Hepatocellular Carcinoma Cell Lines Research Articles

GOLM1 dictates acquired Lenvatinib resistance by a GOLM1-CSN5 positive feedback loop upon EGFR signaling activation in hepatocellular carcinoma.

Lenvatinib is a multiple receptor tyrosine kinases inhibitor (TKI) authorized for first-line treatment of hepatocellular carcinoma (HCC). However, Lenvatinib resistance is common in HCC clinical treatment, highlighting the urgent need to understand mechanisms of resistance. Here, we identified Golgi membrane protein 1 (GOLM1), a type II transmembrane protein originally located in the Golgi apparatus, as a novel regulator of Lenvatinib resistance. We found GOLM1 was overexpressed in Lenvatinib resistant human HCC cell lines, blood and HCC samples. Additionally, GOLM1 overexpression contributes to Lenvatinib resistance and HCC progression in vitro and in vivo. Mechanistically, GOLM1 upregulates CSN5 expression through EGFR-STAT3 pathway. Reversely, CSN5 deubiquitinates and stabilizes GOLM1 protein by inhibiting ubiquitin-proteasome pathway of GOLM1. Furthermore, clinical specimens of HCC showed a positive correlation between the activation of the GOLM1-EGFR-STAT3-CSN5 axis. Finally, GOLM1 knockdown was found to act in synergy with Lenvatinib in subcutaneous and orthotopic mouse model. Overall, these findings identify a mechanism of resistance to Lenvatinib treatment for HCC, highlight an effective predictive biomarker of Lenvatinib response in HCC and show that targeting GOLM1 may improve the clinical benefit of Lenvatinib.

Cytotoxicity, chemical, and nutritional profile evaluation of biomass extracts of the Lemna aequinoctialis (duckweed) aquatic plant

ABSTRACT Lemna aequinoctialis (duckweed) is the smallest and fast-growing aquatic plant species producing protein-rich biomass with high protein nutritional value, phytoremediation capacity, and nutrient removal from wastewater. Duckweed may also be used as a new potential bioreactor for biological products, such as vaccines, antibodies, and pharmaceutical proteins. Based upon the potential importanc of L. aequinoctialis in phytoremediation and as a bioreactor the aim of this study was to (1) characterize the chemical and nutritional profiles of L. aequinoctialis biomass utilizing an integrated multi-trophic aquaculture system (IMTA) and a pond, and (2) investigate the cytotoxic potential of different concentrations of organic extracts and fractions using the MTT bioassay. EDXRF and ICP-MS analyses indicated the presence of trace elements in lower amounts in relation to the biomass of L. aequinoctialis in the lagoon, emphasizing the importance of plant inclusion management to reduce bioaccumulation of these elements. Analysis of mineral profiles, fatty acids, and amino acids indicated a satisfactory nutritional composition for the use of biomass as a bioproduct. Pigment analysis showed a high concentration of carotenoids, especially astaxanthin. After standardizing the controls, the MTT cell viability test was carried out utilizing rat hepatoma cell line (HTC), which are metabolizing cells that were treated with aqueous or ethanolic extracts and the dichloromethane, ethyl acetate, and methanol fractions at different concentrations. No apparent cytotoxic potential was observed following treatments, since there was no significant reduction in cell viability. Therefore, this study provides information regarding the biomass of L. aequinoctialis derived from the IMTA system, which might support further research into the application of this species as a bioproduct.

Microwave synthesis, density functional theory study and antiproliferative activity of the novel spiropyrazole derivatives

Different spiro-pyrazole-oxy-indoline substituted compounds I-IV have been synthesized. In Jaguar, Hybrid DFT using Becke’s three-parameter transfer possibility also,Lee-Yang-Parr correlated practical (B3LYP) viewwith a 6-31 G** basis set were utilized to improve the geometrical characteristics of the hit molecules and reference compound. The MTT test exhibited whether the prepared substance has an inhibitory impact on cell development or not. This was successfully represented that in hepatocellular carcinoma (HEPG-2), colorectal carcinoma (HCT-116), and mammary gland breast cancer cell lines (MCF-7) colorimetric test, the mitochondrial succinate dehydrogenase in live cells transform yellow tetrazolium bromide (MTT) to a purple formazan product. Compound IV was found to be a better cytotoxic action against to the HEPG-2 hepatocellular carcinoma cell line, and it has been seen a strong activity against the colorectal carcinoma cell lines HCT-116 and MCF-7 mammary gland breast cancer cell lines. While the other three compounds I, II, and III showed a moderate cytotoxic action to high action against the same cell lines.

