Inhibited Hepatocellular Carcinoma Cell Growth Research Articles

Development and Characterization of Benzoselenazole Derivatives as Potent and Selective c-MYC Transcription Inhibitors.

Developing c-MYC transcription inhibitors that target the G-quadruplex has generated significant interest; however, few compounds have demonstrated specificity for c-MYC G-quadruplex and cancer cells. In this study, we designed and synthesized a series of benzoazole derivatives as potential G-quadruplex ligand-based c-MYC transcription inhibitors. Surprisingly, benzoselenazole derivatives, which are rarely reported as G-quadruplex ligands, demonstrated greater c-MYC G-quadruplex selectivity and cancer cell specificity compared to their benzothiazole and benzoxazole analogues. The most promising compound, benzoselenazole m-Se3, selectively inhibited c-MYC transcription by specifically stabilizing the c-MYC G-quadruplex. This led to selective inhibition of hepatoma cell growth and proliferation by affecting the MYC target gene network, as well as effective tumor growth inhibition in hepatoma xenografts. Collectively, our study demonstrates that m-Se3 holds significant promise as a potent and selective inhibitor of c-MYC transcription for cancer treatment. Furthermore, our findings inspire the development of novel selenium-containing heterocyclic compounds as c-MYC G-quadruplex-specific ligands and transcription inhibitors.

Suppressing circIDE/miR-19b-3p/RBMS1 axis exhibits promoting-tumour activity through upregulating GPX4 to diminish ferroptosis in hepatocellular carcinoma

ABSTRACT Ferroptosis is a newly characterized form of iron-dependent non-apoptotic cell death, which is closely associated with cancer progression. However, the functions and mechanisms in regulation of escaping from ferroptosis during hepatocellular carcinoma (HCC) progression remain unknown. In this study, we reported that the RNA binding motif single stranded interacting protein 1 (RBMS1) participated in HCC development，and functioned as a regulator of ferroptosis. Clinically, the downregulation of RBMS1 occurred in HCC tissues, and low RBMS1 expression was associated with worse HCC patients survival. Mechanistically, RBMS1 overexpression inhibited HCC cell growth by attenuating the expression of glutathione peroxidase 4 (GPX4)and further facilitated ferroptosis in vitro and in vivo. More importantly, a novel circIDE (hsa_circ_0000251) was identified to elevate RBMS1 expression via sponging miR-19b-3p in HCC cells. Collectively, our findings established circIDE/miR-19b-3p/RBMS1 axis as a regulator of ferroptosis, which could be a promising therapeutic target and prognostic factor.

The Comprehensive Effects of Carassius auratus Complex Formula against Lipid Accumulation, Hepatocarcinogenesis, and COVID-19 Pathogenesis via Stabilized G-Quadruplex and Reduced Cell Senescence.

Carassius auratus complex formula (CACF) is a traditional Chinese medicine known for its antidiabetic effects. Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide, and there are currently no effective therapies for advanced HCC. This study explores the comprehensive effects and possible mechanisms of CACF on HCC. The results show that CACF reduces the viability of hepatoma cells in vitro, while benefiting normal hepatocytes. In addition, CACF inhibits hepatoma cell growth in a zebrafish xenotransplantation model and decreases lipid accumulation, represses inflammation and cell proliferation markers in fatty acid translocase (CD36) transgenic zebrafish, and inhibits the expression of cell proliferation and β-catenin downstream targets in telomerase (tert) transgenic zebrafish models. Ingenuity Pathway Analysis reveals that CACF exerts multiple functions, including reduction of inflammation and inhibition of lipid transporter and PPAR signaling pathway. Surprisingly, CACF also regulates the expression of genes and reduces coronavirus infection and pathogenesis in a zebrafish model. CACF treatment is validated to regulate the expression of genes for anti-coronavirus activity. Mechanistically, CACF stabilizes G-quadruplex and reduces cell senescence associated β-galactosidase activity. In summary, CACF may be a promising therapeutic agent with multiple functions including anticancer, anti-inflammation, and anti-microorganisms in a zebrafish model.

