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PubMed2026

Characterization of a Novel BTD Hypomorphic Variant in a Patient with Complex Neurodevelopmental Delay: Resolving Actionable Metabolic Vulnerabilities Beyond Borderline Plasma Biochemistry.

Plasma biochemistry often presents significant limitations in diagnosing borderline metabolic disorders, particularly within complex neurodevelopmental phenotypes. Here, we present the clinical genomic evaluation of a six-year patient presenting with early-onset hypotonia and severe gastrointestinal complications whose newborn screening panel did not evaluate biotinidase (BTD) activity. While initial baseline plasma biochemistry yielded borderline residual BTD function (46% of the population mean), targeted sequencing identified a novel, compound heterozygous hypomorphic variant (p.Thr459Met) in trans with the common p.Asp424His allele. In vitro functional validation confirmed that p.Thr459Met induces severe protein misfolding and intracellular retention, impairing enzyme secretion. Biotin supplementation triggered a documented and favorable therapeutic improvement, establishing this borderline enzymatic background as an actionable metabolic vulnerability unmasked by chronic gastrointestinal stressors. This study underscores the critical value of functional genomic characterization over static enzymatic biomarkers to identify highly treatable metabolic components within heterogeneous clinical landscapes.

Humans↗

PubMed2026

Biomonitoring of industrial heavy metal pollution via enzymatic and metabolic responses in desert ants (Cataglyphis savignyi) and beetles (Tentyrum sp) as bioindicators.

The current work seeks to evaluate the effectiveness of Cataglyphis saviginyi and Tentyrum sp as indicators of pollution in the city's main industrial regions by analyzing their enzymatic activity and primary metabolites. Soil samples were collected at each site under investigation to analyze soil characteristics and heavy metal content. C. saviginyi and Tentyrum sp were collected across four consecutive seasons (2023-2024) to investigate enzymatic (GPT, GOT, ALP, ACP, LDH) and metabolic (lipid, protein, carbohydrate) biomarkers. The physicochemical properties of the soil differed substantially between the industrial areas and the control site. Soil heavy metal buildup was highest at industrial sites (1 and 4) compared to the control site, with the order being Zn > Cr > Cd > Cu. Heavy metal pollution indices were determined. Increased industrial activity from metal industries, ceramics, and chemical painting companies defines this area, as seen by the high Cdeg, mCd, PI, and PLI values derived for industrial sites 1 and 4. While C. saviginyi and Tentyrum sp deconcentrated and released Cr, Cd, and Zn into the soil via the biological accumulation factor (BAF), Cu acted as a macro-concentrator. Compared with the control site, industrial environments were shown to increase levels of GPT, GOT, LDH, ACP, protein, and carbohydrates in C. saviginyi. However, lipid and ALP activity was suppressed. at industrial sites, Tentyrum sp carbohydrate content was higher than at control sites, but GPT, GOT, ALP, ACP, LDH, protein, and lipid activities were all suppressed. Consequently, enzymatic and metabolic biomarkers proved to be sensitive indicators for assessing industrial heavy metal pollution in desert ecosystems.

Animals↗

PubMed2026

Label-Free Urinary Proteomics Uncovers Immune-Related Non-Invasive Biomarkers for Primary Biliary Cholangitis.

Diagnosis of primary biliary cholangitis (PBC) currently depends on invasive liver biopsy or serum markers with inadequate diagnostic performance. This study aimed to identify non-invasive urinary protein biomarkers for PBC detection. Urine specimens from biopsy-verified PBC patients and healthy controls were processed through ultracentrifugation-based protein extraction, enzymatic digestion, and HPLC-ESI-IT/MS proteomic profiling; protein quantification was completed using Spectronaut v14.8. We identified 194 differentially expressed urinary proteins (109 upregulated, 85 downregulated) and screened 10 immune-related candidates through GO and KEGG enrichment. Pearson correlation further filtered three core proteins, osteopontin (SPP1/OPN), RAMP3 and S100A8, that correlated significantly with key PBC biochemical indices (ALP, GGT, AST, ALT, IgM, p < 0.05). Elevated urinary concentrations of OPN, RAMP3 and S100A8 were validated by ELISA in an independent cohort containing 30 PBC patients and 20 healthy volunteers. In summary, urinary OPN, RAMP3 and S100A8 are markedly increased in PBC patients and hold promise as non-invasive diagnostic biomarkers for PBC; however, their diagnostic specificity against other cholestatic and autoimmune liver diseases remains to be evaluated, and further confirmation in larger multicenter cohorts with disease control groups is warranted.

