Hepatocellular Carcinoma Tumor Tissues Research Articles

Identification of ASF1A and HJURP by global H3–H4 histone chaperone analysis as a prognostic two-gene model in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a malignancy with poor prognosis. Abnormal expression of H3–H4 histone chaperones has been identified in many cancers and holds promise as a biomarker for diagnosis and prognosis. However, systemic analysis of H3–H4 histone chaperones in HCC is still lacking. Here, we investigated the expression of 19 known H3–H4 histone chaperones in HCC. Integrated analysis of multiple public databases indicated that these chaperones are highly expressed in HCC tumor tissues, which was further verified by immunohistochemistry (IHC) staining in offline samples. Additionally, survival analysis suggested that HCC patients with upregulated H3–H4 histone chaperones have poor prognosis. Using LASSO and Cox regression, we constructed a two-gene model (ASF1A, HJURP) that accurately predicts prognosis in ICGC-LIRI and GEO HCC data, which was further validated in HCC tissue microarrays with follow-up information. GSEA revealed that HCCs in the high-risk group were associated with enhanced cell cycle progression and DNA replication. Intriguingly, HCCs in the high-risk group exhibited increased immune infiltration and sensitivity to immune checkpoint therapy (ICT). In summary, H3–H4 histone chaperones play a critical role in HCC progression, and the two-gene (ASF1A, HJURP) risk model is effective for predicting survival outcomes and sensitivity to immunotherapy for HCC patients.

Poorly Differentiated Hepatocellular Carcinoma Cells Avoid Apoptosis by Interacting with T Cells via CD40–CD40 Ligand Linkage

Hepatocellular carcinoma (HCC) is associated with elevated soluble CD40 levels in our previous study. Here we aimed to investigate CD40's role in liver tumor progression. We examined CD40 levels in HCC patient tissues and various HCC cell lines, studying their interaction with CD4+T cells. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumour tissues exhibited high membrane-bound CD40 (mCD40) expression, in contrast to non-tumour areas. Poorly differentiated HCC cell lines showed high expression of mCD40 with low CD40 promoter methylation which were opposite of well-differentiated ones. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, HLFs co-cultured with activated (CD40L+) CD4+ T cells elevated CD40 levels and showed a modest 3.2% of dead cells, then increased to 10.9% underwent pre-neutralizing CD40 condition, while pre-blocking both CD40 and Integrin α5β1 concomitantly caused only 1.9% of cell death. RNA sequencing of co-cultured HLFs with activated CD4+ T cells revealed the upregulation of interferon (IFN) and immune response pathways. Elevated IFN-γ levels in the activated T cell media stimulated the JAK1/STAT3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevents cell death by interacting with CD40L in activated T cells. Targeting CD40 may represent a promising anticancer therapy.

Knockdown of ribosome RNA processing protein 15 suppresses migration of hepatocellular carcinoma through inhibiting PATZ1-associated LAMC2/FAK pathway

BackgroundRibosomal RNA processing protein 15 (RRP15) has been found to regulate the progression of hepatocellular carcinoma (HCC). Nevertheless, the extent to which it contributes to the spread of HCC cells remains uncertain. Thus, the objective of this research was to assess the biological function of RRP15 in the migration of HCC.MethodsThe expression of RRP15 in HCC tissue microarray (TMA), tumor tissues and cell lines were determined. In vitro, the effects of RRP15 knockdown on the migration, invasion and adhesion ability of HCC cells were assessed by wound healing assay, transwell and adhesion assay, respectively. The effect of RRP15 knockdown on HCC migration was also evaluated in vivo in a mouse model.ResultsBioinformatics analysis showed that high expression of RRP15 was significantly associated with low survival rate of HCC. The expression level of RRP15 was strikingly upregulated in HCC tissues and cell lines compared with the corresponding controls, and TMA data also indicated that RRP15 was a pivotal prognostic factor for HCC. RRP15 knockdown in HCC cells reduced epithelial-to-mesenchymal transition (EMT) and inhibited migration in vitro and in vivo, independent of P53 expression. Mechanistically, blockade of RRP15 reduced the protein level of the transcription factor POZ/BTB and AT hook containing zinc finger 1 (PATZ1), resulting in decreased expression of the downstream genes encoding laminin 5 subunits, LAMC2 and LAMB3, eventually suppressing the integrin β4 (ITGB4)/focal adhesion kinase (FAK)/nuclear factor κB kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway.ConclusionsRRP15 promotes HCC migration by activating the LAMC2/ITGB4/FAK pathway, providing a new target for future HCC treatment.