DKK1-CKAP4 signal axis promotes hepatocellular carcinoma aggressiveness.

Hepatocellular carcinoma (HCC) is the most prevalent malignant liver neoplasm. Despite the advances in diagnosis and treatment, the prognosis of HCC patients remains poor. Cytoskeleton-associated membrane protein 4 (CKAP4) is a receptor of the glycosylated secretory protein Dickkopf-1 (DKK1), and the DKK1-CKAP4 axis is activated in pancreatic, lung, and esophageal cancer cells. Expression of DKK1 and CKAP4 has been examined in HCC in independent studies that yielded contradictory results. In this study, the relationship between the DKK1-CKAP4 axis and HCC was comprehensively examined. In 412 HCC cases, patients whose tumors were positive for both DKK1 and CKAP4 had a poor prognosis compared to those who were positive for only one of these markers or negative for both. Deletion of either DKK1 or CKAP4 inhibited HCC cell growth. In contrast to WT DKK1, DKK1 lacking the CKAP4 binding region did not rescue the phenotypes caused by DKK1 depletion, suggesting that binding of DKK1 to CKAP4 is required for HCC cell proliferation. Anti-CKAP4 Ab inhibited HCC growth, and its antitumor effect was clearly enhanced when combined with lenvatinib, a multikinase inhibitor. These results indicate that simultaneous expression of DKK1 and CKAP4 is involved in the aggressiveness of HCC, and that the combination of anti-CKAP4 Ab and other therapeutics including lenvatinib could represent a promising strategy for treating advanced HCC.

Squalene epoxidase promotes hepatocellular carcinoma development by activating STRAP transcription and TGF-β/SMAD signalling.

Squalene epoxidase (SQLE) is a key enzyme involved in cholesterol biosynthesis, but growing evidence also reveals that SQLE is abnormally expressed in some types of malignant tumours, even though the underlying mechanism remains poorly understood. Bioinformatics analysis and RNA sequencing were applied to detect differentially expressed genes in clinical hepatocellular carcinoma (HCC). MTT, colony formation, AnnexinV-FITC/PI, EdU, wound healing, transwell, western blot, qRT-PCR, IHC, F-actin, RNA-sequencing, dual-luciferase reporters, and H&E staining were used to investigate the pharmacological effects and possible mechanisms of SQLE. SQLE expression was specifically elevated in HCC, correlating with poor clinical outcomes. SQLE significantly promoted HCC growth, epithelial-mesenchymal transition, and metastasis both in vitro and in vivo. RNA sequencing and functional experiments revealed that the protumourigenic effect of SQLE on HCC was closely related to the activation of TGF-β/SMAD signalling, but the stimulatory effect of SQLE on TGF-β/SMAD signalling and HCC development is critically dependent on STRAP. SQLE expression is well correlated with STRAP in HCC, and further, to amplify TGF-β/SMAD signalling, SQLE even transcriptionally increased STRAP gene expression mediated by AP-2α. Finally, as a chemical inhibitor of SQLE, NB-598 markedly inhibited HCC cell growth and tumour development. Taken together, SQLE serves as a novel oncogene in HCC development by activating TGF-β/SMAD signalling. Targeting SQLE could be useful in drug development and therapy for HCC.

A novel antimicrobial peptide M1-8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells.

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1-8 (M1-8), a small anti-tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy-lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1-8-induced apoptosis. After exposure to M1-8, human hepatoma HepG2 cells rapidly co-localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1-8-treated HepG2 cells, autophagic protein content increased and the lysosome-autophagosome fusion was inhibited, suggesting that M1-8 can cause apoptosis through autophagy and lysosomes. This result indicates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1-8 in autophagy and lysosomes, which may facilitate the development of M1-8 as a potential cancer therapeutic agent.