Humans↗

PubMed2026

Lipid reprogramming of stratified squamous epithelium by the high-risk HPV E6 and E6/E7 oncoproteins.

INTRODUCTION: High risk human papillomavirus (HPV) infection and genome integration with pronounced expression of the viral E6/E7 oncogenes is the major cause of cervical cancer. Emerging evidence suggests that HPV reprograms host metabolism to support viral persistence and cellular transformation. However, global HPV oncogene-induced lipidomic reprogramming remains poorly understood, particularly at early stages of HPV-induced transformation. OBJECTIVE: We sought to define the regulation of lipid metabolism in squamous epithelia of transgenic mice expressing the HPV16 oncogene E6 alone or in conjunction with E7. METHODS: Untargeted lipidomics was used to identify novel lipid biomarkers in the skin and female reproductive tract (FRT) of HPV16 E6 and E6/E7 transgenic compared to wild-type (WT) mice. To investigate enzymatic dysregulation of lipids by HPV oncogene expression, we employed Lipid Network Explorer (LINEX2), which analyzes lipidomics data through lipid enrichment analysis. We also used the Global Natural Product Social Molecular Networking (GNPS) platform to enhance lipid identification, exploring molecular networking to improve feature annotation. RESULTS: Our lipidomic analysis produced several new observations. First, E6 expression caused a consistent alteration of glycerophospholipids, with particularly significant substrate-product shifts in the phosphatidylcholine (PC) to lysophosphatidylcholine (LPC) pathway in the skin. Second, E6/E7 expression caused a dysregulation of glucosylceramide (GlcCer) biosynthesis. Third, both E6/E7 expressing skin and FRT tissues exhibited a redox imbalance and increased levels of oxidized lipids, including oxylipins and several oxidized PCs. These findings suggest that HPV oncoproteins drive lipid reprogramming, potentially contributing to early HPV-related tumorigenesis. CONCLUSIONS: These findings provide new insights into HPV‑induced lipid reprogramming and establish a framework for future studies examining the functional and clinical relevance of lipid alterations in HPV‑associated cancers.

Animals↗

PubMed2026

From glycosylation to inflammation: insights from NMR-Derived GlycA and GlycB.

Post-translational modifications (PTMs) play a crucial role in increasing proteomic diversity. N-linked glycosylation acts as a key regulatory layer that influences protein stability, trafficking, circulation, and immune responses. Unlike conventional inflammatory biomarkers that measure individual proteins, nuclear magnetic resonance (NMR) spectroscopy identifies the combined signals GlycA and GlycB from glycoproteins, offering an overall view of systemic glycoprotein changes. These signals represent the N-glycosylation patterns of several abundant acute-phase proteins (APPs), giving detailed molecular insights. This review offers a detailed assessment of GlycA and GlycB as mechanistically grounded indicators of liver glycoprotein remodeling and systemic inflammation. GlycA mainly indicates the levels and structural complexity of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues linked to acute-phase glycoproteins and glycan branching. In contrast, GlycB reflects changes in terminal sialylation, which influences glycoprotein half-life, immune recognition via lectins, and inflammatory signaling. Collectively, these biomarkers combine measurements of hepatic APP production with variations in glycan structure, offering mechanistically anchored reporters of hepatic glycoprotein remodeling. We explore the enzymatic pathways responsible for N-glycan branching, fucosylation, and sialylation, as well as the roles of major APP scaffolds in the GlycA and GlycB resonances. We also highlight the emerging clinical significance of these signals across infectious, autoimmune, cardiovascular, metabolic, neurodegenerative, and cancer-related diseases. Rather than serving simply as markers of inflammation, GlycA and GlycB provide mechanistically interpretable readouts of cytokine-driven hepatic glycoprotein remodeling and systemic immune activation, supporting their application in disease risk stratification, longitudinal monitoring, therapeutic response assessment, and precision medicine.

GlycA↗

PubMed2026

Exploring the impact of syndecans in prostate cancer: Stage-specific roles and therapeutic implications.