Comprehensive Analysis of the Expression of Cell Adhesion Molecules Genes in Hepatocellular Carcinoma and their Prognosis, and Biological Significance.

Collagen-related cell adhesion molecules (CAMs) are a major component of the extracellular matrix (ECM) and often accumulate in the liver during chronic liver disease or hepatocellular carcinoma (HCC). In this study we identified several promising collagens related to CAMs that may be of clinical use for the diagnosis and prognosis of HCC. We obtained multi-omics data including RNA sequencing (RNA-Seq) data, microarray data, proteomic data from the TCGA, GEO databases, GTEx, and NODE. Bioinformatics analyses were then performed to investigate correlations between the expression patterns of significant genes and HCC. Tumor tissue and para-cancerous tissue samples from HCC patients were also used to validate the results using RT-PCR. A literature research and LASSO-COX analysis identified three significant collagen-related CAM genes: SERPINH1, DCN, and ITGB1. Immunohistochemistry images in the Human Protein Atlas Project database showed that SERPINH1 and ITGB1 proteins were moderately or highly expressed in HCC tumor tissues compared to para-cancerous tissue, whereas DCN expression was lower in HCC tumor tissue. These results were validated by RT-PCR. Low- and high-risk groups of HCC patients were distinguished by the logistic panel in the TCGA database. These showed significantly different prognosis, clinicopathological features, and immune cell infiltration. Logistic regression was used to construct predictive models based on the individual expression levels of DCN, SERPINH1, and ITGB1. These showed highly accurate diagnostic ability (AUC = 0.987). The current findings suggest that the collagen-related CAMs DCN, SERPINH1, and ITGB1 may be potential therapeutic targets in HCC. Logistic panels of DCN, SERPINH1 and ITGB1 could serve as non-invasive and effective diagnostic biomarkers for HCC. Identifier: NCT03189992. Registered on June 4, 2017. Retrospectively registered (https://clinicaltrials.gov/).

Functional genetic variants of the disulfidptosis-related INF2 gene predict survival of hepatitis B virus-related hepatocellular carcinoma.

Disulfidptosis is a novel form of programmed cell death involved in migration and invasion of cancer cells, but few studies investigated the roles of genetic variants in disulfidptosis-related genes in survival of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We used Cox proportional hazards regression analyses, Kaplan-Meier curves and receiver operating characteristic curves to assess effects of genetic variants in 14 disulfidptosis-related genes on overall survival of 866 HBV-HCC patients. The Bayesian false discovery probability was used for multiple testing corrections. We also investigated biological mechanisms of the significant variants through expression quantitative trait loci analyses using the data from publicly available databases, luciferase reporter assays and differential expression analyses. As a result, we identified two independently functional single nucleotide polymorphisms (SNPs) (INF2 rs4072285 G > A and INF2 rs4444271 A > T) that predicted overall survival of HBV-HCC patients, with adjusted hazard ratios of 1.60 (95% CI = 1.22-2.11, P = 0.001) and 1.50 (95% CI = 1.80-1.90, P < 0.001), respectively, after multiple testing correction. Luciferase reporter assays indicated that both INF2 rs4072285 A and INF2 rs4444271 T alleles increased INF2 mRNA expression levels (P < 0.001) that were also higher in HCC tumor tissues than in adjacent normal tissues (P < 0.001); such elevated INF2 expression levels were associated with a poorer survival of HBV-HCC patients (P < 0.001) in the TCGA database. In summary, this study supported that INF2 rs4072285 and INF2 rs4444271 may be novel biomarkers for survival of HBV-HCC patients.