Schistosoma japonicumsja-let-7 Inhibits the Growth of Hepatocellular Carcinoma Cells via Cross-Species Regulation of Col1α2.

Liver fibrosis, a critical precursor to hepatocellular carcinoma (HCC), results from chronic liver injury and significantly contributes to HCC progression. Schistosomiasis, a neglected tropical disease, is known to cause liver fibrosis; however, this process can be modulated by schistosome-derived miRNAs. Previous studies from our laboratory have demonstrated that Schistosoma japonicum extracellular vesicles (EVs) deliver sja-let-7 to hepatic stellate cells, leading to the inhibition of Col1α2 expression and alleviation of liver fibrosis. Given the well-documented antifibrotic and antiproliferative properties of the let-7 miRNA family, this study aims to preliminarily investigate the effects of the sja-let-7/Col1α2 axis on BALB/c mice and HCC cell line SNU387, providing a basis for the potential application of parasite-derived molecules in HCC therapy. In the present study, schistosome-induced fibrosis datasets were analyzed to identify the role of Col1α2 in extracellular matrix organization. Pan-cancer analysis revealed that Col1α2 is upregulated in various cancers, including HCC, with significant associations with immune cell infiltration and clinical parameters, highlighting its diagnostic importance. Functional assays demonstrated that transfection with sja-let-7 mimics significantly reduced Col1α2 expression, inhibited HCC cell proliferation, migration, and colony formation. These findings suggest that sja-let-7, by targeting Col1α2, has the potential to serve as a therapeutic agent in HCC treatment. This study indicates the pivotal role of Col1α2 in liver fibrosis and HCC, and the promising therapeutic application of helminth-derived miRNAs.

CNDP1 Suppresses the Malignant Behavior of Hepatoma Cell via Restricting PI3K-AKT-mTOR Activation.

Hepatocellular carcinoma (HCC) is a global health problem with increasing morbidity and mortality, and exploring the diagnosis and treatment of HCC at the gene level has been a research hotspot in recent years. In this paper, a series of differentially expressed genes were found from the biochip related to HCC by bioinformatic analysis, then CNDP1 was finally selected for in-depth study according to the function and research progress of each gene. As the rate-limiting enzyme of carnosine hydrolysis, CNDP1 participates in the progress of many diseases, but its function has not been revealed in HCC. In the follow-up study, the low expression of CNDP1 in liver cancer tissues and cells was verified, then the pcDNA3.1-CNDP1 was used to improve the expression level of CNDP1 in HCC cell lines. Furthermore, this paper found that CNDP1 overexpression could significantly suppress cell prolifer-ation, migration, and invasion of HCC cell lines. Mechanismly, the GeneMANIA database predicted that CNDP1 could interact with various proteins that regulate the PI3K-AKT-mTOR signaling pathway, which is overactivated in HCC. And this study showed that CNDP1 overexpression could effectively inhibit the activation of PI3K-AKT-mTOR signaling pathways, more significantly, inhibition of PI3K-AKT-mTOR signaling pathway could disrupt the anti-cancer effect of CNDP1 on HCC. In conclusion, we confirmed that CNDP1 was lowly expressed in HCC tissues and cells, and had potential anti-cancer activity. This discovery will lay a cytological foundation for expanding the biological function of CNDP1 and the diagnosis and treatment of HCC in the future.

