HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells.

BACKGROUND: The role of exosomes derived from HepG2.2.15 cells, which express hepatitis B virus (HBV)-related proteins, in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive. The focus was on comprehending the relationship and influence of differentially expressed microRNAs (DE-miRNAs) within these exosomes. AIM: To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell (HSC) LX2 and the progression of liver fibrosis. METHODS: Exosomes from HepG2.2.15 cells, which express HBV-related proteins, were isolated from parental HepG2 and WRL68 cells. Western blotting was used to confirm the presence of the exosomal marker protein CD9. The activation of HSCs was assessed using oil red staining, whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells. Additionally, we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2'-deoxyuracil staining and western blotting, respectively. DE-miRNAs were analyzed using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were used to annotate the target genes of DE-miRNAs. RESULTS: Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells. A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells. GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation, intracellular signal transduction, negative regulation of apoptosis, extracellular exosomes, and RNA binding. KEGG pathway analysis highlighted ubiquitin-mediated proteolysis, the MAPK signaling pathway, viral carcinogenesis, and the toll-like receptor signaling pathway, among others, as enriched in these targets. CONCLUSION: These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation, proliferation, and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Unraveling the Heterogeneity of CD8+ T-Cell Subsets in Liver Cirrhosis: Implications for Disease Progression.

Background/Aims: : Liver cirrhosis involves chronic inflammation and progressive fibrosis. Among various immune cells, CD8+ T cells are considered a major contributor to hepatic inflammation and fibrosis. However, the exact molecular pathways governing CD8+ T-cell-mediated effects in cirrhosis remain unclear. Methods: : This study analyzed transcriptomic and single-cell sequencing data to elucidate CD8+ T-cell heterogeneity and implications in cirrhosis. Results: : Weighted gene co-expression analysis of bulk RNA-seq data revealed an association between cirrhosis severity and activated T-cell markers like HLA and chemokine genes. Furthermore, single-cell profiling uncovered eight CD8+ T-cell subtypes, notably, effector memory (Tem) and exhausted (Tex) T cells. Tex cells, defined by PDCD1, LAG3, and CXCL13 expression, were increased in cirrhosis, while Tem cells were decreased. Lineage tracing and differential analysis highlighted CXCL13+ Tex cells as a terminal, exhausted subtype of cells with roles in PD-1 signaling, glycolysis, and T-cell regulation. CXCL13+ Tex cells displayed T-cell exhaustion markers like PDCD1, HAVCR2, TIGIT, and TNFRSF9. Functional analysis implicated potential roles of these cells in immunosuppression. Finally, a CXCL13+ Tex-cell gene signature was found that correlated with cirrhosis severity and poorer prognosis of liver cancer. Conclusions: : In summary, this comprehensive study defines specialized CD8+ T-cell subpopulations in cirrhosis, with CXCL13+ Tex cells displaying an exhausted phenotype associated with immune dysregulation and advanced disease. Key genes and pathways regulating these cells present potential therapeutic targets.

Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common cancer worldwide, and sorafenib is a first-line drug for the treatment of advanced liver cancer. Resistance to sorafenib has become a major challenge in the treatment of hepatocellular carcinoma, however, studies have shown that metformin can promote ferroptosis and sorafenib sensitivity. Therefore, the aim of this study was to investigate the promotion of ferroptosis and sorafenib sensitivity by metformin via ATF4/STAT3 in hepatocellular carcinoma cells. METHODS: Hepatocellular carcinoma cells Huh7 and Hep3B and induced sorafenib resistance (SR) Huh7/SR and Hep3B/SR cells were used as in vitro cell models. Cells were injected subcutaneously to establish a drug-resistant mouse model. CCK-8 was used to detect cell viability and sorafenib IC50. Western blotting was used to detect the expression of relevant proteins. BODIPY staining was used to analyze the lipid peroxidation level in cells. A scratch assay was used to detect cell migration. Transwell assays were used to detect cell invasion. Immunofluorescence was used to localize the expression of ATF4 and STAT3. RESULTS: Metformin promoted ferroptosis in hepatocellular carcinoma cells through ATF4/STAT3, decreased sorafenib IC50, increased ROS and lipid peroxidation levels, decreased cell migration and invasion, inhibited the expression of the drug-resistant proteins ABCG2 and P-GP in hepatocellular carcinoma cells, and thus inhibited sorafenib resistance in hepatocellular carcinoma cells. Downregulating ATF4 inhibited the phosphorylated nuclear translocation of STAT3, promoted ferroptosis, and increased the sensitivity of Huh7 cells to sorafenib. Metformin was also shown in animal models to promote ferroptosis and sorafenib sensitivity in vivo via ATF4/STAT3. CONCLUSION: Metformin promotes ferroptosis and sensitivity to sorafenib in hepatocellular carcinoma cells via ATF4/STAT3, and it inhibits HCC progression.