Novel nanoparticle CS-C60-Fe3O4 magnetically induces tissue-specific aggregation and enhances thermal ablation of hepatocellular carcinoma

Metallofullerenes are an important type of metallic nanomaterial with promising applications in several medical fields. Thermal ablation, including radiofrequency ablation (RFA) and microwave ablation (MWA), is an important treatment strategy for advanced hepatocellular carcinoma (HCC). The thermal expansion of fullerenes makes them good adjuncts to thermal ablation treatment of HCC. In this study, we used an innovative method of emulsification and cross-linking to produce CS-C60-Fe3O4 (Chitosan-C60-Fe3O4) nanoparticles, which have the advantages of uniform particle size and high bioavailability, as a kind of novel nano-pharmaceutical. The CS-C60-Fe3O4 nanoparticles were prepared by the cross-linking reaction from chitosan–acetic acid solution, Fe3O4 nanoparticles by Fe2SO4·7H2O and FeCl3·6H2O, and C60. The average particle size of CS-C60-Fe3O4 was 194.3 nm. Because CS-C60-Fe3O4 is magnetic, it can achieve specific and tissue aggregation in HCC tumor tissues. Moreover, compared with normal soluble C60 (EL35-C60), CS-C60-Fe3O4 prolonged the retention time of C60 in the blood of mice. CS-C60-Fe3O4 alone is not cytotoxic to cultured cells or tumor tissues, but when combined with thermal ablation strategies (RFA and MWA), it significantly upregulates the antitumor effects of thermal ablation on HCC tissues, that is, it acts as a sensitiser to thermal ablation. In the presence of thermal ablation, CS-C60-Fe3O4 interfered with iron metabolism in HCC cells and induced ferroptosis of HCC cells in the tumor tissues. These results not only expand our understanding of metallofullerenes but also provide additional options for the treatment of advanced HCC.

GLUD1 inhibits hepatocellular carcinoma progression via ROS-mediated p38/JNK MAPK pathway activation and mitochondrial apoptosis

Glutamate dehydrogenase 1 (GLUD1) is an important enzyme in glutamine metabolism. Previously, we found GLUD1 was down-regulated in tumor tissues of hepatocellular carcinoma (HCC) patients by proteomics study. To explore its role in the progression of HCC, the expressional level of GLUD1 was firstly examined and presented as that both the protein and mRNA levels were down-regulated in tumor tissues compared to the normal liver tissues. GLUD1 overexpression significantly inhibited HCC cells proliferation, migration, invasion and tumor growth both in vitro and in vivo, while GLUD1 knocking-down promoted HCC progression. Metabolomics study of GLUD1 overexpressing and control HCC cells showed that 129 differentially expressed metabolites were identified, which mainly included amino acids, bases, and phospholipids. Moreover, metabolites in mitochondrial oxidative phosphorylation system (OXPHOS) were differentially expressed in GLUD1 overexpressing cells. Mechanistic studies showed that GLUD1 overexpression enhanced mitochondrial respiration activity and reactive oxygen species (ROS) production. Excessive ROS lead to mitochondrial apoptosis that was characterized by increased expression levels of p53, Cytochrome C, Bax, Caspase 3 and decreased expression level of Bcl-2. Furthermore, we found that the p38/JNK MAPK pathway was activated in GLUD1 overexpressing cells. N-acetylcysteine (NAC) treatment eliminated cellular ROS and blocked p38/JNK MAPK pathway activation, as well as cell apoptosis induced by GLUD1 overexpression. Taken together, our findings suggest that GLUD1 inhibits HCC progression through regulating cellular metabolism and oxidative stress state, and provide that ROS generation and p38/JNK MAPK pathway activation as promising methods for HCC treatment.

Suppressing MTERF3 inhibits proliferation of human hepatocellular carcinoma via ROS-mediated p38 MAPK activation

Mitochondrial transcription termination factor 3 (MTERF3) negatively regulates mitochondrial DNA transcription. However, its role in hepatocellular carcinoma (HCC) progression remains elusive. Here, we investigate the expression and function of MTERF3 in HCC. MTERF3 is overexpressed in HCC tumor tissues and higher expression of MTERF3 positively correlates with poor overall survival of HCC patients. Knockdown of MTERF3 induces mitochondrial dysfunction, S-G2/M cell cycle arrest and apoptosis, resulting in cell proliferation inhibition. In contrast, overexpression of MTERF3 promotes cell cycle progression and cell proliferation. Mechanistically, mitochondrial dysfunction induced by MTERF3 knockdown promotes ROS accumulation, activating p38 MAPK signaling pathway to suppress HCC cell proliferation. In conclusion, ROS accumulation induced by MTERF3 knockdown inhibits HCC cell proliferation via p38 MAPK signaling pathway suggesting a promising target in HCC patients.