Pathobiological characterization of a novel duck picornavirus in duck in China

A novel strain of duck picornavirus was isolated from duck tissue in Taian, Shandong Province, in 2017 in our laboratory. The virus was amplified in specific-pathogen-free (SPF) chicken embryos, purified and then analyzed by whole genome sequencing, which revealed a new duck-derived small RNA virus that was designated as Duck/FC22/China/2017 (FC22, GenBank accession no. MN102111) based on its genome structure and phylogenetic relationship. An in-depth study revealed that the virus grew well on the Leghorn male hepatoma (LMH) cell line. After propagation of the virus, SPF ducks were inoculated for pathogenicity tests, and their mental state, growth and development were observed after inoculation; the ducks were dissected to observe the organs and histopathological changes. A TaqMan fluorescence quantitative PCR method was utilized to detect the proliferation and shedding patterns of the virus within the ducks, while the SYBR Green I fluorescence quantitative PCR method was used to assess cytokine expression levels in the organs. The results showed that following inoculation with the FC22 strain, the mental status of the SPF ducks remained unchanged. Mild oedema was observed in some tissue organs during dissection; however, no pathological changes, such as congestion or degeneration, were noted. Histopathological analysis revealed cellular necrosis in organs, including the heart, liver, and bursa of Fabricius, as well as a reduced volume and deep staining of certain neurons in the cerebrum. Following infection, the virus titres in various organs and in cloacal swabs of the SPF ducks peaked on the first day before gradually decreasing daily. By the 10th d, the virus titres in all organs had decreased to less than 101 copies/μL. Additionally, notable alterations were observed in the expression levels of the cytokines IFNα, IL6, IL10, and TNFα. This indicates that the FC22 strain does not cause significant disease in ducks. This report presents the initial study on a recently discovered picornavirus, offering a thorough and methodical examination of its pathogenic characteristics and provides a reference for the clinical evaluation and scientific strategies for the prevention and treatment of this particular picornavirus.

AURKB targets DHX9 to promote hepatocellular carcinoma progression via PI3K/AKT/mTOR pathway.

Aurora kinase B (AURKB) is known to play a carcinogenic role in a variety of cancers, but its underlying mechanism in liver cancer is unknown. This study aimed to investigate the role of AURKB in hepatocellular carcinoma (HCC) and its underlying molecular mechanism. Bioinformatics analysis revealed that AURKB was significantly overexpressed in HCC tissues and cell lines, and its high expression was associated with a poorer prognosis in HCC patients. Furthermore, downregulation of AURKB inhibited HCC cell proliferation, migration, and invasion, induced apoptosis, and caused cell cycle arrest. Moreover, AURKB downregulation also inhibited lung metastasis of HCC. AURKB interacted withDExH-Box helicase 9(DHX9) and targeted its expression in HCC cells. Rescue experiments further demonstrated that AURKB targeting DHX9 promoted HCC progression through the PI3K/AKT/mTOR pathway. Our results suggest that AURKB is significantly highly expressed in HCC and correlates with patient prognosis. Targeting DHX9 with AURKB promotes HCC progression via the PI3K/AKT/mTOR pathway.