Exosome-mediated circTTLL5 transfer promotes hepatocellular carcinoma malignant progression through miR-136-5p/KIAA1522 axis.

Exosomes have been recognized as messengers for intercellular communication in tumor microenvironment. Exosomal circRNAs are reported to be important in tumors. Here, this study identified the potential function of exosomal circular RNA tubulin tyrosine ligase like 5 (circTTLL5) in hepatocellular carcinoma (HCC) progression. The expression of circTTLL5, microRNA (miR)-136-5p and KIAA1522 was detected using qRT-PCR and Western blot assays. Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM) and Western blot. Cell proliferation, apoptosis and metastasis were investigated using cell counting kit-8, colony formation, flow cytometry, would healing, transwell and Western blot assays, respectively. The interaction between miR-136-5p and circTTLL5 or KIAA1522 was confirmed by dual-luciferase reporter and pull-down assays. In vivo experiment was performed using Xenograft models. CircTTLL5 was incorporated into exosomes and highly expressed in HCC tissues and cells. CircTTLL5 knockdown suppressed HCC cell proliferation and metastasis in vitro and impeded tumor growth in mice. CircTTLL5 could be delivered to recipient cells via exosomes, and treatment of circTTLL5-elevated exosomes could attenuate the anticancer effects of circTTLL5 knockdown on HCC in vitro and in vivo. Mechanically, circTTLL5 could sponge miR-136-5p, which controlled its down-stream target KIAA1522. MiR-136-5p inhibition reversed the effects of circTTLL5 knockdown on HCC cells. Besides that, miR-136-5p re-expression inhibited HCC cell growth and metastasis, which was abated by KIAA1522 overexpression. Exosomal circTTLL5 promoted HCC progression through miR-136-5p/KIAA1522 axis, suggesting that blockage of the exosome-mediated transfer of circTTLL5 might be a therapeutic target for HCC.

Overcoming the resistance of hepatocellular carcinoma to PD-1/PD-L1 inhibitor and the resultant immunosuppression by CD38 siRNA-loaded extracellular vesicles

ABSTRACT Extracellular vesicles (EVs) are promising tools for drug delivery across different biological barriers. Here, we evaluated the potential of EVs-mediated delivery of CD38 siRNA on the immunosuppression of hepatocellular carcinoma (HCC). EVs were isolated from bone marrow mesenchymal stem cell culture medium and loaded with CD38 siRNA to prepare EVs/siCD38. Loss-of-function assays were conducted to investigate the biological functions of EVs/siCD38 in HCC cells. Xenograft mouse models were performed for further validation. High CD38 expression was found in HCC. EVs/siCD38 inhibited CD38 enzyme activity, decreased adenosine production, and promoted macrophage repolarization to M1 type, thus inhibiting HCC cell growth and metastasis in vitro as well as tumor growth in mice. Mechanistically, CD38 was upregulated in mice resistant to PD-1/PD-L1 inhibitor and EVs/siCD38 reversed the resistance of tumor to PD-1/PD-L1 inhibitor in vivo. Our results provide functional evidence for the use of EV-mediated delivery of CD38 siRNA to prevent immunosuppression feature of HCC.

SMYD2 Promotes Hepatocellular Carcinoma Progression by Reprogramming Glutamine Metabolism via c-Myc/GLS1 Axis

