Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Multi-Omics profiling identifies aldehyde dehydrogenase 2 as a critical mediator in the crosstalk between Treg-mediated immunosuppression microenvironment and hepatocellular carcinoma.

Dysregulation of the aldehyde dehydrogenase (ALDH) family has been implicated in various pathological conditions, including cancer. However, a systematic evaluation of ALDH alterations and their therapeutic relevance in hepatocellular carcinoma (HCC) remains lacking. Herein, we found that 15 of 19 ALDHs were transcriptionally dysregulated in HCC tissues compared to normal liver tissues. A four gene signature, including ALDH2, ALDH5A1, ALDH6A1, and ALDH8A1, robustly predicted prognosis and defined a high-risk subgroup exhibiting immunosuppressive features like regulatory T cell (Tregs) infiltration. Single-cell profiling revealed selective overexpression of tumor necrosis factor receptor superfamily member 18 (TNFRSF18) on Tregs, upregulated in high-risk HCC patients. We identified ALDH2 as a tumor suppressor in HCC, with three novel phosphorylation sites mediated by protein kinase C zeta that enhanced enzymatic activity. Mechanistically, ALDH2 suppressed Tregs differentiation by inhibiting ß-catenin/TGF-ß1 signaling in HCC. Collectively, our integrated multi-omics analysis defines an ALDH-Tregs-TNFRSF18 axis that contributes to HCC pathogenesis and represents potential therapeutic targets for this aggressive malignancy.

SOAT1 regulates cholesterol metabolism to induce EMT in hepatocellular carcinoma.

Cholesterol metabolism reprogramming is one of the significant characteristics of hepatocellular carcinoma (HCC). Cholesterol increases the risk of epithelial-mesenchymal transition (EMT) in cancer. Sterol O-acyltransferases 1 (SOAT1) maintains the cholesterol homeostasis. However, the exact mechanistic contribution of SOAT1 to EMT in HCC remains unclear. Here we demonstrated that SOAT1 positively related to poor prognosis of HCC, EMT markers and promoted cell migration and invasion in vitro, which was mediated by the increased cholesterol in plasmalemma and cholesterol esters accumulation. Furthermore, we reported that SOAT1 disrupted cholesterol metabolism homeostasis to accelerate tumorigenesis and development in HCC xenograft and NAFLD-HCC. Also, we detected that nootkatone, a sesquiterpene ketone, inhibited EMT by targeting SOAT1 in vitro and in vivo. Collectively, our finding indicated that SOAT1 promotes EMT and contributes to hepatocarcinogenesis by increasing cholesterol esterification, which is suppressed efficiently by nootkatone. This study demonstrated that SOAT1 is a potential biomarker and therapeutic target in NAFLD-HCC and SOAT1-targeting inhibitors are expected to be the potential new therapeutic treatment for HCC.

Interleukin-21 receptor signaling promotes metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma by inducing immunosuppressive IgA+ B cells.

BACKGROUND: Dysregulation of immune surveillance is tightly linked to the development of metabolic dysfunction-associated steatohepatitis (MASH)-driven hepatocellular carcinoma (HCC); however, its underlying mechanisms remain unclear. Herein, we aimed to determine the role of interleukin-21 receptor (IL-21R) in MASH-driven HCC. METHODS: The clinical significance of IL-21R was assessed in human HCC specimens using immunohistochemistry staining. Furthermore, the expression of IL-21R in mice was assessed in the STAM model. Thereafter, two different MASH-driven HCC mouse models were applied between IL-21R-deficient mice and wild type controls to explore the role of IL-21R in MASH-driven HCC. To further elucidate the potential mechanisms by which IL-21R affected MASH-driven HCC, whole transcriptome sequencing, flow cytometry and adoptive lymphocyte transfer were performed. Finally, flow cytometry, enzyme-linked immunosorbent assay, immunofluorescent staining, chromatin immunoprecipitation assay and western blotting were conducted to explore the mechanism by which IL-21R induced IgA+ B cells. RESULTS: HCC patients with high IL-21R expression exhibited poor relapse-free survival, advanced TNM stage and severe steatosis. Additionally, IL-21R was demonstrated to be upregulated in mouse liver tumors. Particularly, ablation of IL-21R impeded MASH-driven hepatocarcinogenesis with dramatically reduction of lipid accumulation. Moreover, cytotoxic CD8+ T lymphocyte activation was enhanced in the absence of IL-21R due to the reduction of immunosuppressive IgA+ B cells. Mechanistically, the IL-21R-STAT1-c-Jun/c-Fos regulatory axis was activated in MASH-driven HCC and thus promoted the transcription of Igha, resulting in the induction of IgA+ B cells. CONCLUSIONS: IL-21R plays a cancer-promoting role by inducing IgA+ B cells in MASH-driven hepatocarcinogenesis. Targeting IL-21R signaling represents a potential therapeutic strategy for cancer therapy.