Syndecans (SDCs) 1-4 are a family of transmembrane heparan sulfate proteoglycans (HSPGs) that regulate cell-cell communication, adhesion, extracellular matrix organization, and signaling pathways involved in tumor biology. In prostate cancer (PCa), accumulating evidence suggests that SDCs contribute to tumor progression, therapeutic resistance, and interactions within the tumor microenvironment. However, their specific, stage-dependent roles remain incompletely understood. This review provides an integrated synthesis of current experimental and clinical evidence on SDC1-SDC4 in PCa, complemented by exploratory analyses of publicly available transcriptomic, genomic, and proteomic datasets. In contrast, copy-number alteration (CNA) strata dichotomized by the mean for SDC1, SDC2, and SDC4 showed differences in progression-free interval. Specific CNA subclasses and relationships between CNA values and SDC mRNA or protein abundance could not be determined. Proteomic pseudotime analysis further suggested that SDC4 expression increases during PCa progression, supporting its potential involvement in advanced disease. We discuss the regulation and modulation of SDCs by androgen deprivation therapy (ADT), enzymatic shedding, integrin-mediated signaling, extracellular matrix interactions, lipid signaling pathways, and microRNA networks. In particular, SDC1-microRNA interactions may influence PCa cell proliferation, cellular senescence, epithelial-mesenchymal transition (EMT), and intracellular signaling pathways. Overall, this review highlights SDCs as context-dependent regulators of PCa biology with potential relevance as biomarkers or therapeutic targets. However, clinical translation will require independent validation, standardized assays, compartment-resolved analyses, and mechanistic confirmation.

Prognosis↗

PubMed2026

Lactylome Reprogramming Mediates Therapeutic Response and Adaptation to Neoadjuvant Chemotherapy in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) exhibits high prevalence in China and poor prognosis despite neoadjuvant chemotherapy (NACT), with significant chemoresistance development. Tumor-associated metabolic reprogramming and NACT-induced cellular stress promote lactate accumulation, which serves as a precursor for lysine lactylation (Kla), a post-translational modification potentially regulating cancer progression. We hypothesized that systematic characterization of the lactylome in response to NACT could reveal critical molecular mechanisms underlying treatment and identify new therapeutic vulnerabilities in ESCC. Herein, through comprehensive proteomic and lactylome profiling of tumor and adjacent normal adjacent tissues from 31 ESCC patients (with or without NACT treatment), we identified 8281 proteins and 1836 Kla sites across 62 samples. NACT induced substantial lactylome alterations with 307 differentially expressed Kla sites predominantly in nonhistone proteins involved in DNA damage response and metabolic pathways. Our data revealed that while NACT-induced suppression of energy metabolism, coupled with upregulated 3-hydroxy-3-methylglutaryl reductase degradation 1 complex expression, may exert potential proapoptotic effects, the activation of ribosome biogenesis and increased nucleoprotein lactylation triggered tumor-protective mechanisms. Mechanistically, we demonstrated that DNA damage and elevated lactate levels induced poly(ADP-ribose) polymerase 1 K654 lactylation, enhancing its enzymatic activity and augmenting poly(ADP-ribosyl)ation of downstream targets, potentially playing a pivotal role in chemotherapy resistance-associated pathways. This comprehensive tissue-level landscape of Kla dynamics in ESCC response to chemotherapy establishes Kla as a critical regulatory mechanism in treatment response, potentially offering novel therapeutic targets and predictive biomarkers for personalized treatment strategies.

Humans↗

PubMed2026

Decreased TXNRD1 is associated with resistance to tagraxofusp in blastic plasmacytoid dendritic cell neoplasms, as seen in phase II.

Tagraxofusp is a CD123-targeted therapy comprised of a recombinant human interleukin-3 (IL-3) fused to a truncated diphtheria toxin payload. It is the first approved treatment specifically for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN). To identify biomarkers of response, bone marrow samples from 12 BPDCN patients who were treated with tagraxofusp in the pivotal phase II trial (NCT02113982) were profiled longitudinally using a gene panel and single-cell RNA sequencing. Residual tumor cells following tagraxofusp expressed lower levels of TXNRD1 that would reduce the efficacy of tagraxofusp. In support of this, enzymatic inhibition of TXNRD1 resulted in higher viability of CAL-1 BPDCN cells following tagraxofusp. Responders had either wild-type or missense TET2 mutations, while transient and non-responders had at least one truncating TET2 mutation. Examples of these mutations within the catalytic domain of TET2 were constructed and transduced into cells. Missense and truncating mutants displayed reduced sensitivities to hypomethylating agents and prolonged S-phase stasis. These results suggest that the levels of TXNRD1 interact with intrinsic TET2 truncating mutations within the bone marrow to modulate patient response to tagraxofusp.

Female↗

PubMed2026

Standardization Challenges in Glycated Albumin Measurement: Methodological Heterogeneity and Quantification Discrepancies.