A hsa_circ_001726 axis regulated by E2F6 contributes to metastasis of hepatocellular carcinoma

BackgroundCircRNAs participate in the development of hepatocellular carcinoma (HCC). This work aims to explore the key tumor promoting circRNA as a gene therapy target.MethodsThe differentially expressed gene circRNAs in HCC tumor tissues was identified by mining GSE121714 dataset. EdU staining, wound healing, transwell invasion assay, TUNEL staining and western blotting examined proliferation, migration, invasion, apoptosis and epithelial mesenchymal transition (EMT). Xenograft mouse model and orthotopic transplantation tumor mouse model were constructed to verify the role of hsa_circ_001726 in growth and metastasis of HCC. The relationship among CCT2, E2F6, hsa_circ_001726, miR-671-5p and PRMT9 was identified by RNA-fluorescence in situ hybridization, luciferase reporter assay and RNA Immunoprecipitation.ResultsEleven differentially expressed circRNAs were found in HCC tumors. Among them, hsa_circ_001726 was highly expressed in HCC tumors and cells, which was transcribed from CCT2. As a transcription factor of CCT2, E2F6 knockdown inactivated CCT2 promoter and reduced hsa_circ_001726 expression. Moreover, hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway. Additionally, hsa_circ_001726 deficiency repressed malignant phenotypes of HCC cells, including proliferation, migration, invasion, EMT and apoptosis. In vivo, hsa_circ_001726 deficiency reduced tumor growth and lung metastasis of HCC in xenograft mouse models and orthotopic transplantation tumor mouse models.ConclusionHsa_circ_001726 functioned as an oncogene in HCC, which was derived from CCT2 and regulated by E2F6. Hsa_circ_001726 elevated PRMT9 expression by sponging miR-671-5p, and then activated Notch signaling pathway, thereby accelerating malignant phenotypes of HCC. Therefore, targeting hsa_circ_001726 may be a new avenue for HCC treatment.

Correlation Between Low Cytoplasmic Expression of XBP1 and the Likelihood of Surviving Hepatocellular Carcinoma.

Our objectives in this study were to (i) evaluate the clinical significance of X-box-binding protein 1 (XBP1) expression in cases of hepatocellular carcinoma (HCC) and (ii) assess the potential of XBP1 to be used as a prognostic biomarker. The expression of XBP1 protein in 267 HCC tissue specimens was measured using immunohistochemistry in order to characterize the associations among XBP1 expression, clinicopathological factors and survival outcomes. Survival analysis using follow-up data was used to assess the prognostic value of XBP1 in cases of HCC. Immunohistochemistry revealed a significant decrease in cytoplasmic XBP1 protein expression in HCC tumor tissue. Immunoreactivity results showed that low cytoplasmic XBP1 expression was significantly associated with vascular invasion, as well as poor 5-year overall survival and long-term disease-specific (DSS) and disease-free (DFS) survival rates. Kaplan-Meier survival curves further confirmed a significant association between low cytoplasmic XBP1 protein expression and poor DSS and DFS. Univariate and multivariate analyses revealed that XBP1 expression, tumor differentiation, vascular invasion, tumor stage, and the rate of recurrence were linked to DSS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DSS. Our analysis also revealed that XBP1 expression, tumor differentiation, vascular invasion, and T classification were linked to DFS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DFS. Low cytoplasmic XBP1 protein expression may play an important role in the pathogenesis of HCC, which suggests that XBP1 could potentially be targeted to benefit therapeutic strategies for HCC.

AGXT2 Suppresses the Proliferation and Dissemination of Hepatocellular Carcinoma Cells by Modulating Intracellular Lipid Metabolism.