MiR-21-5p promotes sorafenib resistance and hepatocellular carcinoma progression by regulating SIRT7 ubiquitination through USP24.

OBJECTIVE: To validate the mechanism by which miR-21-5p mediates autophagy in drug-resistant cells in hepatocellular carcinoma (HCC), aggravating sorafenib resistance and progression of HCC. METHODS: HCC cells were treated with sorafenib to establish sorafenib-resistant cells, and nude mice were subcutaneously injected with hepatoma cells to establish animal models. RT-qPCR was used to determine the level of miR-21-5p, and Western blotting was used to determine the level of related proteins. Cell apoptosis, cell migration, the level of LC3 were accessed. Immunohistochemical staining was used for detection of Ki-67 and LC3. A dual-luciferase reporter assay certified that miR-21-5p targets USP42, and a co-immunoprecipitation assay validated the mutual effect between USP24 and SIRT7. RESULTS: miR-21-5p and USP42 were highly expressed in HCC tissue and cells. Inhibition of miR-21-5p or knockdown of USP42 inhibited cell proliferation and cell migration, upregulated the level of E-cadherin, and downregulated the level of vimentin, fibronectin and N-cadherin. Overexpression of miR-21-5p reversed the knockdown of USP42. Inhibition of miR-21-5p downregulated the ubiquitination level of SIRT7, downregulated the levels of LC3II/I ratio and Beclin1, and upregulated the expression of p62. The tumor size in the miR-21-5p inhibitor group was smaller, and Ki-67 and LC3 in tumor tissue were reduced, while the overexpression of USP42 reversed the effect of the miR-21-5p inhibitor. CONCLUSION: miR-21-5p promotes deterioration and sorafenib resistance in hepatocellular carcinoma by upregulating autophagy levels. Knockdown of miR-21-5p inhibits the development of sorafenib-resistant tumors by USP24-mediated SIRT7 ubiquitination.

Exosome-derived circCCAR1 promotes CD8 + T-cell dysfunction and anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND: Circular RNAs (circRNAs) can be encapsulated into exosomes to participate in intercellular communication, affecting the malignant progression of a variety of tumors. Dysfunction of CD8 + T cells is the main factor in immune escape from hepatocellular carcinoma (HCC). Nevertheless, the effect of exosome-derived circRNAs on CD8 + T-cell dysfunction needs further exploration. METHODS: The effect of circCCAR1 on the tumorigenesis and metastasis of HCC was assessed by in vitro and in vivo functional experiments. The function of circCCAR1 in CD8 + T-cell dysfunction was measured by enzyme-linked immunosorbent assay (ELISA), western blotting and flow cytometry. Chromatin immunoprecipitation, biotinylated RNA pull-down, RNA immunoprecipitation, and MS2 pull-down assays were used to the exploration of mechanism. A mouse model with reconstituted human immune system components (huNSG mice) was constructed to explore the role of exosomal circCCAR1 in the resistance to anti-PD1 therapy in HCC. RESULTS: Increased circCCAR1 levels existed in tumor tissues and exosomes in the plasma of HCC patients, in the culture supernatant and HCC cells. CircCCAR1 accelerated the growth and metastasis of HCC in vitro and in vivo. E1A binding protein p300 (EP300) and eukaryotic translation initiation factor 4A3 (EIF4A3) promoted the biogenesis of circCCAR1, and Wilms tumor 1-associated protein (WTAP)-mediated m6A modification enhanced circCCAR1 stability by binding insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3). CircCCAR1 acted as a sponge for miR-127-5p to upregulate its target WTAP and a feedback loop comprising circCCAR1/miR-127-5p/WTAP axis was formed. CircCCAR1 is secreted by HCC cells in a heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1)-dependent manner. Exosomal circCCAR1 was taken in by CD8 + T cells and caused dysfunction of CD8 + T cells by stabilizing the PD-1 protein. CircCCAR1 promoted resistance to anti-PD1 immunotherapy. Furthermore, increased cell division cycle and apoptosis regulator 1 (CCAR1) induced by EP300 promoted the binding of CCAR1 and ß-catenin protein, which further enhanced the transcription of PD-L1. CONCLUSIONS: The circCCAR1/miR-127-5p/WTAP feedback loop enhances the growth and metastasis of HCC. Exosomal circCCAR1 released by HCC cells contributes to immunosuppression by facilitating CD8 + T-cell dysfunction in HCC. CircCCAR1 induces resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for HCC patients.

