Nuclear factor interleukin-3-regulated protein (NFIL3) is a transcription factor involved in the regulation of circadian rhythms and immune crosstalk. The transcriptional repressive and activational roles that influence cytokine expressions and tumor microenvironment have made NFIL3 a therapeutic target for several diseases. While protein-level functions of NFIL3 are widely explored, the molecular details regarding phospho-signals regulating NFIL3 and their functional significance are underexplored. To interpret the phosphoregulatory patterns of NFIL3, we conducted an extensive literature survey for mass-spectrometry-based cellular phosphoproteomics datasets with representation of NFIL3 phosphosites. From the compiled list of 656 cellular profiles and 116 datasets with differential expression, Ser301 and Ser353 of NFIL3 were identified as the predominant phosphosites, accounting for the majority of NFIL3 phosphosignals. Further, we explored the coregulated phosphosites in the interacting partners and transcription factors associated with NFIL3. With our integrative phosphoproteomics and the IL-4 dependent transcriptome dataset analysis, we report Ser301 and Ser353 as positively co-occurring phosphorylations in NFIL3 that essentially regulate its transcriptional activity. Further, we also unveil Mitogen-activated protein kinase 14 (MAPK14) as a high-confidence upstream kinase that can phosphorylate Ser301. This study identifies novel coregulation expression patterns and highlights regulatory phosphosites, establishing the foundation for future exploration of the NFIL3-associated dark phosphoproteome. NFIL3 has emerged as a promising therapeutic target in a range of diseases, including inflammatory disorders, autoimmune conditions, and cancers, owing to its central role in regulating circadian rhythms, cytokine expression, and immune cell differentiation. However, the lack of information on kinases phosphorylating NFIL3 and its regulatory mechanisms hinders understanding of how phosphorylation influences its signal-dependent transcriptional activity. To address this, our study systematically investigates the phosphoregulatory landscape of NFIL3 by global phosphoproteomics datasets. Furthermore, by integrating phosphorylation events with transcriptomic changes, we highlight how phosphorylation of NFIL3 may influence its transcriptional output, laying the groundwork for future mechanistic and therapeutic explorations.
Insulin-like growth factor binding protein 6 (IGFBP6) is thought to be associated with the progression of atherosclerosis (AS). However, whether IGFBP6 is involved in ferroptosis, a key biological process, in the progression of AS remains unclear. In this study, IGFBP6 expression was downregulated in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). Overexpression of IGFBP6 inhibited reactive oxygen species (ROS) production, reduced Fe2+ concentration and and inflammatory factor levels, and increased Glutathione (GSH) content in ox-LDL-treated HUVECs. Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, reversed IGFBP6 silencing induced ferroptosis of HUVECs. Mechanistic studies revealed that IGFBP6 is a target of Methyltransferase-like protein 3 (METTL3) that mediates N6-methyladenosine (m6A) modification, and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) recognizes and promotes METTL3-m6A-mediated IGFBP6 mRNA degradation. Downregulated IGFBP6 reduces its protein level by interacting with GPX4, thereby promoting ferroptosis in HUVECs. In addition, APOE-/- mice fed with a high-fat diet were used as an AS animal model, and the results showed that endothelial cell specific METTL3 knockdown or IGFBP6 overexpression hindered the development of AS in mice. In conclusion, knockdown of METTL3 inhibited the degradation of IGFBP6 mRNA in a YTHDF2 dependent manner, thereby inhibiting GPX4-mediated ferroptosis and alleviating AS progression.Insight Box This study provided new molecular targets for the effective treatment of AS. In this study, we constructed an AS cell model and an AS mouse model to explore whether METTL3 affects ferroptosis of HUVECs by regulating IGFBP6 expression through m6A modification through gain of function and loss of function experiments of METTL3 and IGFBP6, thus revealing the molecular mechanism of its involvement in the occurrence and development of AS. Results showed that METTL3 induced GPX4-mediated ferroptosis in HUVECs through m6A-YTHDF2-dependent downregulation of IGFBP6, thereby promoting the progression of AS. These findings suggest that interfering with the METTL3/YTHDF2/IGFBP6/GPX4 axis with small molecules or gene therapy may be a new strategy to intervene the occurrence and development of AS.