Alanine glyoxylate aminotransferase (AGXT) family members are crucial in cancer processes, but their role in hepatocellular carcinoma (HCC) metabolism is unclear. This study investigates AGXT2's function in HCC. AGTX2 expression was studied using bioinformatics, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), Western blot, and Enzyme-linked immunosorbent assay (ELISA). A lentivirus-induced AGTX2 overexpression cell model was analyzed with RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS). Cholesterol levels were confirmed by Oil Red O staining. AGTX2 effects were evaluated through cell cycle analysis, wound healing, and transwell migration assays.Tumorigenic effects were observed in NOD-SCID IL2Rγnull (NTG) mice in subcutaneous experiments. Protein interaction was examined through co-immunoprecipitation methods. We observed a significant reduction in AGXT2 mRNA and protein levels in both HCC tumor tissues and serum samples from patients with liver cancer, which was associated with a worse prognosis. The activation of AGXT2 has been shown to effectively decrease cholesterol levels in liver cancer cells, serving as an antagonist in the cholesterol metabolism pathway. An increase in low density lipoprotein receptor (LDLR) mRNA was noted in cells overexpressing AGXT2, accompanied by a decrease in LDLR protein and an elevation in proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein levels. Molecular docking and co-immunoprecipitation experiments further elucidated the interaction between AGXT2 and LDLR proteins. AGXT2 was observed to suppress the migratory and invasive capabilities of HCC cells, inducing cell cycle arrest in the G2/M phase. AGXT2 activation inhibited subcutaneous liver cancer tumor growth in NTG mice. AGXT2 was found to lower cholesterol levels in liver cancer cells, possibly through interactions with the LDLR protein and modulation of PCSK9-mediated LDLR degradation. This mechanism may impede cholesterol transport to liver cancer cells, thereby suppressing their growth and metastasis.

Investigation of the feasibility of NRAV as a biomarker for hepatocellular carcinoma.

The long non-coding RNA, Negative Regulator of Antiviral Response (NRAV) has been identified as a participant in both respiratory virus replication and immune checkpoints, however, its involvement in pan-cancer immune regulation and prognosis, particularly those of hepatocellular carcinoma (HCC), remains unclear. To address this knowledge gap, we analyzed expression profiles obtained from The Cancer Genome Atlas (TCGA) database, comparing normal and malignant tumor tissues. We found that NRAV expression is significantly upregulated in tumor tissues compared to adjacent nontumor tissues. Kaplan-Meier (K-M) analysis revealed the prognostic power of NRAV, wherein overexpression was significantly linked to reduced overall survival in a diverse range of tumor patients. Furthermore, noteworthy associations were observed between NRAV, immune checkpoints, immune cell infiltration, genes related to autophagy, epithelial-mesenchymal transition (EMT), pyroptosis, tumor mutational burden (TMB), and microsatellite instability (MSI) across different cancer types, including HCC. Moreover, NRAV upregulation expression was associated with multiple pathological stages by clinical observations. Furthermore, our investigation revealed a substantial elevation in the expression of NRAV in both HCC tumor tissues and cells compared to normal tissues and cells. The inhibition of NRAV resulted in the inhibition of cell proliferation, migration, and invasion in HCC cells, while also influencing the expression of CD274 (PD-L1) and CD44, along with various biomarkers associated with EMT, autophagy, and pyroptosis. The aforementioned results propose NRAV as a promising prognostic biomarker for HCC.

The extract of an herbal medicine Chebulae fructus inhibits hepatocellular carcinoma by suppressing the Apelin/APJ system.

Introduction: Hepatocellular carcinoma (HCC) has been a highly common and pathological disease worldwide, while current therapeutic regimens have limitations. Chebulae Fructus, a common herbal medicine in Asia, has been documented to exert potential therapeutic effects on HCC in ancient medicine clinical practice. However, the molecular mechanism underlying its inhibitory effects on HCC requires further investigation. Methods: In this study, the anti-HCC effect of the aqueous extract of Chebulae Fructus (CFE) on human HCC and its underlying mechanism were evaluated. Assays including CCK8, EdU staining, crystal violet staining, cell clone formation, flow cytometry, wound healing, and transwell were used in vitro. The cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models were used in vivo. Transcriptomics analysis, qRT-PCR, ELISA, IHC staining, and Western blotting were employed to determine the mechanism of action of CFE. Results: The results demonstrate that CFE effectively suppressed the proliferation and activity of HepG2 and PLC/PRF/5 HCC cells. CFE also induced apoptosis, and suppressed the migration and invasion abilities of these cells. Furthermore, CFE exhibited inhibitory effects on tumor growth in both H22 and PLC/PRF/5 mouse models, as well as in an HCC PDX model which is derived from patient tumor samples. Moreover, it was identified that CFE treatment specifically suppressed the Apelin/APJ system in HCC cells and tumor tissues. To investigate the role of the Apelin/APJ system in mediating the effects of CFE treatment, an APJ overexpressed cell model is established. Interestingly, it was found that the overexpression of APJ significantly diminished the inhibitory effects of CFE on HCC in vitro. Discussion: Collectively, this study provides compelling evidence that CFE exerts significant anti-HCC effects in cell and animal models. Moreover, our findings suggest that the Apelin/APJ system may play a vital role in the therapeutic effects of CFE against HCC.