ALDH1L2 drives HCC progression through TAM polarization

Background & AimsDysregulation of one-carbon metabolism is considered an early hallmark of mitochondrial dysfunction and cancer metabolism. ALDH1L2 belongs to the aldehyde dehydrogenase family and plays an important role in tumor progression. However, little is known about the precise role and underlying mechanisms of ALDH1L2 in hepatocellular carcinoma (HCC). MethodsImmunohistochemistry, Western blotting and immunofluorescence staining were used to evaluate ALDH1L2 expression in HCC samples (n=90) and cell lines (n=9). A series of in vitro and in vivo assays were performed to explore the role and molecular mechanism of ALDH1L2 in HCC progression. ResultsALDH1L2 upregulation is associated with poor prognosis in HCC (HR 1.923; 95% CI 1.03-3.59; p=0.04). ALDH1L2 promotes tumor cell proliferation and metastasis by activating NRF2/IL-6/STAT3 signaling. ALDH1L2 promotes mitochondrial respiration, increases ATP production and protects HCC cells from reactive oxygen species (ROS)-induced cellular damage via NRF2 stabilization. NRF2 also directly binds to the ALDH1L2 promoter and increases ALDH1L2 transcription, thereby establishing a positive feedback loop to maintain the function of ALDH1L2. The interaction between tumor-associated macrophages (TAMs) and ALDH1L2-overexpressing HCC cells further promotes HCC progression. In addition, ALDH1L2 knockdown enhances the anti-HCC activity of the tyrosine kinase inhibitor sorafenib. ConclusionsThese findings provide the first evidence indicating that ALDH1L2 is directly involved in tumor progression by interacting with TAMs through the Jak2/STAT3 signaling pathway and that ALDH1L2 may be a target molecule for HCC therapy. Impact and implicationsThis research highlights that ALDH1L2 could serve as a predictive and prognostic marker in HCC. We found that a positive feedback loop between ALDH1L2 and NRF2 promotes HCC progression by activating the IL-6/Jak2/STAT3 signaling axis and TAM polarization. In addition, we found that ALDH1L2 knockdown enhances the anti-HCC effect of sorafenib.

MiR-424-5p suppresses tumor growth and progression by directly targeting CHEK1 and activating cell cycle pathway in Hepatocellular Carcinoma

ObjectivesThe aim of this study is to elucidate the functional mechanism of the miRNA-424-5p/CHEK1 pathway in hepatocellular carcinoma (HCC), thereby offering novel insights for the development of targeted therapeutic strategies for HCC. MethodsWe employed a combination of bioinformatics analysis and data from the GEO to construct a regulatory network between miRNA and mRNA. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to assess the expression levels of miR-424-5p and CHEK1. Protein expression of CHEK1 was determined using Western blot analysis. The targeting relationship between miR-424-5p and CHEK1 was validated through a dual-luciferase reporter assay. Furthermore, the effects of miR-424-5p on HCC cell proliferation, migration, and invasion were evaluated using the Cell Counting Kit-8 assay, wound healing assay, and Transwell invasion assay, respectively. Apoptosis of HCC cells was measured by flow cytometry. ResultsBioinformatics analysis revealed that miR-424-5p was significantly downregulated, while CHEK1 was upregulated respectively in GEO dataset. Furthermore, this inverse expression pattern was observed in both HCC tissues and cell lines. Specifically, downregulation of miR-424-5p was found to promote the proliferation, migration, and invasion of HCC cells, while also inhibiting their apoptosis. The dual-luciferase reporter assay confirmed a direct targeting relationship between miR-424-5p and CHEK1. Inhibition of miR-424-5p was shown to counteract the suppressive effects on HCC cell proliferation, migration, and invasion that result from CHEK1 silencing. Additionally, experimental verification indicated that the activation of the cell cycle pathway is implicated in the oncogenic function of miR-424-5p/CHEK1 in HCC. ConclusionsThe present study demonstrates that miR-424-5p exerts a suppressive effect on HCC cell proliferation, migration, and invasion by downregulating the expression of CHEK1. This finding may offer a theoretical foundation for improving the prognosis and developing novel therapeutic strategies for HCC patients.

Detection of low-copy proteins in proteomic studies: issues and solutions.

Detection of low-copy proteins in complex biological samples is one of the most important issues of modern proteomics. The main reason for inefficient detection of low protein concentrations is the insufficient sensitivity of mass spectrometric detectors and the high dynamic range of protein concentrations. In this study we have investigated the possibilities and limitations of a targeted mass spectrometric analysis using the reconstructed system of standard proteins UPS1 (Universal Proteomic Standard 1) as an example. The study has shown that the sensitivity of the method is affected by the concentration of target proteins of the UPS1 system, as well as by a high level of biological noise modelled by proteins of whole E. coli cell lysate. The limitations of the method have been overcome by concentrating and pre-fractionating the sample peptides in a reversed phase chromatographic system under alkaline elution conditions. Proteomic analysis of the biological sample (proteins of the human hepatocellular carcinoma cell line HepG2 encoded by genes of human chromosome 18) showed an increase in the sensitivity of the method as compared to the standard targeted mass spectrometric analysis. This culminated in registration of 94 proteins encoded by genes located on human chromosome18.