BACKGROUND: Glycated albumin (GA) is a valuable biomarker for monitoring glycemic status. However, measurement standardization is challenged by methodological heterogeneity, where different analytical principles and target measurands cause quantification discrepancies. This study systematically compared prevailing methodologies to identify a robust reference measurement procedure for widespread standardization. METHODS: We compared a targeted bottom-up proteomics method (ID-LC-MS/MS) with an enzymatic assay and the Japan Society of Clinical Chemistry (JSCC) reference method. A cohort of 129 donor serum specimens and certified reference materials (JCCRM-611) were analyzed to assess methodological comparability. Furthermore, GA concentration-dependent glycation kinetics at the Lys-525 site of albumin was examined. RESULTS: The optimized targeted bottom-up proteomics method showed a strong correlation (r = 0.986) with both the enzymatic assay and JSCC reference method. However, a progressively increasing negative systematic bias was observed at higher GA levels, confirming that Lys-525 underestimates GA at higher levels. In addition, with increasing overall GA concentration, the glycation ratio at the Lys-525 site consistently declined compared to the total glycated lysine residues. CONCLUSIONS: The standardization of GA measurements requires a precise, universally accepted definition to address analytical discrepancies. The present results indicate that quantification targeting all glycated lysine residues (as in the JSCC method) aligns more closely with biologically relevant GA values than site-specific measurement at Lys-525, which shows greater bias at higher concentrations. Therefore, further GA standardization would focus on adopting total glycated lysine residues on albumin as the preferred measurand definition, to improve detection accuracy and clinical comparability.

Serum Albumin↗

PubMed2025

Mass spectrometry-based ligand binding assays in biomedical research.

INTRODUCTION: Ligand binding assays combining immunoaffinity enrichment steps with mass spectrometry (MS) readout have gained attention as a highly specific and sensitive tool for protein quantification. These techniques typically combine enzymatic fragmentation of the sample or enriched protein with capture on the protein or peptide-level for quantification. Antibodies ensure specific target recognition, while MS offers quantitative accuracy with isotopically labeled internal standards. This dual approach supports a broad dynamic range, enabling protein measurements from picomolar to nanomolar levels. These methods have diverse applications, from quantifying signaling proteins in basic research to biomarker monitoring in clinical trials and analyzing the pharmacokinetics of therapeutic proteins. AREAS COVERED: This review delves into the diverse workflows of immunoaffinity-MS, shedding light on the innovative strategies employed, their practical applications, efficacy, and inherent limitations in the realm of protein quantification. EXPERT OPINION: Immunoaffinity-MS has transformed protein analysis, but widespread adoption is hindered by complex workflows, high instrument costs, and limited capture molecule availability. Efforts to enhance automation, standardize workflows, and advance technological innovation aim to overcome these barriers. Improvements in mass spectrometer sensitivity, advances in recombinant capture technologies, and support from public initiatives are poised to further improve the reliability and accessibility of this method.

Mass Spectrometry↗

PubMed2026

Faecalibacterium harmsenii sp. nov., an abundant but previously overlooked Faecalibacterium in the human gut.

Faecalibacterium is one of the most abundant anaerobes in the human colon. At the genus level, this bacterium shows a strong positive association with human health. Expanding collections of isolates and metagenome-assembled genomes have revealed its species diversity, yet species-level functions remain so far underexplored. Here, we describe a novel species, Faecalibacterium harmsenii. In addition, we reclassify another isolate as a member of the recently reported Faecalibacterium langellae species. Despite close genomic relatedness, these isolates exhibit distinct physiological and biochemical traits, including differences in carbohydrate utilization, stress tolerance, enzymatic activity, Gram-staining and fatty acid composition. Our present comparative genomics analyses further uncover extensive functional diversity and plasticity across type strains, with F. harmsenii being distinguished by an expanded carbohydrate gene repertoire and reduced defense systems, mobile genetic elements and antibiotic resistance genes. Extending to the species, we identify species-specific ecological niches across hosts and differential sensitivities to human diseases, highlighting certain species as reliable biomarkers of gut health. Together, these findings refine our understanding of Faecalibacterium diversity and provide a framework for its use in microbiome-based diagnostics and therapeutic development.

Faecalibacterium harmsenii↗

PubMed2026

Personalised Nutraceutical Treatment Guided by MTHFR Genotype in Mental Health: A Retrospective Cohort Study.