Artificial Intelligence-assisted Raman Spectroscopy for Liver cancer diagnosis

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, represents a global health challenge due to its complexity and the limitations of current diagnostic techniques. By combining Raman spectroscopy and Artificial Intelligence (AI), we have succeeded in classifying tumor cells. In fact, we have performed a first Raman spectral analysis based on the characterization and differentiation between uncultured primary human liver cells derived from resected HCC tumor tissue and the adjacent non-tumor counterpart. Biochemical analysis of the collected Raman spectra revealed that there is more DNA in the nuclei of the tumor cells than in non-tumor cells. We then develop three machine learning approaches, including multivariate models and neural networks, to rapidly automate the recognition and classification of the Raman spectra of both cells. To evaluate the performance of the developed AI models, we prepared and analyzed two additional cell samples with a ratio of 4:1 and 3:1 between tumor and non-tumor cells and compared the obtained results with the nominal percentages (accuracy of 80 and 60%, respectively). These results confirm that the models are able to make classifications at the level of a single spectrum, indicating the possibility of rapidly analysing and classifying a primary HCC cell.

Deubiquitination of CIB1 by USP14 promotes lenvatinib resistance via the PAK1-ERK1/2 axis in hepatocellular carcinoma.

Background: Lenvatinib is the most common multitarget receptor tyrosine kinase inhibitor for the treatment of advanced hepatocellular carcinoma (HCC). Acquired resistance to lenvatinib is one of the major factors leading to the failure of HCC treatment, but the underlying mechanism has not been fully characterized. Methods: We established lenvatinib-resistant cell lines, cell-derived xenografts (CDXs) and patient-derived xenografts (PDXs) and obtained lenvatinib-resistant HCC tumor tissues for further study. Results: We found that ubiquitin-specific protease 14 (USP14) was significantly increased in lenvatinib-resistant HCC cells and tumors. Silencing USP14 significantly attenuated lenvatinib resistance in vitro and in vivo. Mechanistically, USP14 directly interacts with and stabilizes calcium- and integrin-binding protein 1 (CIB1) by reversing K48-linked proteolytic ubiquitination at K24, thus facilitating the P21-activated kinase 1 (PAK1)-ERK1/2 signaling axis. Moreover, in vivo adeno-associated virus 9 mediated transduction of CIB1 promoted lenvatinib resistance in PDXs, whereas CIB1 knockdown resensitized the response of PDXs to lenvatinib. Conclusions: These findings provide new insights into the role of CIB1/PAK1-ERK1/2 signaling in lenvatinib resistance in HCC. Targeting CIB1 and its pathways may be a novel pharmaceutical intervention for the treatment of lenvatinib-resistant HCC.

The identification of N6-methyladenosine-related miRNAs predictive of hepatocellular carcinoma prognosis and immunotherapy efficacy.