Tumor-Derived Immunoglobulin-Like Transcript 4 Promotes Postoperative Relapse via Inducing Vasculogenic Mimicry through MAPK/ERK Signaling in Hepatocellular Carcinoma

Traditional anti-angiogenesis drugs are usually unsatisfactory in hepatocellular carcinoma (HCC) treatment. Therefore, it is urgent to find new precise therapeutic targets and further develop more effective drugs for the treatment of HCC. Vasculogenic mimicry (VM) is different from classical endothelium-dependent angiogenesis and associated with poor prognosis in patients with malignant tumor. However, the mechanism underlying VM occurrence is complex and not fully defined. Immunoglobulin-like transcript (ILT) 4, as a negative regulator of immune response, was recently found expressed in many solid tumors. However, whether and how ILT4 regulate VM remains unclear. In this study, we found VM enriched in HCC tissues especially in tissues from patients who experience relapse within 5 years after surgery. Similarly, ILT4 expression level was also higher in HCC tissues from patients who experience relapse within 5 years after surgery. Linear regression analysis revealed a positive correlation between the expression of ILT4 and VM density. Further, Overexpression/knockdown of ILT4 expression upregulated/downregulated VM related marker, three-dimensional tube formation, the migration and invasion of HCC cell lines in vitro. Mechanistic studies showed that ILT4 promoted VM formation via MAPK/ERK signaling. In conclusion, this study provides a rationale and mechanism for ILT4-mediated postoperative relapse via inducing VM in HCC. The related molecular pathways can be used as novel therapeutic targets for the inhibition of HCC angiogenesis and postoperative relapse.

P11-07 In vitro effects of polystyrene microplastics, alone or in combination with environmental pollutants, on viability and lipid content of a human hepatocarcinoma cell line

P11-07 In vitro effects of polystyrene microplastics, alone or in combination with environmental pollutants, on viability and lipid content of a human hepatocarcinoma cell line

Kinesin Family Member C1: Function in liver hepatocellular carcinoma and potential target for chemotherapeutic

MiR-105 exerts inhibitory effects on the development and progression of various cancers, including breast cancer, lung cancer, and gastric cancer. Through GEO data analysis, we observed decreased expression of miR-105 in liver cancer tissues compared to adjacent tissues. Furthermore, miR-105 downregulates KIFC1 expression levels by targeting its 3' UTR. KIFC1 (Kinesin Family Member C1), a Protein Coding gene, may play a role in mitotic metaphase plate polymerization and mitotic spindle assembly. However, our findings suggest that this gene could serve as a potential chemotherapeutic target for Liver hepatocellular carcinoma (LIHC). We obtained the LIHC dataset from the TCGA database and genotype Tissue Expression Project (GTEx) normal tissue data for differential analysis. Additionally, we utilized the cBioPortal database, tumor immune single-cell center (TISCH) database, gene set enrichment analysis (GSEA), and R software to investigate the possible functions and mechanisms of KIFC1. These findings were further validated through experiments such as immunohistochemistry and wound healing assays. Our results indicate that KIFC1 might be involved in DNA repair and cell cycle regulation in LIHC cells which subsequently impacts tumor cell proliferation; moreover, miR-105 influences hepatoma cell line proliferation via its interaction with KIFC1. Collectively, these results highlight the potential therapeutic significance of targeting KIFC1 for chemotherapy treatment in LIHC patients.