BACKGROUND & AIMS: One-carbon metabolism plays a central role in neurotransmitter synthesis, methylation capacity, and neurobiological resilience. Variants in the methylenetetrahydrofolate reductase (MTHFR) gene can reduce enzymatic activity, affecting folate- and methionine-cycle functions and potentially influencing biological pathways relevant to mood and anxiety disorders. Personalised nutraceutical treatment strategies, particularly those addressing methylation capacity through targeted B-vitamin, folate, and adjunctive metabolic interventions are increasingly implemented in integrative clinical practice, yet evidence regarding their clinical outcomes remains limited. METHODS: We conducted a retrospective cohort study of 50 adults attending an integrative general practice clinic for anxiety and/or depression. All received personalised nutraceutical treatment informed by clinical assessment, laboratory testing and, for 37/50 patients, MTHFR genotyping. Psychological distress was measured using the Kessler-10 (K10) scale at baseline and approximately three months later. Secondary analyses evaluated whether outcomes differed by MTHFR genotype, whether specific supplements (e.g., L-methylfolate and SAMe) were associated with greater improvement, whether biomarker changes correlated with symptom change, and the safety/tolerability profile. RESULTS: Across the full cohort, mean K10 scores significantly decreased by four points over the treatment period, with 72% of patients showing clinical improvement. Reductions in psychological distress were seen across all MTHFR genotypes, including individuals with homozygous variant genotypes. Supplement-specific analyses showed improvement among those receiving methylfolate or SAMe, although the differences were not statistically significant. Following nutraceutical treatment, biomarker analyses demonstrated significant increases in serum vitamin B12 and modest reductions in homocysteine, but biomarker shifts did not correlate strongly with K10 change. No serious adverse events or clinically significant abnormalities in liver or renal function were identified. CONCLUSIONS: In this real-world primary care cohort, personalised nutraceutical treatment, grounded in one-carbon metabolism support and applied alongside usual care, was associated with clinically meaningful reductions in psychological distress. Outcomes were comparable across MTHFR genotypes when treatments were appropriately tailored, suggesting that genotype and biomarker-informed nutraceutical strategies may mitigate potential metabolic disadvantages. These findings support further controlled research into precision nutraceutical psychiatry for anxiety and depression. Secondary analyses of genotype subgroup, specific supplements, and biomarker-outcome associations are reported alongside Benjamini-Hochberg FDR-adjusted p-values and should be interpreted as hypothesis-generating.

Humans↗

PubMed2018

High-Throughput Metabolomics by 1D NMR.

Metabolomics deals with the whole ensemble of metabolites (the metabolome). As one of the -omic sciences, it relates to biology, physiology, pathology and medicine; but metabolites are chemical entities, small organic molecules or inorganic ions. Therefore, their proper identification and quantitation in complex biological matrices requires a solid chemical ground. With respect to for example, DNA, metabolites are much more prone to oxidation or enzymatic degradation: we can reconstruct large parts of a mammoth's genome from a small specimen, but we are unable to do the same with its metabolome, which was probably largely degraded a few hours after the animal's death. Thus, we need standard operating procedures, good chemical skills in sample preparation for storage and subsequent analysis, accurate analytical procedures, a broad knowledge of chemometrics and advanced statistical tools, and a good knowledge of at least one of the two metabolomic techniques, MS or NMR. All these skills are traditionally cultivated by chemists. Here we focus on metabolomics from the chemical standpoint and restrict ourselves to NMR. From the analytical point of view, NMR has pros and cons but does provide a peculiar holistic perspective that may speak for its future adoption as a population-wide health screening technique.

Animals↗

PubMed2026

Development of a High-Sensitivity Glycoproteomics Approach for Fc-Specific Quantification of IgG Core Fucosylation in Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers complex neuroinflammatory cascades that involve sustained immune activation and dysregulated antibody effector functions. Immunoglobulin G (IgG) Fc N-glycosylation, particularly core fucosylation, critically modulates immune signaling through altered Fcγ receptor (FcγR) interactions; however, its role in TBI remains unexplored. Here, we developed a high-sensitivity, mass spectrometry-based glycoproteomics method for the systematic analysis of IgG Fc core fucosylation dynamics following TBI. The approach integrates Fc-specific enzymatic truncation with GlycINATOR (EndoS2) and tryptic digestion, followed by high-resolution LC-MS/MS profiling, enabling confident identification of truncated Fc glycopeptides. Furthermore, a targeted parallel reaction monitoring (PRM) strategy allowed direct quantification of core fucosylated and afucosylated glycopeptides from 10 μg of crude serum protein, eliminating the need for IgG purification. Our results reveal time-dependent and subclass-specific remodeling of IgG Fc fucosylation postinjury, characterized by an overall reduction in fucosylated species and a relative increase in afucosylation. Collectively, this study establishes a scalable analytical platform for Fc-specific glycosylation profiling and identifies IgG core fucosylation as a candidate molecular indicator of immune dysregulation in TBI, providing new insights into post-traumatic immune regulation.