Background Benzo[a]pyrene diol epoxide (BPDE), a key carcinogenic metabolite of the ubiquitous environmental pollutant benzo[a]pyrene, is a well-established etiological agent in lung cancer. However, a systems-level understanding of its molecular targets and pathogenic mechanisms remains incomplete. This study aimed to identify and characterize the core toxicity targets linking BPDE exposure to lung carcinogenesis using an integrative network toxicology approach.Methods Putative BPDE targets were retrieved from SwissTargetPrediction and TargetNet. Lung cancer-associated genes were compiled from GeneCards and TCGA datasets. The intersection yielded candidate genes, which were analyzed via protein-protein interaction (PPI) network construction, functional enrichment, and machine learning (LASSO, XGBoost, Random Forest, Boruta) to identify core biomarkers. A nomogram was developed for risk prediction. The key target CA2 was further validated for prognostic value, differential expression across multiple cohorts (TCGA, GEO), and associated biological pathways via gene set enrichment analysis (GSEA) and pathway activity scoring. Molecular docking was performed to simulate the BPDE-CA2 interaction.Results Thirty-one overlapping targets were identified. Enrichment analysis implicated processes like inflammation, oxidative stress response, and cancer-related pathways (e.g. p53, cell cycle). Machine learning convergence highlighted four core biomarkers: PARP1, ALOX5, CA9, and CA2. Among these, CA2 demonstrated significant prognostic value, consistent downregulation in lung tumor tissues across independent datasets, and distinct molecular phenotypes: its high expression correlated with immune activation and suppressed VEGF signaling, while low expression was linked to enhanced cell cycle activity. Molecular docking revealed a stable binding interaction between BPDE and CA2.Conclusion This integrative study identifies a set of core targets, prominently featuring CA2, that potentially mediate BPDE-induced lung carcinogenesis. CA2 downregulation is associated with poor prognosis, a pro-proliferative phenotype, and may be directly modulated by BPDE, positioning it as a critical molecular link between environmental exposure and lung cancer development. Insight Paragraph This study provides a systems-level molecular bridge connecting environmental carcinogen exposure to lung cancer pathogenesis. By integrating network toxicology, machine learning, and multi-omics validation, we move beyond single-gene associations to identify a core set of toxicity targets, most notably CA2. We demonstrate that CA2 downregulation, potentially via direct interaction with BPDE, is linked to poor prognosis and a pro-proliferative phenotype. This integrative approach offers a novel mechanistic insight, positioning CA2 as a critical node at the intersection of environmental exposure, disrupted metabolism, and tumor progression, with implications for risk prediction and targeted prevention strategies.Conclusion This integrative study identifies a set of core targets, prominently featuring CA2, that potentially mediate BPDE-induced lung carcinogenesis. CA2 downregulation is associated with poor prognosis, a pro-proliferative phenotype, and may be directly modulated by BPDE, positioning it as a critical molecular link between environmental exposure and lung cancer development. Insight Paragraph This study provides a systems-level molecular bridge connecting environmental carcinogen exposure to lung cancer pathogenesis. By integrating network toxicology, machine learning, and multi-omics validation, we move beyond single-gene associations to identify a core set of toxicity targets, most notably CA2. We demonstrate that CA2 downregulation, potentially via direct interaction with BPDE, is linked to poor prognosis and a pro-proliferative phenotype. This integrative approach offers a novel mechanistic insight, positioning CA2 as a critical node at the intersection of environmental exposure, disrupted metabolism, and tumor progression, with implications for risk prediction and targeted prevention strategies.