A necroptosis-regulated model from single-cell analysis that predicts survival and identifies the Pivotal role of MAGEA6 in hepatocellular carcinoma

ObjectiveHepatocellular carcinoma (HCC) ranks as the third leading cause of cancer-related deaths, constituting 75%–85 % of all primary liver cancers. The objective of this study was to develop a necroptosis-related gene signature using single-cell and bulk RNA sequencing to predict HCC patient prognoses. MethodsA total of 25 key necroptosis regulators were identified from previous literature. We evaluated the necroptosis scores of different cell types using single-cell sequencing data from HCC and analyzed 168 necroptosis-related genes. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset served as the training set for establishing a novel necroptosis-related gene risk signature, employing univariate and multivariate Cox regression analyses. Additionally, the study examined the model's relevance in immunity and immunotherapy, and predicted chemosensitivity in HCC patients based on the gene signature. The key genes were validated by the biological experiments. ResultsCompared to other cell types, hepatoma cells displayed the lowest necroptosis scores. A new six-gene necroptosis-related signature (S100A11, MAGEC2, MAGEA6, CTP2C9, SOX4, AKR1B10) was developed using the TCGA database and validated in the ICGC database. Patients in the high-risk category had poorer prognoses, with the risk score serving as an independent prognostic indicator beyond other clinical factors. These high-risk patients also exhibited greater immune infiltration but demonstrated a weaker anti-tumor response due to elevated expression of immune checkpoints. Pathways involving hypoxia, glycolysis, and P53, as well as the frequency of P53 somatic mutations, were notably heightened in the high-risk group. Additionally, the six genes in the model showed significantly different mRNA expression in hepatoma cell lines compared to normal hepatocytes, with the role of MAGEA6 in liver cancer being elucidated through critical experiments. ConclusionsThis study successfully developed a six-gene necroptosis-related signature to predict prognoses in HCC patients. It further explored the roles of necroptosis in hepatoma cells and the tumor microenvironment.

Identification of a novel 10-hydroxyevodiamine prodrug as a potent topoisomerase inhibitor with improved aqueous solubility for treatment of hepatocellular carcinoma

Natural product evodiamine (Evo) and its synthetic derivatives represent an attractive dual Topo 1/2 inhibitors with broad-spectrum antitumor efficacy. However, the clinical applications of these compounds have been impeded by their poor aqueous solubility. Herein, a series of water-soluble 10-substituted-N(14)-phenylevodiamine derivatives were designed and synthesized. The most potent compound 45 featuring a quaternary ammonium salt fragment achieved robust aqueous solubility and nanomolar potency against a panel of human hepatoma cell lines Huh7, HepG2, SK-Hep-1, SMMC-7721, and SMMC-7721/DOX (doxorubicin-resistant cell). Further studies revealed that 45 could inhibit Topo 1 and Topo 2, induce apoptosis, arrest the cell cycle at the G2/M stage and inhibit the migration and invasion. Compound 45 exhibited potent antitumor activity (TGI = 51.1 %, 10 mg/kg) in the Huh7 xenograft model with acceptable safety profile. In addition, a 21-day long-term dose toxicity study confirmed that the maximum tolerated dose of compound 45 was 20 mg/kg. Overall, this study presented a promising Evo-derived candidate for the treatment of hepatocellular carcinoma.

Knockdown and Overexpression Experiments to Investigate the Inhibitory Mechanism of Fuzheng Xiaozheng Prescription, an Effective Chinese Herbal Formula for the Clinical Treatment of Hepatocellular Carcinoma.

Fuzheng Xiaozheng prescription (FZXZP) is an effective formula for the treatment of different kinds of chronic liver diseases. However, its potential molecular mechanisms in treating hepatocellular carcinoma (HCC) have not been investigated thoroughly. The aim of this study is to elucidate the targets and intrinsic mechanisms of FZXZP and their active components for the treatment of HCC. The efficacy of FZXZP against HCC was clarified through a rat HCC model and HCC cell culture. Network pharmacology and molecular docking were utilized to predict the mechanism of action and effector components of FZXZP. The key mechanism and targets were verified by the construction of overexpression and knockout cell models. The results showed that FZXZP greatly delayed the development of HCC in vivo experiments, as evidenced by biochemical evaluations, H&E analyses and growth inhibition of HCC. FZXZP dramatically inhibited cell viability and proliferative capacity and induced the apoptosis of hepatoma cells in vitro. Moreover, network pharmacology analyses demonstrated that the EGFR family and apoptosis-related targets were found to be the most significant in bioinformatics analysis. Furthermore, the EGFR/STAT3 signal axis might be the most likely target of FZXZP in anti-HCC due to the fact that it could be down-regulated by FZXZP with an upward trend of Bax, Caspase-3, Caspase-8, Caspase-9 and an inverse trend of Bcl2. Importantly, the above targeted signal axis was finally validated by our knockdown and overexpression analyses. Meanwhile, flow cytometry and TUNEL staining also revealed that FZXZP significantly induced apoptosis in the EGFR-overexpressing HCC cell line. The molecular docking results revealed that the key effector components of FZXZP that exerted the above regulatory roles were wogonin and glycitein. All of these results suggest that FZXZP could significantly delay HCC development by inhibiting proliferation and promoting apoptosis of HCC cells, and the EGFR/STAT3 signal axis might be a critical signal axis of FZXZP in suppressing HCC progression.