Fluorescence imaging sheds light on the immune evasion mechanisms of hepatic stellate cells mediated by superoxide anion.

Whether and how the reactive oxygen species generated by hepatic stellate cells (HSCs) promote immune evasion of hepatocellular carcinoma (HCC) remains mysterious. Therefore, investigating the function of superoxide anion (O2•-), the firstly generated reactive oxygen species, during the immune evasion become necessary. In this work, we establish a novel in situ imaging method for visualization of O2•- changes in HSCs based on a new two-photon fluorescence probe TPH. TPH comprises recognition group for O2•- and HSCs targeting peptides. We observe that O2•- in HSCs gradually rose, impairing the infiltration of CD8+ T cells in HCC mice. Further studies reveal that the cyclin-dependent kinase 4 is deactivated by O2•-, and then cause the up-regulation of PD-L1. Our work provides molecular insights into HSC-mediated immune evasion of HCC, which may represent potential targets for HCC immunotherapy.

Mitochondria-Derived Vesicles, Sterile Inflammation, and Pyroptosis in Liver Cancer: Partners in Crime or Innocent Bystanders?

Alterations in cellular signaling, chronic inflammation, and tissue remodeling contribute to hepatocellular carcinoma (HCC) development. The release of damage-associated molecular patterns (DAMPs) upon tissue injury and the ensuing sterile inflammation have also been attributed a role in HCC pathogenesis. Cargoes of extracellular vesicles (EVs) and/or EVs themselves have been listed among circulating DAMPs but only partially investigated in HCC. Mitochondria-derived vesicles (MDVs), a subpopulation of EVs, are another missing link in the comprehension of the molecular mechanisms underlying the onset and progression of HCC biology. EVs have been involved in HCC growth, dissemination, angiogenesis, and immunosurveillance escape. The contribution of MDVs to these processes is presently unclear. Pyroptosis triggers systemic inflammation through caspase-dependent apoptotic cell death and is implicated in tumor immunity. The analysis of this process, together with MDV characterization, may help capture the relationship among HCC development, mitochondrial quality control, and inflammation. The combination of immune checkpoint inhibitors (i.e., atezolizumab and bevacizumab) has been approved as a synergistic first-line systemic treatment for unresectable or advanced HCC. The lack of biomarkers that may allow prediction of treatment response and, therefore, patient selection, is a major unmet need. Herein, we overview the molecular mechanisms linking mitochondrial dysfunction, inflammation, and pyroptosis, and discuss how immunotherapy targets, at least partly, these routes.

Discovery of potent and novel dual NAMPT/BRD4 inhibitors for efficient treatment of hepatocellular carcinoma.

The NAPRT-induced increase in NAD+ levels was proposed as a mechanism contributing to hepatocellular carcinoma (HCC) resistance to NAMPT inhibitors. Thus, concurrently targeting NAMPT and NAPRT could be considered to overcome drug resistance. A BRD4 inhibitor downregulates the expression of NAPRT in HCC, and the combination of NAMPT inhibitors with BRD4 inhibitors simultaneously blocks NAD+ generation via salvage and the PH synthesis pathway. Moreover, the combination of the two agents significantly downregulated the expression of tumor-promoting genes and strongly promoted apoptosis. The present work identified various NAMPT/BRD4 dual inhibitors based on the multitargeted drug rationale. Among them, compound A2, which demonstrated the strongest effect, exhibited potent inhibition of NAMPT and BRD4 (IC50 = 35 and 58 nM, respectively). It significantly suppressed the growth and migration of HCC cells and facilitated their apoptosis. Furthermore, compound A2 also manifested a robust anticancer effect in HCCLM3 xenograft mouse models, with no apparent toxic effects. Our findings in this study provide an effective approach to target NAD+ metabolism for HCC treatment.

Ficolin 3 promotes ferroptosis in HCC by downregulating IR/SREBP axis-mediated MUFA synthesis.