Background Diabetes mellitus (DM) and its cardiovascular complications represent one of the most prevalent causes of death worldwide, closely linked to chronic inflammatory states. Inflammatory Burden Index (IBI) is a composite inflammatory marker that comprehensively assesses systemic inflammation levels. Currently, the prognostic association of IBI with outcomes in individuals living with DM and prediabetes (pre-DM) remains unclear. Objective To investigate association of IBI with all-cause and cardiovascular mortalities in individuals living with DM and pre-DM. Methods Utilizing data from NHANES 1999-2010, we enrolled 8738 participants. Kaplan-Meier analysis and weighted multivariate Cox proportional hazards models were employed to examine relationship of IBI tertiles with mortality risk. RCS analysis tested for non-linear associations, while subgroup analyses evaluated effect modification. Sensitivity analysis was performed by excluding deaths occurring during the early follow-up period. Results During a median 11.7 years of follow-up, 2726 all-cause deaths (31.2%) and 893 cardiovascular deaths (10.2%) were recorded. Results demonstrated significant associations of IBI with mortality risks in individuals living with pre-DM and DM. In fully adjusted model, compared with the lowest IBI tertile (T1), the highest tertile (T3) was associated with a 42% elevated risk of all-cause mortality (HR = 1.42, 95%CI: 1.24-1.63, P < 0.001) and a 38% elevated risk of cardiovascular mortality (HR = 1.38, 95%CI: 1.08-1.76, P = 0.011). RCS analysis revealed an overall significant association of IBI with mortality risks without evidence of non-linearity (P-non-linear>0.05). No significant interaction effects were seen in subgroup analyses. After excluding deaths occurring within the first 1 and 2 years of follow-up in sensitivity analyses, the above associations remained generally consistent. Conclusion Elevated IBI was significantly associated with elevated risks of all-cause and cardiovascular mortalities in persons with DM and pre-DM, suggesting that IBI may help identify subjects at higher mortality risk within this population.
Purpose This study used scRNA-seq to explore the underlying mechanisms of decidual natural killer cells affecting recurrent miscarriage (RM).Methods We obtained the GSE214607 scRNA-seq dataset from Gene Expression Omnibus (GEO) database, containing data from 5 normal and 3 RM pregnancy samples. The 'Seurat' and 'Harmony' R package was used for the scRNA-seq analysis. Differentially expressed genes among cell subpopulations were identified using FindAllMarkers function, and the 'Monocle2' package was employed to construct pseudo-time trajectories. Finally, the 'CellChat' package was used to analyze ligand-receptor interactions between cells.Results Our analysis revealed altered immune cell composition in RM, with reduced decidual NK (dNK), decidual stromal, and epithelial glandular cells but increased Mono/Macro proportions. Two dNK subclusters were identified: PAEP-high (linked to growth factor signaling) and LGALS1-high (involved in glycolysis and platelet function). Despite similar subcluster frequencies, RM samples showed elevated interferon response genes (e.g. IFITM1, ISG15) and diminished migration/activation signals. Pseudo-time analysis highlighted progressive gene expression changes during RM. CellChat revealed reduced Vascular Endothelial Growth Factor (VEGF) signaling, indicating impaired angiogenesis, and enhanced TNF, MHC-II, and FAS/FASL interactions, suggesting increased inflammation and trophoblast apoptosis. These findings implicate dNK dysfunction in RM and suggest potential therapeutic targets.Conclusions Our findings demonstrate dNK cell dysfunction in RM, marked by heightened interferon signaling and aberrant crosstalk with trophoblasts. Integrative analysis of public transcriptomic data suggests that dNK cell-directed interventions represent a putative therapeutic avenue for RM. Insight RM linked to decidual immune imbalance with reduced dNK and increased monocytes/macrophages. Two dNK subclusters (PAEP-high, LGALS1-high) with specific gene expression pattern in RM. RM shows impaired angiogenesis, inflammation, and trophoblast apoptosis in dNK cell-mediated signaling pathways. Dysfunctional dNK cells drive RM, revealing potential therapeutic targets for treatment.Conclusions Our findings demonstrate dNK cell dysfunction in RM, marked by heightened interferon signaling and aberrant crosstalk with trophoblasts. Integrative analysis of public transcriptomic data suggests that dNK cell-directed interventions represent a putative therapeutic avenue for RM. Insight RM linked to decidual immune imbalance with reduced dNK and increased monocytes/macrophages. Two dNK subclusters (PAEP-high, LGALS1-high) with specific gene expression pattern in RM. RM shows impaired angiogenesis, inflammation, and trophoblast apoptosis in dNK cell-mediated signaling pathways. Dysfunctional dNK cells drive RM, revealing potential therapeutic targets for treatment.