Metabolic reprogramming, such as alterations in glutamine metabolism or glycolysis, is the hallmark of hepatocellular carcinoma (HCC). However, the underlying mechanisms are still incompletely elucidated. Previous studies have identified that methyltransferase SET and MYND domain-containing protein 2(SMYD2) is responsible for the pathogenesis of numerous types of cancer. Here, we innovatively uncover how SMYD2 regulates glutamine metabolism in HCC cells and promotes HCC progression. We identified that SMYD2 expression is upregulated in HCC tissues, which correlates with unfavorable clinical outcomes. Our in vitro and in vivo results showed that the depletion of SMYD2 inhibits HCC cell growth. Mechanistically, c-Myc methylation by SMYD2 increases its protein stability through the ubiquitin-proteasome system. We showed SMYD2 depletion destabilized c-Myc protein by increasing the conjugated K48-linked polyubiquitin chain. SMYD2 increased c-Myc expression and further upregulated glutaminase1 (GLS1), a crucial enzyme that catalyzes the conversion of glutamine to glutamic acid, in HCC cells. GLS1 plays an important role in SMYD2-mediated HCC progression and glutamine metabolism regulation. The knockdown of SMYD2 inhibited glutamine metabolism in HCC cells and overcame their chemoresistance to sorafenib. Collectively, our findings demonstrated a novel mechanism of how SMYD2 promotes HCC progression by regulating glutamine metabolism through the c-Myc/GLS1signaling, implicating the therapeutic potential of targeting SMYD2 in HCC patients.

Research progress on the P2X7 receptor in liver injury and hepatocellular carcinoma.

Purinergic ligand-gated ion channel 7 receptor (P2X7 receptor) is an adenosine triphosphate (ATP)-gated ion channel that is widely distributed on the surfaces of immune cells and tissues such as those in the liver, kidney, lung, intestine, and nervous system. Hepatocellular carcinoma (HCC) is one of the most common malignancies with increasing incidence and mortality. Although many treatments for liver cancer have been studied, the prognosis for liver cancer is still very poor. Therefore, new liver cancer treatments are urgently needed. P2X7 receptor activation can secrete proinflammatory factors through the P2X7 receptor-NLRP3 signaling pathway, thereby affecting the progression of liver injury. The P2X7 receptor may be a target for growth inhibition of HCC cells and may affect the invasion and migration of HCC cells through the PI3K/AKT and AMPK signaling pathways. In recent years, P2X7 receptor antagonists or inhibitors have attracted widespread attention as therapeutic targets for hepatocellular carcinoma and liver injury. Therefore, this review covers the basic concepts of the P2X7 receptor and role of the P2X7 receptor in liver cancer and liver injury, providing new potential therapeutic targets for hepatocellular carcinoma and liver injury.

DDX24 regulates the chemosensitivity of hepatocellular carcinoma to sorafenib via mediating the expression of SNORA18

ABSTRACT Sorafenib (SFN) is a multi-kinase inhibitor drug for the treatment of advanced hepatocellular carcinoma (HCC), but its limited efficacy is a major obstacle to the clinical outcomes of patients with HCC. We aimed to explore a novel molecular mechanism underlying the chemosensitivity of HCC to SFN, and to identify a promising therapeutic target for HCC treatment. In this study, bioinformatic analysis revealed that DDX24 was associated with poor survival in HCC cases, and significantly related to the pathways modulating tumor development. DDX24 regulated HCC cell proliferation and migration potentials. Moreover, reduction of DDX24 promoted the sorafenib-mediated inhibition of HCC cell growth and migration, the elevation of sorafenib-induced HCC cell apoptosis. DDX24 overexpression suppressed the inhibitory effect of SFN on cell proliferation and migration and reduced the apoptosis induced by SFN. Further, DDX24, combined with SFN treatment, presented a synergistic enhancement of the sensitivity of SFN to the growth and migration of HCC cells via AKT/ERK and the epithelial-mesenchymal transition (EMT) pathways, and that it modulated apoptosis via the caspase/PARP pathway. Mechanistically, SNORA18 served as a target gene for DDX24, regulating the chemosensitivity of sorafenib-treated HCC cells. Furthermore, SNORA18 knockdown or overexpression could partially reverse the inhibition or elevation of cell viability, colony formation and migration induced by DDX24 in sorafenib-treated HCC cells, respectively. Collectively, our results suggest that DDX24 regulates the chemosensitivity of HCC to SFN by mediating the expression of SNORA18, which may act as an effective therapeutic target for improving SFN efficiency in HCC treatment.