BACKGROUND: Targeting ferroptosis has been identified as a promising approach for the development of cancer therapies. Monounsaturated fatty acid (MUFA) is a type of lipid that plays a crucial role in inhibiting ferroptosis. Ficolin 3 (FCN3) is a component of the complement system, serving as a recognition molecule against pathogens in the lectin pathway. Recent studies have reported that FCN3 demonstrates inhibitory effects on the progression of certain tumors. However, whether FCN3 can modulate lipid metabolism and ferroptosis remains largely unknown. METHODS: Cell viability, BODIPY-C11 staining, and MDA assay were carried out to detect ferroptosis. Primary hepatocellular carcinoma (HCC) and xenograft models were utilized to investigate the effect of FCN3 on the development of HCC in vivo. A metabonomic analysis was conducted to assess alterations in intracellular and HCC intrahepatic lipid levels. RESULTS: Our study elucidates a substantial decrease in the expression of FCN3, a component of the complement system, leads to MUFA accumulation in human HCC specimens and thereby significantly promotes ferroptosis resistance. Overexpression of FCN3 efficiently sensitizes HCC cells to ferroptosis, resulting in the inhibition of the oncogenesis and progression of both primary HCC and subcutaneous HCC xenograft. Mechanistically, FCN3 directly binds to the insulin receptor ß (IR-ß) and its pro-form (pro-IR), inhibiting pro-IR cleavage and IR-ß phosphorylation, ultimately resulting in IR-ß inactivation. This inactivation of IR-ß suppresses the expression of sterol regulatory element binding protein-1c (SREBP1c), which subsequently suppresses the transcription of genes related to de novo lipogenesis (DNL) and lipid desaturation, and consequently downregulates intracellular MUFA levels. CONCLUSIONS: These findings uncover a novel regulatory mechanism by which FCN3 enhances the sensitivity of HCC cells to ferroptosis, indicating that targeting FCN3-induced ferroptosis is a promising strategy for HCC treatment.

DCAF1 interacts with PARD3 to promote hepatocellular carcinoma progression and metastasis by activating the Akt signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is a fatal malignancy with poor prognosis due to lack of effective clinical interference. DCAF1 plays a vital role in regulating cell growth and proliferation, and is involved in the progression of various malignancies. However, the function of DCAF1 in HCC development and the underlying mechanism are still unknown. This study aimed to explore the effect of DCAF1 in HCC and the corresponding molecular mechanism. METHODS: Quantitative real-time PCR, Western blot and immunostaining were used to determine DCAF1 expression in tumor tissues and cell lines. Subsequently, in vitro and in vivo experiments were conducted to explore the function of DCAF1 in tumor growth and metastasis in HCC. Coimmunoprecipitation, mass spectrometry and RNA sequencing were performed to identify the underlying molecular mechanisms. RESULTS: In this study, we found that DCAF1 was observably upregulated and associated with poor prognosis in HCC. Knockdown of DCAF1 inhibited tumor proliferation and metastasis and promoted tumor apoptosis, whereas overexpressing DCAF1 yielded opposite effects. Mechanistically, DCAF1 could activate the Akt signaling pathway by binding to PARD3 and enhancing its expression. We also found that the combined application of DCAF1 knockdown and Akt inhibitor could significantly suppress subcutaneous xenograft tumor growth. CONCLUSIONS: Our study illustrates that DCAF1 plays a crucial role in HCC development and the DCAF1/PARD3/Akt axis presents a potentially effective therapeutic strategy for HCC.

