylation level were significantly correlated with the prognosis of patients with HNSCC. The expression of SMAD7 was related to the level of immune cell infiltration in HNSCC. CONCLUSIONS SMAD7 promotes the proliferation, migration, and invasion of HNSCC cells by regulating the expression of VCAM-1. It may be a potential tumor biomarker and therapeutic target for HNSCC.
Humans ; Smad7 Protein/metabolism* ; Prognosis ; Squamous Cell Carcinoma of Head and Neck ; Head and Neck Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Signal Transduction ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Computational Biology

OBJECTIVES: This study aims to investigate the impact of glutathione S-transferase (GST) on the environmental adaptability of Streptococcus mutans (S. mutans). METHODS: A GST knockout strain ΔgsT was constructed. Transcriptomic sequencing was performed to analyze the gene expression differences between the wild-type S. mutans UA159 and its GST knockout strain ΔgsT. Comprehensive functional assessments, including acid tolerance assays, hydrogen peroxide challenge assays, nutrient limitation growth assays, and fluorescence in situ hybridization, were conducted to evaluate the acid tolerance, antioxidant stress resistance, growth kinetics, and interspecies competitive ability of ΔgsT within plaque biofilms. RESULTS: Compared with the wild-type S. mutans, 198 genes in ΔgsT were significantly differentially expressed and enriched in pathways related to metabolism, stress response, and energy homeostasis. The survival rate of ΔgsT in acid tolerance assays was markedly reduced (P<0.01). After 15 min of hydrogen peroxide challenge, the survival rate of ΔgsT decreased to 38.12% (wild type, 71.75%). Under nutrient-limiting conditions, ΔgsT exhibited a significantly lower final OD₆₀₀ value than the wild-type strain (P<0.05). In the biofilm competition assays, the proportion of S. mutans ΔgsT in the mixed biofilm (8.50%) was significantly lower than that of the wild type (16.89%) (P<0.05). CONCLUSIONS GST enhances the acid resistance, oxidative stress tolerance, and nutrient adaptation of S. mutans by regulating metabolism-related and stress response-related genes.
Streptococcus mutans/enzymology* ; Biofilms ; Glutathione Transferase/physiology* ; Adaptation, Physiological ; Hydrogen Peroxide/pharmacology* ; Gene Expression Regulation, Bacterial ; Oxidative Stress ; Metabolic Reprogramming

There is a large gap between production and demand of plant oil in China, which leads to the heavy reliance on imports. Diacylglycerol acyltransferase (DGAT) and phospholipid: diacylglycerol acyltransferase (PDAT) are two key enzymes responsible for the synthesis of triacylglycerol, thereby affecting the yield and quality of plant oil. This paper comprehensively reviews the research progress in DGAT and PDAT in terms of their biological functions in plant oil synthesis, the molecular mechanisms of regulating plant lipid metabolism, growth, and development under stress, and their roles in driving oil synthesis under the background of synthetic biology. Furthermore, future research and application of DGAT and PDAT are prospected. This review aims to provide a basis for deeply understanding the molecular mechanism of plant oil synthesis and improving the quality and productivity of oil crops by the utilization of DGAT and PDAT genes.
Diacylglycerol O-Acyltransferase/physiology* ; Plant Oils/metabolism* ; Acyltransferases/metabolism* ; Lipid Metabolism/genetics* ; Gene Expression Regulation, Plant ; Triglycerides/biosynthesis*