α-Fetoprotein fragment synergizes with sorafenib to inhibit hepatoma cell growth and migration and promote the apoptosis.

Alpha fetoprotein (AFP) is associated with hepatocellular carcinoma (HCC) by stimulating the proliferation, metastasis and drug resistance. The application of AFP fragments to inhibit the malignant behaviours induced by AFP is a new strategy for the treatment of HCC. In an effort to design, screen and discover drugs, we attempted to express different human AFP fragments (AFP220-609 , AFP390-609 and AFP460-609 ) in a Bac-to-Bac system. We found that the AFP390-609 fragment was highly expressed in the system. Then, we assessed the bioactivity of the fragment in the human liver cancer cell line Bel7402, and the results indicated that the AFP fragment synergized with sorafenib to inhibit the hepatoma cell growth and migration and promote the apoptosis. This study provides a method to produce significant AFP fragments to screen AFP inhibitors for use in HCC therapy.

Identification of the prognostic, diagnostic, and biological significance of the miR-148a-3p/cathepsin A axis in hepatocellular carcinoma.

A comprehensive analysis of the prognostic, diagnostic, and biological significance of miR-148a-3p and cathepsin A (CTSA) in hepatocellular carcinoma (HCC) was performed using bioinformatics algorithms with The Cancer Genome Atlas(TCGA) data. miR-148a-3p and CTSA gene expression in HCC tissues and nontumor specimens was analyzed using TCGA database with R software. CTSA staining analysis was validated using the Human Protein Atlas database. Prognostic, diagnostic, gene set enrichment, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and immune infiltration analyses were implemented using the TCGA database with R software. Based on TCGA data and our cohort populations, CTSA expression was significantly elevated in HCC tissues compared with nontumor specimens. A significant negative correlation between miR-148a-3p and CTSA was observed in the TCGA data and our cohort population. Mechanistically, CTSA was a direct gene target of miR-148a-3p. Both CTSA and miR-148a-3p could serve as prognostic and diagnostic indicators in HCC. miR-148a-3p expression was significantly and negatively correlated with the StromalScore, ImmuneScore, and ESTIMATEScore in patients with liver cancer. miR-148a-3p mimic-mediated apoptosis and the inhibition of HCC cell growth and migration were counteracted by CTSA overexpression. The miR-148a-3p/CTSAaxis was implicated in immune cell infiltration and carcinogenesis of HCC. miR-148a-3p and CTSA might be prospective molecular targets to enhance the potency of immunotherapy in HCC.

CircSOD2 Contributes to Tumor Progression, Immune Evasion and Anti-PD-1 Resistance in Hepatocellular Carcinoma by Targeting miR-497-5p/ANXA11 Axis.

Circular RNAs (circRNAs) can function as functional molecules in hepatocellular carcinoma (HCC). Herein, circRNA superoxide dismutase 2 (circSOD2) was researched in HCC progression and immune system. The real-time polymerase chain reaction (qRT-PCR) was used for quantification of circSOD2, microRNA-497-5p (miR-497-5p) and Annexin A11 (ANXA11). Cell assays were performed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) and colony formation assays for proliferation, flow cytometry for apoptosis and cell cycle, wound healing assay for migration and transwell assay for migration/invasion. ANXA11 and metastatic protein levels were measured by western blot. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to analyze target binding. CD8+ T cell immunity was assessed by Immunohistochemistry (IHC) assay, and the effect of circSOD2 on programmed cell death 1 (PD-1) immune checkpoint inhibitors (anti-PD-1) therapy was evaluated by mice xenograft assay. CircSOD2 was upregulated in HCC tissues and cells. Knockdown of circSOD2 resulted in HCC cell growth inhibition, apoptosis promotion, cell cycle arrest and metastasis suppression. Mechanically, circSOD2 promoted HCC development by acting as a miR-497-5p sponge and miR-497-5p played a tumor-inhibitory role in HCC cells by targeting ANXA11. Moreover, circSOD2 induced upregulation of ANXA11 expression by interacting with miR-497-5p. Also, the promoting effects of circSOD2 on immune evasion and anti-PD-1 resistance were related to miR-497-5p/ANXA11 axis. This study elucidated the pivotal function of circSOD2 in HCC progression and immunosuppression by mediating miR-497-6p/ANXA11 axis. CircSOD2/miR-497-5p/ANXA11 axis was a novel view of circRNA research in HCC.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUNDThe phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIMTo confirm the effect of XHP on HCC and the possible mechanisms involved.METHODSThe chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTSThe following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSIONXHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