PLAG1 interacts with GPX4 to conquer vulnerability to sorafenib induced ferroptosis through a PVT1/miR-195-5p axis-dependent manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC), yet its effectiveness is often constrained. Emerging studies reveal that sorafenib triggers ferroptosis, an iron-dependent regulated cell death (RCD) mechanism characterized by lipid peroxidation. Our findings isolate the principal target responsible for ferroptosis in HCC cells and outline an approach to potentially augment sorafenib's therapeutic impact on HCC. METHODS: We investigated the gene expression alterations following sgRNA-mediated knockdown induced by erastin and sorafenib in HCC cells using CRISPR screening-based bioinformatics analysis. Gene set enrichment analysis (GSEA) and the "GDCRNATools" package facilitated the correlation studies. We employed tissue microarrays and cDNA microarrays for validation. Ubiquitination assay, Chromatin immunoprecipitation (ChIP) assay, RNA immunoprecipitation (RIP) assay, and dual-luciferase reporter assay were utilized to delineate the specific mechanisms underlying ferroptosis in HCC cells. RESULTS: Our study has revealed that pleiomorphic adenoma gene 1 (PLAG1), a gene implicated in pleomorphic adenoma, confers resistance to ferroptosis in HCC cells treated with sorafenib. Sorafenib leads to the opposite trend of protein and mRNA levels of PLAG1, which is not caused by affecting the stability or ubiquitination of PLAG1 protein, but by the regulation of PLAG1 at the transcriptional level by its upstream competitive endogenous long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1). Data from 139 HCC patients showed a significant positive correlation between PLAG1 and GPX4 levels in tumor samples, and PLAG1 is instrumental in redox homeostasis by driving the expression of glutathione peroxidase 4 (GPX4), the enzyme that reduces lipid peroxides (LPOs), which further leads to ferroptosis inhibition. CONCLUSIONS: Ferroptosis is a promising target for cancer therapy, especially for patients resistant to standard chemotherapy or immunotherapy. Our findings indicate that PLAG1 holds therapeutic promise and may enhance the efficacy of sorafenib in treating HCC.

PSMD9 promotes the malignant progression of hepatocellular carcinoma by interacting with c-Cbl to activate EGFR signaling and recycling.

BACKGROUND: Mounting evidences shows that the ubiquitin‒proteasome pathway plays a pivotal role in tumor progression. The expression of 26S proteasome non-ATPase regulatory subunit 9 (PSMD9) is correlated with recurrence and radiotherapy resistance in several tumor types. However, the role and mechanism of PSMD9 in hepatocellular carcinoma (HCC) progression remain largely unclear. METHODS: PSMD9 was identified as a prognosis-related biomarker for HCC based on analysis of clinical characteristics and RNA-seq data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the JP Project of the International Cancer Genome Consortium (ICGC-LIRI-JP). PSMD9 expression was analyzed in cancer tissues and adjacent noncancerous tissues via immunohistochemistry and Western blotting. Multiple in vivo and in vitro experimental techniques (such as CCK-8, colony formation, EdU, and Transwell assays; flow cytometry; Western blotting; quantitative RT-PCR; Coimmunoprecipitation assay and immunofluorescence confocal imaging) were used to assess the functions of PSMD9 in the pathogenesis of HCC. RESULTS: We found that the expression of PSMD9 was upregulated and associated with a poor prognosis in HCC patients. PSMD9 promoted HCC cell proliferation, migration, invasion and metastasis. Knockdown of PSMD9 significantly inhibited HCC cell proliferation by inducing G1/S cell cycle arrest and apoptosis. Mechanistically, we demonstrated that PSMD9 promoted HCC cell proliferation and metastasis via direct interaction with the E3 ubiquitin ligase c-Cbl, suppresses EGFR ubiquitination, influenced EGFR endosomal trafficking and degradation and subsequently activated ERK1/2 and Akt signaling. In addition, we showed that PSMD9 knockdown sensitized HCC cells to the tyrosine kinase inhibitor erlotinib in vitro and in vivo. CONCLUSIONS: Collectively, our results indicate that PSMD9 drives HCC progression and erlotinib resistance by suppressing c-Cbl mediated EGFR ubiquitination and therefore can be a potential therapeutic target for HCC.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Hsa_circ_0051908 Promotes Hepatocellular Carcinoma Progression by Regulating the Epithelial-Mesenchymal Transition Process.

Materials and Methods: Hsa_circ_0051908 expression was determined using RT-qPCR. HCC cell proliferation, apoptosis, invasion, and migration were assessed using CCK-8 assay, EdU staining, TUNEL staining, flow cytometry, and transwell assay. The molecular mechanism was analyzed using western blotting. In addition, the role of hsa_circ_0051908 in tumor growth was evaluated in vivo. Results: Hsa_circ_0051908 expression was increased in both HCC tissues and cell lines. The proliferation, migration, and invasion of HCC cells were significantly decreased after hsa_circ_0051908 knockdown, while cell apoptosis was notably increased. Furthermore, we found that hsa_circ_0051908 silencing downregulated vimentin and Snail and upregulated E-cadherin. In vivo, hsa_circ_0051908 silencing significantly inhibited the growth of the tumor. Conclusions: Our data provide evidence that hsa_circ_0051908 promotes HCC progression partially by mediating the epithelial-mesenchymal transition process, and it may be used for HCC treatment.

