Background and aims: Microvascular invasion (MVI) is a crucial pathological hallmark of hepatocellular carcinoma (HCC), intricately associated with poor outcomes, early recurrence and intrahepatic metastasis following surgical resection and transplantation. However, the intricate tumor microenvironment (TME) and transcriptional programs underlying MVI in HCC remain poorly understood. Approach and results: We performed single-cell RNA sequencing of 46,789 individual cells from 10 samples of MVI+ (MVI present) and MVI- (MVI absent) HCC patients. We conducted comprehensive and comparative analyses to characterize cellular and molecular features associated with MVI, and validated key findings using external bulk, single-cell and spatial transcriptomic datasets, coupled with multiplex immunofluorescence assays. The comparison identified specific subtypes of immune and stromal cells critical to the formation of the immunosuppressive and pro-metastatic microenvironment in MVI+ tumors, including cycling T cells, LAMP3+ dendritic cells, TREM2+ macrophages, myofibroblasts, and arterial i endothelial cells. MVI+ malignant cells are characterized by high proliferation rates while MVI-malignant cells exhibit an inflammatory milieu. Additionally, we identified the MDK-dominated interaction between TREM2+ macrophages and malignant cells as a contributor to MVI formation and tumor progression. Notably, we unveiled a spatially co-located multicellular community exerting a dominant role in shaping the immunosuppressive microenvironment of MVI and correlating with unfavorable prognosis. Conclusions: This study provides a comprehensive single-cell atlas of MVI in HCC, shedding light on the complex multicellular ecosystem and molecular features associated with MVI. These findings deepen our understanding of the underlying mechanisms driving MVI and provide valuable insights for improving clinical diagnosis and developing more effective treatment strategies. Export

Hepatocellular carcinoma (HCC) stands as the most prevalent and treatment‐resistant malignant tumour, characterized by a dismal prognosis. Croton acylation (CA) has recently gained attention as a critical factor in cancer pathogenesis. This study sought to rapidly identify prognostic features of HCC linked to CA. Differential analysis was conducted between tumour tissues and adjacent non‐tumour tissues in the TCGA‐LIHC and GSE76427 datasets to uncover differentially expressed genes (DEG1 and DEG2). The intersection of DEG1 and DEG2 highlighted DEGs with consistent expression patterns. Single‐sample gene set enrichment analysis scores were calculated for 18 lysine crotonylation‐related genes (LCRGs) identified in prior research, showing significant differences between tumour and normal groups. Subsequently, weighted gene co‐expression network analysis was employed to identify key module genes correlated with the LCRG score. Candidate genes were identified by overlapping consistently expressed DEGs with key module genes. Prognostic features were identified, and risk scores were determined via regression analysis. Patients were categorized into risk groups based on the optimal cutoff value. Gene set enrichment analysis (GSEA) and immunoassays were also performed. The prognostic features were further validated using reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR). A total of 88 candidate genes were identified from 1179 consistently expressed DEGs and 4200 key module genes. Seven prognostic features were subsequently identified: TMCO3, RAP2A, ITGAV, ZFYVE26, CHST9, HMGN4, and KLHL21. GSEA revealed that DEGs between risk groups were primarily associated with chylomicron metabolism, among other pathways. Additionally, activated CD4+ T cells demonstrated the strongest positive correlation with risk scores, and most immune checkpoints showed significant differences between risk groups, with ASXL1 exhibiting the strongest correlation with risk scores. The Tumour Immune Dysfunction and Exclusion score was notably higher in the high‐risk group. Moreover, in both the TCGA‐LIHC and ICGC‐LIRI‐JP datasets, the expression of other prognostic features was elevated in tumour tissues, with the exception of CHST9. RT‐qPCR confirmed the increased expression of TMCO3, RAP2A, ITGAV, ZFYVE26, and HMGN4. This study establishes a risk model for HCC based on seven crotonylation‐associated prognostic features, offering a theoretical framework for the diagnosis and treatment of HCC.

Cystatin SN, a specific cysteine protease inhibitor, is thought to be involved in various malignant tumors. Therefore, we evaluated the role of cystatin SN in hepatocellular carcinoma (HCC). Notably, cystatin SN was elevated in tumorous samples and cells. Moreover, overexpression of cystatin SN was correlated with tumor diameter and TNM stage. Cox multivariate analysis displayed that cystatin SN was an independent prognosis indicator and that high cystatin SN level was associated with a dismal prognosis. Moreover, cystatin SN enhancement facilitated the proliferation, migratory, and invasive potential of Huh7 and HCCLM3 cells, whereas cystatin SN knockdown caused the opposite effect. Cystatin SN also modulated the epithelial‐mesenchymal transition progression through the PI3K/AKT pathway. In vivo cystatin SN promoted HCCLM3 cell growth and metastasis in xenograft mice model. Thus, cystatin SN was involved in HCC progression and could be a latent target for HCC treatment.