MicroRNA-148a Inhibits Hepatocellular Carcinoma Cell Growth via Epithelial-to-Mesenchymal Transition and PI3K/AKT Signaling Pathways by Targeting Death Receptor-5.

The purpose of this study was to investigate the role of microRNA-148a (miR-148a) in hepatocellular carcinoma (HCC) metastasis and explore its potential mechanism in HCC cells. Expression levels of miR-148a were measured using qRT-PCR in 120 HCC tissue samples and two HCC cell lines. Migration and invasion assays were used to determine the role of miR-148a in HCC cells. Flow cytometry was used to access the effect of miR-148a on cell cycle of HCC cells. Western blot was performed to analyze the effect of miR-148a on epithelial-to-mesenchymal transition (EMT) and PI3K/AKT signaling pathways in HCC cells. Luciferase reporter assay was conducted to explore the downstream targets and biological function of miR-148a in HCC cells. The results showed that level of miR-148a was significantly downregulated in both HCC tissue and plasma samples in HCC patients. A higher level of miR-148a was positively correlated with better survival time and prognosis of HCC patients. Transfection of miR-148a inhibited the proliferation, migration and invasion of HCC cell lines. Transfection of miR-148a arrested HCC cells at S phase and promoted apoptosis of HCC cells. Death receptor-5 (DR-5) was identified as a direct target of miR-148a in HCC cell lines. Western blot and qRT-PCR analyses showed that miR-148a upregulated EMT and downregulated PI3K/AKT signaling pathways in HCC cell lines. In conclusion, data in the current study indicate that miR-148a inhibits HCC cells growth via downregulation of EMT and PI3K/AKT signaling pathways by targeting death receptor. These data suggest that miR-148a may serve as a therapeutic target for HCC cancer therapy in the future.

Long non-coding RNA (lncRNA) CYTOR promotes hepatocellular carcinoma proliferation by targeting the microRNA-125a-5p/LASP1 axis

ABSTRACT This study investigated the function of long non-coding RNA (lncRNA) cytoskeleton regulator RNA (CYTOR) in hepatocellular carcinoma (HCC). In HCC, the expression of CYTOR and microRNA (miR)-125a-5p were measured by quantitative real-time PCR (qRT-PCR). The expression of actin skeletal protein 1 (LASP1) was evaluated by Western blot analysis. Flow cytometry assays, transwell assays, colony formation assay, and cell counting kit-8 (CCK-8) assay were used to evaluate the roles of miR-125a-5p and CYTOR in HCC cells. The target genes of CYTOR and miR-125a-5p were identified by bioinformatics analysis and Luciferase assay. CYTOR was upregulated in HCC cell lines, and knockdown of CYTOR inhibited HCC cell growth. MiR-125a-5p was downregulated in HCC cells and a target of CYTOR in regulating HCC progression. Furthermore, LASP1 was a downstream target of miR-125a-5p. Finally, CYTOR was found to be involved in HCC progression in vivo. CYTOR promotes HCC development by regulating the miR-125a-5p/LASP1 axis.