Expression of Fascin-1 and its diagnostic value in liver cancer.

Although some studies have reported on the expression and clinical significance of Fascin-1 (FSCN1) in liver cancer, the clinical application and differential diagnosis value of FSCN1 in liver cancer are still unclear. The aim of this study was to analyze the expression level of FSCN1 protein in liver cancer tissues and explore its diagnostic and application value in differentiating between hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). The immunehistochemical analysis was used to detect the expression of FSCN1 in 108 cases of HCC, 26 cases of ICC, 23 cases of liver cirrhosis, and 11 cases of normal liver tissues. The differences in the positive expression rate and strong positive expression rate of FSCN1 among different groups were analyzed. The positive rate of FSCN1 in normal liver tissues, liver cirrhosis, HCC, and ICC tissues was 0.0% (0/11), 0.0% (0/23), 13.9% (15/108), and 92.3% (24/26), respectively, while the strong positive rate was 0.0% (0/11), 0.0% (0/23), 0.9% (1/108), and 69.2% (18/26), respectively. Both the positive rate and strong positive rate of FSCN1 in ICC tissues were significantly higher than those in HCC, liver cirrhosis, and normal liver tissues. Additionally, the positive rate of FSCN1 in moderately to poorly differentiated HCC tissues was 18.8% (15/80), significantly higher than in well-differentiated HCC (0.0%, 0/28) (P = 0.031). In liver cancer, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FSCN1 positive prediction for ICC were 92.3%, 86.1%, 61.5%, and 97.9%, respectively, whereas the sensitivity, specificity, PPV, and NPV of FSCN1 strong positive prediction for ICC were 69.2%, 99.1%, 94.7%, and 93.0%, respectively. These results suggest that FSCN1 may play an important role in the occurrence and progression of liver cancer, and it can be used as a novel diagnostic marker for ICC.

Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

A comprehensive review of phytoconstituents in liver cancer prevention and treatment: targeting insights into molecular signaling pathways.

Primary liver cancer is a type of cancer that develops in the liver. Hepatocellular carcinoma is a primary liver cancer that usually affects adults. Liver cancer is a fatal global condition that affects millions of people worldwide. Despite advances in technology, the mortality rate remains alarming. There is growing interest in researching alternative medicines to prevent or reduce the effects of liver cancer. Recent studies have shown growing interest in herbal products, nutraceuticals, and Chinese medicines as potential treatments for liver cancer. These substances contain unique bioactive compounds with anticancer properties. The causes of liver cancer and potential treatments are discussed in this review. This study reviews natural compounds, such as curcumin, resveratrol, green tea catechins, grape seed extracts, vitamin D, and selenium. Preclinical and clinical studies have shown that these medications reduce the risk of liver cancer through their antiviral, anti-inflammatory, antioxidant, anti-angiogenic, and antimetastatic properties. This article discusses the therapeutic properties of natural products, nutraceuticals, and Chinese compounds for the prevention and treatment of liver cancer.

Ethanol responsive lnc171 promotes migration and invasion of HCC cells via mir-873-5p/ZEB1 axis.

BACKGROUNDS: Long nonconding RNAs (lncRNAs) have been found to be a vital regulatory factor in the development process of human cancer, and could regarded as diagnostic or prognostic biomarkers for human cancers. Here, we aim to confirm the expression and molecular mechanism of RP11-171K16.5 (lnc171) in hepatocellular carcinoma (HCC). METHODS: Screening of differentially expressed lncRNAs by RNA sequencing. Expression level of gene was studied by quantitative real-time PCR (qRT-PCR). The effects of lnc171, mir-873-5p, and ethanol on migration and invasion activity of cells were studied used transwell assay, and luciferase reporter assay was used to confirm the binding site. RESULTS: RNA sequencing showed that lnc171 was markedly up-regulated in HCC. siRNA-mediated knockdown of lnc171 repressed the migration and invasion ability of HCC cells. Bioinformatic analysis, dual luciferase reporter assay, and qRT-PCR indicated that lnc171 interacted with mir-873-5p in HCC cells, and Zin-finger E-box binding homeobox (ZEB1) was a downstream target gene of mir-873-5p. In addition, lnc171 could enhance migration and invasion ability of HCC cells by up-regulating ZEB1 via sponging mir-873-5p. More interestingly, ethanol stimulation could up-regulate the increase of lnc171, thereby regulating the expression of competing endogenous RNA (ceRNA) network factors which lnc171 participated in HCC cells. CONCLUSIONS: Our date demonstrates that lnc171 was a responsive factor of ethanol, and plays a vital role in development of HCC via binding of mir-873-5p.