Hepatocellular carcinoma (HCC) has a high degree of malignancy and poor prognosis. N6-methyladenosine (m6A) modifications and microRNAs (miRNAs) play pivotal roles in tumorigenesis and development. However, the role of m6A-related miRNAs in HCC has not been clarified yet. This study aimed to identify the role of m6A-miRNAs in HCC prognosis through bioinformatics analysis. The clinicopathological information and RNA sequencing data of 369 HCC tumor tissues and 49 tumor-adjacent tissues were downloaded from the TCGA database. A total of 23 m6A regulators were extracted to evaluated the m6A-related miRNAs using Pearson's correlation analysis. Then, we selected prognosis-related m6A-miRNAs using a univariate Cox regression model and used the consensus cluster analysis to explore the characteristics of the m6A-miRNAs. The coefficient of the least absolute shrinkage and selection operator (LASSO) Cox regression was applied to construct a prognostic risk score model. The receiver operated characteristic (ROC) analysis was applied to evaluate the prognostic value of the signature. The biological functions of targeted genes were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Then, to validate the potential predictive value for prognosis, the miRNA expression profiles from the GSE76903 and GSE6857 were used. Single sample Gene Set Enrichment Analysis (ssGSEA) and Estimation of Stromal and Immune cells in Malignant Tumor tissues using Expression data (ESTIMATE) were applied to assess the immune microenvironment of HCC. Additionally, a meta-analysis was used to verify the prognostic value of the m6A-microRNAs. RT-PCR was applied to validated the expression of miRNAs in HCC tissues. Cell viability, transwell assay and RNA m6A dot blot assays of HCC cells was applied to access the function of miR-17-5p. The expression of 48 m6A-related miRNAs was identified and 17 prognostic m6A-miRNAs was discovered. The expression profile of those 17 miRNAs was divided into three clusters, and these clusters were associated with the tumor microenvironment (TME) and prognosis. The nine m6A-related miRNA signature was associated with the prognosis of HCC, the AUC of the ROC was 0.771(TCGA dataset), 0.788(GSE76903) and 0.646(GSE6857). The TME and the expression of immune checkpoint molecules were associated with the risk score. The meta-analysis also validated the prognostic value of the m6A-related miRNAs (miR182-5p (HR:1.58, 95%CI:1.04-2.40) and miR-17-5p (HR:1.58, 95%CI: 1.04-2.40)). The expression of miR-17-5p was upregulated in HCC tissues and miR-17-5p showed an oncogenic role in HCC cells. The clinical innovation is the use of m6A-miRNAs as biomarkers for predicting prognosis regarding immunotherapy response in HCC patients.

Comprehensive analysis of m6A reader YTHDF2 prognosis, immune infiltration, and related regulatory networks in hepatocellular carcinoma

BackgroundN6-Methyladenosine (m6A) RNA modification is the most prevalent internal modification pattern in eukaryotic mRNAs and plays critical roles in diverse physiological and pathological processes. However, the expression of m6A regulator YTHDF2, its prognostic value, its biological function, its correlation with tumor microenvironment (TME) immune infiltrates, and related regulatory networks in hepatocellular carcinoma (HCC) remain determined. MethodsTCGA, GTEx, and GEO databases were used to investigate the expression profile of YTHDF2 in HCC. We performed differentially expressed genes (DEGs) analysis and constructed a PPI network to explore the biological processes of YTHDF2 in HCC. Kaplan-Meier curves and Cox regression analysis were used to assess the prognostic value of YTHDF2 and then a clinical prognostic nomogram was constructed. Additionally, ssGSEA was performed to assess the correlation between YTHDF2 and immune infiltration levels. The TISIDB database was applied to explore the expression of YTHDF2 in immune and molecular subtypes of HCC. GSEA identifies the YTHDF2-related signaling pathways. Finally, we utilized miRNet and starBase database to construct regulatory networks for HCC based on lncRNA-miRNA and miRNA-YTHDF2 interactions. ResultsYTHDF2 was significantly upregulated in HCC tumor tissues compared with the adjacent normal tissues. HCC patients in the high YTHDF2 expression group had poorer survival. Multivariate Cox analysis suggested that YTHDF2 may be a new independent prognostic indicator for HCC patients, with the prognostic nomogram exhibiting satisfactory results. YTHDF2 expression was significantly correlated with TME immune cell-infiltrating characteristics. Strong correlations were also shown in immune subtypes, molecular subtypes and immune checkpoints. Further analysis revealed that the combination of YTHDF2 expression and immune cell score was considerably associated with survival outcome in HCC patients. GESA analysis demonstrated that high YTHDF2 expression is associated with multiple biological processes and oncogenic pathways. Moreover, 14 possible regulatory networks were constructed, which are associated with HCC progression. ConclusionOur findings revealed that YTHDF2 may serve as a promising prognostic biomarker for HCC and may regulate the tumor immune microenvironment to provide effective therapeutic strategies.