Estrogen receptor-α36 is involved in diallyl sulfide-induced inhibition of malignant growth of HepG2 and Huh7 hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly malignant disease that currently lacks effective treatment. Epidemiological studies have suggested the preventive role of raw garlic intake in different tumors, such as HCC. Although diallyl sulfide (DAS), the main component of garlic extracts, has been reported to inhibit the growth of HCC cells, the underlying mechanism remains elusive. This study aimed to investigate the inhibitory effect of DAS on the growth of HepG2 and Huh7 hepatocellular carcinoma cells and its underlying mechanism. HepG2 and Huh7 cells were treated with DAS and nude mice were intrahepatically injected with human HCC HepG2 cells and maintained with or without DAS administration for 28 days. MTS and clonogenic assays revealed that DAS inhibited the growth and clonogenicity of HepG2 and Huh7 hepatocellular carcinoma cells. Furthermore, DAS inhibited the growth of xenograft tumors accompanied by a decreased rate of pathological karyomitosis as observed by H&E staining. The expression levels of estrogen receptor-α36 (ER-α36) and epidermal growth factor receptor (EGFR) in HepG2 and Huh7 cells and in xenograft tumors derived from HepG2 cells after DAS treatment were detected by immunohistochemistry and western blotting. We found that DAS disrupted the positive regulatory loop between ER-α36 and EGFR, and decreased the phosphorylation of AKT at Ser 473 both in vivo and in vitro. DAS also induced cell apoptosis, as evidenced by Hoechst and TUNEL staining. Western blotting revealed activation of caspase3, increased BAX and decreased Bcl-2 expression. However, the ER-α36 expression knockdown attenuated DAS-induced ERK and AKT phosphorylation in HCC cells. DAS was also able to inhibit ER-α36-mediated activation of the MAPK/ERK signaling induced by estrogen. Thus, our results indicate that ER-α36 signaling is involved in DAS-induced inhibition of HCC cell growth both in vitro and in vivo.

Circular RNA circ_0073181 contributes to tumorigenesis by regulating protein tyrosine phosphatase receptor type E via miR-548p in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major world public problem in the world, with high morbidity and mortality rates. Circular RNA (circRNA) circ_0073181 has been reported to be related to HCC development. However, the mechanism of circ_0073181 in HCC is far from being addressed. Circ_0073181, microRNA-548p (miR-548p) and protein tyrosine phosphatase receptor type E (PTPRE) level were detected by real-time quantitative PCR (RT-qPCR). Cell proliferation, migration, invasion and apoptosis were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine, wound healing, transwell and flow cytometry assay. Protein levels of proliferating cell nuclear antigen, Bcl-2 related X protein (Bax) and PTPRE were examined by western blot assay. The binding relationship between miR-548p and circ_0073181 or PTPRE was predicted by circular RNA interactome and targetScan and then verified by a dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The biologic role of circ_0073181 on HCC tumor growth was examined by the xenograft tumor model in vivo. Circ_0073181 and PTPRE were upregulated, and miR-548p was decreased in HCC tissues and cells. Furthermore, circ_0073181 knockdown could boost proliferation, migration, invasion and repress apoptosis of HCC cells in vitro. The mechanical analysis suggested that circ_0073181 could regulate PTPRE expression by sponging miR-548p. In addition, circ_0073181 knockdown suppressed cell growth of HCC in vivo. Circ_0073181 silencing could inhibit HCC cell growth and metastasis partly by regulating the miR-548p/ PTPRE axis, providing a promising therapeutic target for the HCC treatment.

Scutellaria barbata D.Don and Oldenlandia diffusa (Willd.) Roxb crude extracts inhibit hepatitis-B-virus-associated hepatocellular carcinoma growth through regulating circRNA expression

Ethnopharmacological relevanceScutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. Aim of the studyThe aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. Materials and methodsWe determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet–visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. ResultsThe extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. ConclusionsThe extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.