Ferroptosis is a form of regulated cell death, which is dependent on iron metabolism imbalance and characterized by lipid peroxidation. Ferroptosis plays a crucial role in various pathological processes. Studies have shown that the occurrence of ferroptosis is closely associated with the progression of hepatocellular carcinoma (HCC). Ferroptosis is involved in regulating the lipid metabolism, iron homeostasis, mitochondrial metabolism, and redox processes in HCC. Additionally, ferroptosis plays a key role in HCC tumor immunity by modulating the phenotype and function of various immune cells in the tumor microenvironment, affecting tumor immune escape and progression. Ferroptosis-induced lipid peroxidation and oxidative stress can promote the polarization of M1 macrophages and enhance the pro-inflammatory response in tumors, inhibiting immune suppressive cells such as myeloid-derived suppressor cells and regulatory T cells to disrupt their immune suppression function. The regulation of expression of ferroptosis-related molecules such as GPX4 and SLC7A11 not only affects the sensitivity of tumor cells to immunotherapy but also directly influences the activity and survival of effector cells such as T cells and dendritic cells, further enhancing or weakening host antitumor immune response. Targeting ferroptosis has demonstrated significant clinical potential in HCC treatment. Induction of ferroptosis by nanomedicines and molecular targeting strategies can directly kill tumor cells or enhance antitumor immune responses. The integration of multimodal therapies with immunotherapy further expands the application of ferroptosis targeting as a cancer therapy. This article reviews the relationship between ferroptosis and antitumor immune responses and the role of ferroptosis in HCC progression from the perspective of tumor immune microenvironment, to provide insights for the development of antitumor immune therapies targeting ferroptosis.
Ferroptosis ; Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/metabolism* ; Tumor Microenvironment/immunology* ; Lipid Peroxidation ; Immunotherapy ; Oxidative Stress ; Iron/metabolism* ; Lipid Metabolism ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Macrophages/immunology* ; Amino Acid Transport System y+

As the central organ of metabolism, the liver plays a pivotal role in the regulation of the synthesis and metabolism of various nutrients within the body. Ferroptosis, as a newly discovered type of programmed cell death caused by the accumulation of iron-dependent lipid peroxides, is involved in the physiological and pathological processes of a variety of acute and chronic liver diseases. Ferroptosis can accelerate the pathogenetic process of acute liver injury, metabolic associated fatty liver disease, alcoholic liver disease, viral hepatitis, and autoimmune hepatitis; while it can slower disease progression in advanced liver fibrosis and hepatocellular carcinoma. This suggests that targeted regulation of ferroptosis may impact the occurrence and development of various liver diseases. This article reviews the latest research progress of ferroptosis in various liver diseases, including acute liver injury, metabolic associated fatty liver disease, alcoholic liver disease, viral hepatitis, autoimmune hepatitis, liver fibrosis and hepatocellular carcinoma. It aims to provide insights for the prevention and treatment of acute and chronic liver diseases through targeting ferroptosis.
Humans ; Liver Diseases/etiology* ; Ferroptosis/physiology* ; Liver Neoplasms/pathology* ; Carcinoma, Hepatocellular/pathology* ; Liver Cirrhosis/etiology* ; Liver/pathology* ; Hepatitis, Autoimmune/metabolism* ; Liver Diseases, Alcoholic/metabolism*

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.
Humans ; Animals ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; B7-H1 Antigen/metabolism* ; Ligands ; Liver Neoplasms/pathology* ; RNA, Small Interfering/metabolism* ; Neoplastic Stem Cells/physiology* ; Cell Line, Tumor ; Cell Proliferation

OBJECTIVE: Programmed cell death 6 (PDCD6), a Ca ²⁺-binding protein, has been reported to be aberrantly expressed in all kinds of tumors. The aim of this study was to explore the role and mechanism of PDCD6 in hepatocellular carcinomas (HCCs). METHODS: The expression levels of PDCD6 in liver cancer patients and HCC cell lines were analyzed using bioinformatics and Western blotting. Cell viability and metastasis were determined by methylthiazol tetrazolium (MTT) and transwell assays, respectively. And Western blotting was used to test related biomarkers and molecular pathway factors in HCC cell lines. LY294002, a PI3K inhibitor inhibiting AKT, was used to suppress the AKT/GSK3β/β-catenin pathway to help evaluate the role of this pathway in the HCC carcinogenesis associated with PDCD6. RESULTS: The analysis of The Cancer Genome Atlas Database suggested that high PDCD6 expression levels were relevant to liver cancer progression. This was consistent with our finding of higher levels of PDCD6 expression in HCC cell lines than in normal hepatocyte cell lines. The results of MTT, transwell migration, and Western blotting assays revealed that overexpression of PDCD6 positively regulated HCC cell proliferation, migration, and invasion. Conversely, the upregulation of PDCD6 expression in the presence of an AKT inhibitor inhibited HCC cell proliferation, migration, and invasion. In addition, PDCD6 promoted HCC cell migration and invasion by epithelial-mesenchymal transition. The mechanistic investigation proved that PDCD6 acted as a tumor promoter in HCC through the AKT/GSK3β/β-catenin pathway, increasing the expression of transcription factors and cellular proliferation and metastasis. CONCLUSION PDCD6 has a tumor stimulative role in HCC mediated by AKT/GSK3β/β-catenin signaling and might be a potential target for HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Proto-Oncogene Proteins c-akt/metabolism* ; beta Catenin/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Cell Line ; Cell Proliferation ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Calcium-Binding Proteins/metabolism* ; Apoptosis Regulatory Proteins/genetics*

Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.
Humans ; Carcinoma, Hepatocellular/pathology* ; Cell Hypoxia ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Liver Neoplasms/pathology* ; Signal Transduction/genetics* ; tRNA Methyltransferases/metabolism*

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC. METHODS: We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/β-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25. RESULTS: USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/β-catenin pathway (β-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo . CONCLUSIONS In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the β-catenin signaling pathway.
Animals ; Mice ; beta Catenin/genetics* ; Carcinoma, Hepatocellular/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic ; Liver Neoplasms/pathology* ; Ubiquitin Thiolesterase/metabolism* ; Wnt Signaling Pathway/genetics*

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; TOR Serine-Threonine Kinases/metabolism* ; Apoptosis ; Autophagy ; Cell Proliferation ; Cell Line, Tumor ; STAT3 Transcription Factor/metabolism*

Lamin B1 (LMNB1) is highly expressed in liver cancer tissues, and its influence and mechanism on the proliferation of hepatocellular carcinoma cells were explored by knocking down the expression of the protein. In liver cancer cells, siRNAs were used to knock down LMNB1. Knockdown effects were detected by Western blotting. Changes in telomerase activity were detected by telomeric repeat amplification protocol assay (TRAP) experiments. Telomere length changes were detected by quantitative real-time polymerase chain reaction (qPCR). CCK8, cloning formation, transwell and wound healing were performed to detect changes in its growth, invasion and migration capabilities. The lentiviral system was used to construct HepG2 cells that steadily knocked down LMNB1. Then the changes of telomere length and telomerase activity were detected, and the cell aging status was detected by SA-β-gal senescence staining. The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments, subsequent histification staining of tumors, SA-β-gal senescence staining, fluorescence in situ hybridization (FISH) for telomere analysis and other experiments. Finally, the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues, and its relationship with clinical stages and patient survival. Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity, cell proliferation, migration and invasion abilities. Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity, shortened telomere length, senesced cells, reduced cell tumorigenicity and KI-67 expression. Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival. In conclusion, LMNB1 is overexpressed in liver cancer cells, and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.
Animals ; Mice ; Telomerase/metabolism* ; Carcinoma, Hepatocellular/genetics* ; Liver Neoplasms/genetics* ; Telomere Shortening ; In Situ Hybridization, Fluorescence ; Mice, Nude ; Telomere/pathology* ; Carcinogenesis

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNA^Gln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Eukaryotic Initiation Factor-4A/genetics* ; Cell Line ; RNA, Transfer/metabolism* ; RNA ; Cell Proliferation

Objective: To study using isotope-labeled relative and absolute quantitative proteomics methodologies to screen for salivary biological markers as a simple, non-invasive tool for identifying hepatitis B-related HCC at an early stage. Methods: Saliva samples were collected to extract salivary proteins. Isotope-labeled relative and absolute quantitative proteomics were used to analyze the differentially expressed proteins between the hepatocellular carcinoma (HCC) and non-HCC groups. Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays were used to verify differential proteins and identify markers in liver cancer tissues and saliva. Statistical analysis was used to analyze the diagnostic efficiency of salivary biomarkers. Results: 152 differentially expressed salivary proteins were screened out between the HCC and non-HCC groups. Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays validated that the expressions of α-1-acid glycoprotein 1 (ORM1) and alpha-fetoprotein (AFP) were significantly increased in HCC (P < 0.05). There was a significant correlation between salivary AFP and serum AFP (P < 0.05). HCC was diagnosed when salivary α-1-acid glycoprotein 1 combined with AFP. The area under the receiver operating characteristic curve was 0.8726 (95% confidence interval: 0.8104 ~ 0.9347), the sensitivity was 78.3%, and the specificity was 88%. Conclusion: Salivary AFP and α-1-acid glycoprotein 1 can serve as potential biomarkers for hepatitis B-related hepatocellular carcinoma.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Liver Neoplasms/pathology* ; alpha-Fetoproteins/metabolism* ; Biomarkers ; Hepatitis B ; ROC Curve ; Glycoproteins ; Biomarkers, Tumor