NPC1 promotes autophagy with tumor promotion and acts as a prognostic model for hepatocellular carcinoma

Backgroundmore and more studies have indicated that autophagy plays a crucial role in hepatocellular carcinoma (HCC) in recent years. Hence, our study aimed to establish a prognostic signature for HCC based on autophagy-related genes (ARGs) in order to predict the prognosis of HCC. MethodsAll original gene-expression data and clinical information were downloaded from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and Gene Expression Omnibus (GEO). ARGs were obtained from the Human Autophagy Database (HADb). Univariate Cox regression analysis, Least absolute shrinkage and selection operator (LASSO) and Principal Component Analysis (PCA) analysis were performed to identify and validate the validity and reliability of our eight-gene signature, Gene Set Enrichment Analysis (GSEA) was used to perform enrichment analysis by comparing high-risk and low-risk groups in KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) gene sets. Finally, we verified the gene (NPC1) by functional experiments in vitro and in vivo. Results8 ARGs were identified for establishing an eight-gene signature. Then, we validated our eight-gene signature in training, internal, external, and entire testing cohorts. Besides, we also explored the relationships between the eight-gene signature and immune infiltration or immune checkpoints. We also identified NPC1 was closely related to Activated CD4 T cell and Type I IFN Response, and higher expressed level of HCC patients was more sensitive to CTLA4 and TNFRSF9 immune checkpoint inhibitors. NPC1 is highly expressed in HCC cells and tumor tissues, which promotes the proliferation, migration, and invasion of tumor cells by activating autophagy.. Conclusion8 ARGs were used to establish a gene signature to predict the prognosis of HCC. we inferred that NPC1 can promote late autophagy, it could be a future novel therapeutic target of HCC.

Transcriptome analysis of tertiary lymphoid structures (TLSs)-related genes reveals prognostic value and immunotherapeutic potential in cancer

Abstract Objectives Tertiary lymphoid structures (TLSs) are increasingly recognized as pivotal formations in antitumor immunity, with many mature TLSs relating to favorable prognosis in a variety of cancers. Analysis of the transcriptome data provided an opportunity to investigate the relationship between TLSs and the clinical outcomes of patients. Methods We performed transcriptome analysis to examine the expression patterns of 79 TLS-related genes within 32 cancers from The Cancer Genome Atlas, correlating these patterns to the clinical outcomes of patients. To improve the analysis, we developed a novel prognostic signature (TLSig) and validated its robust performance via multiple independent cancer cohorts. Results Our results demonstrated that the expression of TLS-related genes exhibited significant heterogeneity across cancers; and that TLSig was significantly associated with patient prognosis within most cancer types. Specifically, TLSig expressed potential prediction of whether patients would benefit from immunotherapy and presented significant interactions with epithelial to mesenchymal transition (EMT) and the cell cycle. Moreover, a difference in the distribution of TLSs between tumor and nontumor tissues was observed, which was validated in hepatocellular carcinoma (HCC) samples. We further investigated the potential mechanism for the difference in the distribution of TLSs between tumor and nontumor tissues of HCC, and observed the connection between TLSs and human leucocyte antigen (HLA). Conclusions In summary, our results demonstrated the prognostic value of TLSs in predicting patient prognosis and provided new insights into the molecular mechanism of TLSs in cancer.

LASS2 enhances p53 protein stability and nuclear import to suppress liver cancer progression through interaction with MDM2/MDMX

LASS2 functions as a tumor suppressor in hepatocellular carcinoma (HCC), the most common type of primary liver cancer, but the underlying mechanism of its action remains largely unknown. Moreover, details on its role and the downstream mechanisms in Cholangiocarcinoma (CCA) and hepatoblastoma (HB), are rarely reported. Herein, LASS2 overexpression was found to significantly inhibit proliferation, migration, invasion and induce apoptosis in hepatoma cells with wild-type (HB cell line HepG2) and mutated p53 (HCC cell line HCCLM3 and CCA cell line HuCCT1). Gene set enrichment analysis determined the enrichment of the differentially expressed genes caused by LASS2 in the p53 signaling pathway. Moreover, the low expression of LASS2 in HCC and CCA tumor tissues was correlated with the advanced tumor-node-metastasis (TNM) stage, and the protein expression of LASS2 positively correlated with acetylated p53 (Lys373) protein levels. At least to some extent, LASS2 exerts its tumor-suppressive effects in a p53-dependent manner, in which LASS2 interacts with MDM2/MDMX and causes dual inhibition to disrupt p53 degradation by MDM2/MDMX. In addition, LASS2 induces p53 phosphorylation at ser15 and acetylation at lys373 to promote translocation from cytoplasm to nucleus. These findings provide new insights into the LASS2-induced tumor suppression mechanism in liver cancer and suggest LASS2 could serve as a potential therapeutic target for liver cancer.