Background: The protein-coding gene RAB22A, a member of the RAS oncogene family, is amplified or overexpressed in certain cancers. However, its action mechanism in hepatocellular carcinoma (HCC) remains unclear. Here, we aimed to examine the connection between RAB22A and survival prognosis in HCC and explore the biological significance of RAB22A.Methods: A database-based pan-cancer expression analysis of RAB22A was performed. Kaplan–Meier analysis and Cox regression were performed to evaluate the association between RAB22A expression and survival prognosis in HCC. Using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), various potential biological functions and regulatory pathways of RAB22A in HCC were discovered. Tumor immune infiltration was studied using the single sample gene set enrichment analysis (ssGSEA) method. N6-methyladenosine modifications and the regulatory network of competitive endogenous RNA (ceRNA) were verified in the TCGA cohort.Results: RAB22A was upregulated in HCC samples and cell lines. A high RAB22A expression in HCC was strongly correlated with sex, race, age, weight, TNM stage, pathological stage, tumor status, histologic grade, TP53 mutation status, and alpha fetal protein (AFP) levels. Overexpression of RAB22A indicated a poor prognosis was related to overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI). GO and KEGG analyses revealed that the differentially expressed genes related to RAB22A might be involved in the proteasomal protein catabolic process, ncRNA processing, ribosome ribosomal subunit, protein serine/threonine kinase activity, protein serine kinase activity, Endocytosis, and non-alcoholic fatty liver disease. GSEA analyses revealed that the differentially expressed genes related to RAB22A might be involved in the T cell receptor, a co-translational protein, that binds to the membrane, axon guidance, ribosome, phagocytosis, and Eukaryotic translation initiation. RAB22A was correlated with N6-methyladenosine expression in HCC and established RAB22A-related ceRNA regulatory networks. Finally,RAB22A expression was positively connected the levels of infiltrating with T helper cells, Tcm cells, and Th2 cells,In contrast, we observed negatively correlations with cytotoxic cells, DCs, and pDCs cells.Moreover,RAB22A expression showed a strong correlation with various immunomarkergroups in HCC.Conclusions: RAB22A is a potential therapeutic target for improving HCC prognosis and is closely related to immune cell infiltration.

Background: The relationship between sodium intake and the incidence and mortality of non-alcoholic fatty liver disease (NAFLD) is underexplored in nutritional epidemiology, highlighting the need for further research.Methods: This longitudinal cohort study analyzed data from 13,853 Participants aged 20 and older from the National Health and Nutrition Examination Survey (NHANES) (2003–2018), including 4,465 participants with NAFLD. We collected comprehensive data on mortality, dietary sodium intake, and relevant covariates. Logistic regression assessed the relationship between sodium consumption and NAFLD incidence, while Cox regression and smooth curve fitting explored sodium intake’s link to all-cause mortality among Participants with NAFLD.Results: After adjusting for confounders, logistic regression revealed a positive association between higher sodium intake and NAFLD incidence (OR = 1.16, 95% CI = 1.11, 1.21). Adjusted odds ratios for the second (Q2), third (Q3), and fourth (Q4) quartiles of sodium intake were 0.91, 1.23, and 1.52, respectively. Smooth curve fitting and threshold analysis revealed a non-linear association between sodium intake and NAFLD risk, with an inflection point at 2.49 g/d, above which NAFLD risk significantly increased. In Cox regression, sodium intake was inversely correlated with all-cause mortality in Participants with NAFLD (HR = 0.87, 95% CI = 0.80, 0.96), with adjusted hazard ratios for Q2, Q3, and Q4 being 0.79, 0.66, and 0.63, respectively. A nonlinear model indicated a threshold effect, revealing a correlation between dietary sodium intake and mortality risk (p = 0.001). We identified a threshold intake of 3.5 grams per day (equivalent to 8.9 grams of sodium chloride): below this, each unit increase in sodium intake was associated with a 16% reduction in mortality risk (HR = 0.84, 95% CI = 0.80, 0.90). For intakes above this threshold, no significant relationship with mortality risk was observed (HR = 0.99, 95% CI = 0.90, 1.08).Conclusion: This study suggests that higher sodium intake in individuals with NAFLD is associated with increased disease incidence but decreased all-cause mortality. The dose–response relationship between sodium intake and mortality risk exhibited a nonlinear pattern, with a critical inflection point around 3.5 grams per day.

Background and Aim: Lysine lactylation (Kla) has emerged as a novel posttranslational modification implicated in various disease processes, yet its role in diabetic cardiomyopathy (DCM) pathogenesis remains unknown. The objective of this study was to ascertain whether protein lactylation is involved in DCM progression. Methods: Proteomic and lactate analysis via liquid chromatography with tandem mass spectrometry was performed on the heart tissues of db/m mice (as the control group) and db/db mice (as the DCM group). Subsequently, a series of bioinformatics analyses was employed to analyze the Kla site and Kla‐modified proteins in the two groups. Results: Bioinformatics analysis revealed a greater abundance of Kla sites in the DCM group than in the control group. In addition, subcellular localization analysis indicated that Kla‐modified proteins were predominantly located in the cytoplasm and mitochondria. Protein lactylation modification mainly occurred on histone H2, and in comparison to the control group, modification of the H4C1‐K32 site was notably elevated in the DCM group. Furthermore, 113 significantly modified Kla sites were associated with 78 modified proteins in the DCM group, whereas 37 significantly modified Kla sites were associated with 25 modified proteins in the control group. These Kla‐modified proteins participated in biological processes and pathways related to glucose metabolism and DCM. Finally, five candidate sites were identified using random forest, LASSO regression, support vector machine–recursive feature elimination, and logistic regression: A2ASS6_K928_Ttn, A2ASS6_K13499_Ttn, Q61425_K212_Hadh, Q8K2B3_K517_Sdha, and Q9R0Y5_K100_Ak1. Conclusions: Our findings suggest that protein lactate modification in the lactylome and proteome could be a promising treatment for DCM. This provides a reliable basis for further investigating the roles of Kla and Kla‐modified proteins to develop new and effective therapeutic targets for treating DCM.