Analysis of angiogenesis-related subtypes of hepatocellular carcinoma and tumor microenvironment infiltration feature in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is a highly vascularized tumor, and angiogenesis plays an important role in its progression. However, the role of angiogenesis in cell infiltration in the tumor microenvironment (TME) remains unclear. METHODS: We evaluated the associations of 35 angiogenesis-related genes (ARGs) with the clinicopathological features of 816 HCC patients. In addition, we assessed the associations between the ARGs and TME cell infiltration. A nomogram was constructed to determine the prognostic value of ARGs for HCC. The ARG score was used to distinguish angiogenic subtypes of HCC, and its usefulness for predicting the prognosis and treatment response of HCC patients was evaluated. RESULTS: We distinguished three ARG clusters differing in terms of TME cell infiltration, immune cell activation status, clinicopathological features, and clinical outcomes. There were significant associations of ARG expression with tumor immunity, the epithelial-mesenchymal transition (EMT), and transforming growth factor-ß expression. An ARG score model was constructed to generate a risk score for each patient based on differentially expressed genes between clusters. Furthermore, a high ARG score was associated with high expression of CTLA-4 and PD-L1/PD-1, and a low Tumor Immune Dysfunction and Exclusion score, indicating the usefulness of the ARG score for selecting patients for immunotherapy. Considering the relationship between ARGs and tumor immunity, immunotherapy combined with vascular-targeted therapy may be the best treatment for HCC. CONCLUSIONS: ARGs play an important role in TME diversity and complexity in HCC patients. The ARG score of HCC predicts TME invasion and can guide immunotherapy.

Long noncoding RNA NEAT1 changes exosome secretion and microRNA expression carried by exosomes in hepatocellular carcinoma cells.

BACKGROUND: This study aimed to investigate the roles and functions of nuclear-enriched abundant transcript 1 (NEAT1) in exosome secretion and exosomal microRNA (miRNA) changes in hepatocellular carcinoma (HCC) cells. METHODS: HepG2 and HuH-7 cells were divided into two groups: Lv-control (which were infected with lentivirus without NEAT1 expression) and Lv-NEAT1 (which were infected with lentivirus with NEAT1 overexpression). Each group was used to study cell function (proliferation, invasion, and apoptosis) and exosome secretion by nanoparticle tracking analysis (NTA), electron microscopy, and nanoflow cytometry (nanoFCM). Different levels of messenger RNA (mRNA), miRNA, and exosomal miRNA were detected by RNA sequencing. Next, potential target RNAs were verified by reverse transcription polymerase chain reaction (RT-PCR). Changed exosomal miRNAs were found and miRNA mimics were used to study cell function in NEAT1-overexpression and NEAT1-knockdown HCC cells. RESULTS: The data showed that NEAT1-overexpression promoted exosome secretion. The overexpression of NEAT1 altered global genes, including exosome-related genes. Compared with the control group, we observed that several miRNAs changed in the exosomes secreted by NEAT1-overexpressing cells. Our study found that these changed exosomal miRNAs played a suppressor role in HCC. Transfection of miR-634, miR-638, and miR-3960 reversed the enhanced invasion and proliferation in HCC cells with a high level of NEAT1 expression. CONCLUSIONS: These results suggested that NEAT1 regulates exosome-related genes, which might be associated with increasing exosome secretion by NEAT1-overexpressing cells. Furthermore, NEAT1 promotes cell invasion and proliferation via downregulation of miR-634, miR-638, and miR-3960 in exosomes. This study may provide potential targets for exosome-mediated miRNA transfer in HCCs with a high level of NEAT1 expression therapy.

Long noncoding RNA NEAT1 promotes cell proliferation and invasion by regulating hnRNP A2 expression in hepatocellular carcinoma cells.

Growing evidence demonstrates that long noncoding RNAs (lncRNAs) are involved in the progression of various cancers, including hepatocellular carcinoma (HCC). The role of nuclear-enriched abundant transcript 1 (NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, has not been fully explored in HCC. We aimed to determine the expression, roles and functional mechanisms of NEAT1 in the proliferation and invasion of HCC. Based on real-time polymerase chain reaction data, we suggest that NEAT1 is upregulated in HCC tissues compared with noncancerous liver tissues. The knockdown of NEAT1 altered global gene expression patterns and reduced HCC cell proliferation, invasion and migration. RNA immunoprecipitation and RNA pull-down assays confirmed that U2AF65 binds to NEAT1. Furthermore, the study indicated that NEAT1 regulated hnRNP A2 expression and that this regulation may be associated with the NEAT1-U2AF65 protein complex. Thus, the NEAT1-hnRNP A2 regulation mechanism promotes HCC pathogenesis and may provide a potential target for the prognosis and treatment of HCC.