Construction of a prognostic model for disulfidptosis-related long noncoding RNAs in R0 resected hepatocellular carcinoma and analysis of their impact on malignant behavior

BackgroundDisulfidptosis is an emerging form of cellular death resulting from the binding of intracellular disulfide bonds to actin cytoskeleton proteins. This study aimed to investigate the expression and prognostic significance of hub disulfidptosis-related lncRNAs (DRLRs) in R0 resected hepatocellular carcinoma (HCC) as well as their impact on the malignant behaviour of HCC cells.MethodsA robust signature for R0 resected HCC was constructed using least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression and was validated in an independent internal validation cohort to predict the prognosis of R0 HCC patients. Comprehensive bioinformatics analysis was performed on the hub DRLRs (KDM4A-AS1, MKLN1-AS, and TMCC1-AS1), followed by experimental validation using quantitative real-time polymerase chain reaction (qRT‒PCR) and cellular functional assays.ResultsThe signature served as an independent prognostic factor applicable to R0 HCC patients across different age groups, tumour stages, and pathological characteristics. Gene Ontology (GO) and gene set enrichment analysis (GSEA) revealed hub pathways associated with this signature. The high-risk group presented an increased abundance of M0 macrophages and activated memory CD4 T cells as well as elevated macrophage and major histocompatibility complex (MHC) class I expression. High-risk R0 HCC patients also presented increased tumour immune dysfunction and exclusion scores (TIDEs), mutation frequencies, and tumour mutational burdens (TMBs). Drug sensitivity analysis revealed that high-risk patients were more responsive to drugs, including GDC0810 and osimertinib. High expression levels of the three hub DRLRs were detected in R0 HCC tissues and HCC cell lines. Functional assays revealed that the three hub DRLRs enhanced HCC cell proliferation, migration, and invasion.ConclusionsA signature was constructed on the basis of three DRLRs, providing novel insights for personalized precision therapy in R0 HCC patients.Graphical

Reduced aggregation of the Leghorn Male Hepatoma (LMH) cell line in suspension by supplementing dextran sulfate in the media.

The study aimed to improve the efficiency of Leghorn Male Hepatoma (LMH) cells for animal virus vaccine production by transitioning from adherent to suspension culture and evaluating the effects of dextran sulfate (DS) on preventing cell aggregation. The goal was to enhance cell growth, viability, and glucose metabolism and to develop efficient suspension-adapted LMH cells for large-scale vaccine production. LMH cells previously cultured in an adherent state were transferred to 125 mL Erlenmeyer flasks to conduct suspension culture. Cell culture performance, including cell density, viability, and glucose metabolism, during the cultures was measured, along with an assessment of cell aggregation. Additionally, mRNA expression levels of genes associated with cell adhesion and apoptosis were monitored. DS supplementation in suspension culture enhanced cell viability and growth, with higher cell densities and viabilities compared to control media. Additionally, DS supplementation reduced glucose consumption and waste production, indicating improved metabolic efficiency. DS also delayed cell aggregation, possibly by downregulating integrin expression and promoting anti-apoptotic gene expression. However, even after 2 months, cell aggregation persisted in both control and DS-supplemented cultures, suggesting further optimization is needed for LMH cell adaptation to suspension culture. DS supplementation in LMH cell suspension cultures led to notable improvements in cell growth, viability, and glucose metabolism, while also decreasing the cell aggregation.