Inhibition of the transmembrane transporter ABCB1 overcomes resistance to doxorubicin in patient-derived organoid models of HCC.

BACKGROUND: Transarterial chemoembolization is the first-line treatment for intermediate-stage HCC. However, the response rate to transarterial chemoembolization varies, and the molecular mechanisms underlying variable responses are poorly understood. Patient-derived hepatocellular carcinoma organoids (HCCOs) offer a novel platform to investigate the molecular mechanisms underlying doxorubicin resistance. METHODS: We evaluated the effects of hypoxia and doxorubicin on cell viability and cell cycle distribution in 20 patient-derived HCCO lines. The determinants of doxorubicin response were identified by comparing the transcriptomes of sensitive to resistant HCCOs. Candidate genes were validated by pharmacological inhibition. RESULTS: Hypoxia reduced the proliferation of HCCOs and increased the number of cells in the G0/G1 phase of the cell cycle, while decreasing the number in the S phase. The IC50s of the doxorubicin response varied widely, from 29nM to >1µM. Doxorubicin and hypoxia did not exhibit synergistic effects but were additive in some HCCOs. Doxorubicin reduced the number of cells in the G0/G1 and S phases and increased the number in the G2 phase under both normoxia and hypoxia. Genes related to drug metabolism and export, most notably ABCB1, were differentially expressed between doxorubicin-resistant and doxorubicin-sensitive HCCOs. Small molecule inhibition of ABCB1 increased intracellular doxorubicin levels and decreased drug tolerance in resistant HCCOs. CONCLUSIONS: The inhibitory effects of doxorubicin treatment and hypoxia on HCCO proliferation are variable, suggesting an important role of tumor-cell intrinsic properties in doxorubicin resistance. ABCB1 is a determinant of doxorubicin response in HCCOs. Combination treatment of doxorubicin and ABCB1 inhibition may increase the response rate to transarterial chemoembolization.

Exploring the role of CDCA4 in liver hepatocellular carcinoma using bioinformatics analysis and experiments.

Liver hepatocellular carcinoma (LIHC) encompasses diverse therapeutic approaches, among which targeted therapy has gained significant prominence in recent years. The identification of numerous targets and the increasing clinical application of targeted drugs have greatly improved LIHC treatment. However, the precise role of CDCA4 (Cell Division Cycle Associated 4), as well as its underlying mechanisms and prognostic implications in LIHC, remains unclear. CDCA4 expression levels in LIHC were analyzed using multiple databases including the cancer genome atlas (TCGA), gene expression profiling interactive analysis (GEPIA), and ULCAN, as well as the datasets E_TABM_36, GSE144269, GSE14520, and GSE54236. The prognostic value of CDCA4 was then evaluated. Subsequently, the association between CDCA4 and immune cells was investigated. Enrichment analysis (GSEA) was utilized to investigate the functional roles and pathways linked to CDCA4. Additionally, the methylation patterns and drug sensitivity of CDCA4 were examined. A predictive model incorporating immune genes related to CDCA4 was developed. The TISCH dataset was used to investigate the single-cell expression patterns of CDCA4. Finally, validation of CDCA4 expression levels was conducted through RT-PCR, Western blotting, and immunohistochemistry. CDCA4 exhibited significant overexpression in LIHC and demonstrated significant correlations with clinical features. High expression of CDCA4 is associated with a poorer prognosis. Analysis of immune infiltration and enrichment revealed its association with the immune microenvironment. Furthermore, its expression is correlated with methylation and mutation patterns. CDCA4 is associated with 19 drugs. Prognostic models utilizing CDCA4 demonstrate favorable effectiveness. T cell subtypes were found to be associated with CDCA4 through single-cell analysis. The conclusive experiment provided evidence of significant upregulation of CDCA4 in LIHC. The high expression of CDCA4 in LIHC is associated with prognostic significance and is highly expressed in T cell subtypes, providing a new therapeutic target and potential therapeutic strategy for LIHC.