Brain Injuries, Traumatic↗

PubMed2025

The biological relevance of potentially toxic metals in freshwater fish.

Trace elements are essential for a number of physiological functions including oxygen transfer, enzymatic reactions and antioxidant protection of the animal organism. Elevated concentrations outside the physiological optimum, on the other hand, can cause undesirable health complications, disrupt metabolic pathways, reproductive capacity, or oxidative balance. The negative anthropogenic impacts on the environment are alarming and the impacts on the aquatic environment have been increasing disproportionately in recent years. Against this background, all potential threats to biota need to be explained and better understood, the possible risks need to be better informed and understood, and a balance needs to be struck between the fundamental nature and the harmful effects of these metals. This mini-review examines the roles of potentially toxic metals including cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn) in fish physiology. This document also elucidates the mechanisms underlying the assessment of regulatory processes, the potential negative consequences of overexposure, the interactions of these metals on fish health, and in the environmental context.

biomarker↗

PubMed2025

Proteomic profiling identifies miR-423-5p as a modulator of oncogenic metabolism in HCC.

BACKGROUND: Hepatocellular carcinoma (HCC) remains a significant clinical challenge due to limited diagnostic and therapeutic options. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), play key roles in cancer biology. Our previous findings showed that miR-423-5p enhances anti-cancer effects on HCC patients treated with sorafenib by promoting autophagy. Here, we investigated the molecular mechanisms underlying miR-423-5p function through a comprehensive proteomic approach. METHODS: We generated an HCC cell line stably overexpressing miR-423-5p via lentiviral transduction. Total proteins were extracted from SNU-387 cells, enzymatically digested into peptides, and subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/M). Raw spectral data were processed and quantified using MaxQuant. Differentially expressed proteins (DEPs) were defined based on fold-change (|log2FC| ≥ 1) and false discovery rate (FDR < 0.05). The full proteomic dataset is available via the ProteomeXchange repository (identifier: PXD064869). Functional enrichment analysis of DEPs were performed using DAVID and Reactome. To assess clinical relevance, predicted and validated miR-423-5p targets were integrated with The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma (LIHC) dataset using GEPIA platform. Survival analyses were performed using the Kaplan-Meier method. RESULTS: Proteomic profiling identified 698 DEPs in miR-423-5p-overexpressing cells compared to controls with significant enrichment in metabolic pathways, related to purine/pyrimidine metabolism and gluconeogenesis. Integration with bioinformatic predictions and miRTarBase validation identified 43 DEPs as potential direct targets of miR-423-5p. Among these, seven proteins (ACACA, ANKRD52, DVL3, MCM5, MCM7, RRM2, SPNS1, and SRM) were significantly associated with patient prognosis in the TCGA-LIHC cohort. These targets were downregulated in miR-423-5p-overexpressing cells but upregulated in advanced-stage HCC tissues, suggesting a potential role for miR-423-5p in the regulation of HCC pathogenesis. Stage-specific expression analysis showed increased levels from stage I to III, followed by a decline at stage IV. Notably, we experimentally confirmed miR-423-5p-mediated suppression of MCM7, DVL3, IMPDH1, and SRM (SPEE), supporting their functional involvement in HCC progression. CONCLUSION: Overall, our findings support a tumour-suppressive role for miR-423-5p in HCC, mediated by modulation of metabolic pathways and suppression of oncogenic proteins. These results suggest that miR-423-5p and its downstream effectors may serve as promising biomarkers and potential therapeutic targets in HCC. HIGHLIGHTS: miR-423-5p acts as a tumor suppressor in HCC by targeting key nodes of pro-tumorigenic signalling. miR-423-5p significantly altered metabolic pathways, including purine/pyrimidine metabolism and gluconeogenesis. Seven miR-423-5p targets correlate with poor prognosis in TCGA-LIHC patients and are downregulated in miR-423-5p overexpressing HCC cells. miR-423-5p over-expression induces a significant downregulation of MCM7, DVL3, IMPDH1, SPEE in HCC cell models. miR-423-5p limits tumor metabolic plasticity, suggesting therapeutic potential.

MicroRNAs↗