Periplocin improves the sensitivity of oxaliplatin-resistant hepatocellular carcinoma cells by inhibiting M2 macrophage polarization.

The aim of this research was to investigate the impact of periplocin (PPLN) on oxaliplatin (OXA) resistance in hepatocellular carcinoma (HCC) cells and offer insights for improving clinical treatment of HCC. The IC50 value of HCC cell lines against OXA was detected by the CCK-8 assay, and an OXA-resistant HepG2 cell line (HepG2/OXA) was constructed. THP-1 cells were induced into M1 or M2 macrophages, and M2 macrophage-conditioned medium (M2-CM) was prepared. M1 and M2 macrophage polarization were detected using RT-qPCR and flow cytometry. CCK-8, EdU staining, clone formation assay, flow cytometry, and western blotting were used to assess the proliferation and apoptosis of HepG2/OXA cells treated with PPLN and M2-CM. Additionally, a nude mouse subcutaneous graft tumor model was constructed. PPLN enhanced the sensitivity of HepG2/OXA cells to OXA, reduced their clone-forming ability, and promoted their apoptosis. Notably, PPLN hindered M0 macrophage polarization to M2 macrophages, while M1 polarization remained unaffected. The proliferation-inhibiting and apoptosis-promoting effects of OXA+PPLN on HepG2/OXA cells were significantly attenuated by the addition of M2-CM, suggesting that PPLN improves the OXA sensitivity of HepG2/OXA cells by hindering M2 macrophage polarization. Furthermore, PPLN inhibited M2 macrophage polarization and improved the OXA sensitivity of HepG2/OXA cells in vivo. In conclusion, PPLN inhibited the proliferation of HepG2/OXA cells, promoted their apoptosis, and inhibited M2 macrophage polarization both in vivo and in vitro, which in turn enhanced the OXA sensitivity of HepG2/OXA cells.

MiR-29a-laden extracellular vesicles efficiently induced apoptosis through autophagy blockage in HCC cells

BackgroundIn spite of significant advancements in theraputic modalities for hepatocellular carcinoma (HCC), there is still a high annual mortality rate with a rising incidence. Major challenges in the HCC clinical managment are related to the development of therapy resistance, and evasion of tumor cells apoptosis which leading unsatisfactory outcomes in HCC patients. Previous investigations have shown that autophagy plays crucial role in contributing to drug resistance development in HCC. Although, miR-29a is known to counteract authophagy, increasing evidence revealed a down-regulation of miR-29a in HCC patients which correlates with poor prognosis. Beside, evidences showed that miR-29a serves as a negative regulator of autophagy in other cancers. In the current study, we aim to investigate the impact of miR-29a on the autophagy and apoptosis in HCC cells using extracellular vesicles (EVs) as a natural delivery system given their potential in the miRNA delivery both in vitro and in vivo. MethodHuman Wharton’s Jelly mesenchymal stromal cell-derived extracellular vesicles were lately isolated through 20,000 or 110,000 × g centrifugation (EV20K or EV110K, respectively), characterized by western blot (WB), scanning electron microscopy (SEM), and dynamic light scattering (DLS). miR-29a was subsequently loaded into these EVs and its loading efficiency was evaluated via RT-qPCR. Comprehensive in vitro and in vivo assessments were then performed on Huh-7 and HepG2 cell lines. ResultsEV20K-miR-29a treatment significantly induces cell apoptosis and reduces both cell proliferation and colony formation in Huh-7 and HepG2 cell lines. In addition, LC3-II/LC3-I ratio was increased while the expression of key autophagy regulators TFEB and ATG9A were downregulated by this treatment. These findings suggest an effective blockade of autophagy by EV20K-miR-29a leading to apoptosis in the HCC cell lines through concomitant targeting of critical mediators within each pathway.