Inhibits Hepatocellular Carcinoma Development Research Articles

SMYD5 is a ribosomal methyltransferase that catalyzes RPL40 lysine methylation to enhance translation output and promote hepatocellular carcinoma.

While lysine methylation is well-known for regulating gene expression transcriptionally, its implications in translation have been largely uncharted. Trimethylation at lysine 22 (K22me3) on RPL40, a core ribosomal protein located in the GTPase activation center, was first reported 27 years ago. Yet, its methyltransferase and role in translation remain unexplored. Here, we report that SMYD5 has robust in vitro activity toward RPL40 K22 and primarily catalyzes RPL40 K22me3 in cells. The loss of SMYD5 and RPL40 K22me3 leads to reduced translation output and disturbed elongation as evidenced by increased ribosome collisions. SMYD5 and RPL40 K22me3 are upregulated in hepatocellular carcinoma (HCC) and negatively correlated with patient prognosis. Depleting SMYD5 renders HCC cells hypersensitive to mTOR inhibition in both 2D and 3D cultures. Additionally, the loss of SMYD5 markedly inhibits HCC development and growth in both genetically engineered mouse and patient-derived xenograft (PDX) models, with the inhibitory effect in the PDX model further enhanced by concurrent mTOR suppression. Our findings reveal a novel role of the SMYD5 and RPL40 K22me3 axis in translation elongation and highlight the therapeutic potential of targeting SMYD5 in HCC, particularly with concurrent mTOR inhibition. This work also conceptually broadens the understanding of lysine methylation, extending its significance from transcriptional regulation to translational control.

Histone demethylase KDM5D represses the proliferation, migration and invasion of hepatocellular carcinoma through the E2F1/TNNC1 axis.

This study focused on investigating the mechanism in which the KDM5D/E2F1/TNNC1 axis affected hepatocellular carcinoma (HCC) development. At first, we determined HCC cell proliferation, migration, invasion, and apoptosis, as well as SOD activity, MDA content, and ROS level. ChIP assay was subsequently conducted to examine H3K4me3 modification in the E2F1 promoter region and the binding of E2F1 to the TNNC1 promoter region after KDM5D overexpression. Meanwhile, we performed western blot for testing KDM5D, H3K4me3, and E2F1 expression after KDM5D overexpression in Huh-7 cells. The binding of transcription factor E2F1 to the TNNC1 promoter region was assessed by dual luciferase reporter gene assay. We further observed the tumor growth ability in nude mice transplanted tumor models. Overexpressed KDM5D suppressed HCC proliferation, migration, and invasion, promoted the apoptosis, suppressed SOD activity, elevated MDA content and ROS level, and promoted ferroptosis. KDM5D suppressed H3K4me3 modification in the E2F1 promoter region and suppressed E2F1 expression in HCC cells. Reduced KDM5D, H3K4me3, and E2F1 expression was found after KDM5D overexpression in Huh-7 cells. Overexpressing E2F1 reversed the inhibitory effects of KDM5D on HCC cell proliferative, migratory, and invasive behaviors. KDM5D repressed TNNC1 transcription by inhibiting E2F1 binding to the TNNC1 promoter. In vivo KDM5D overexpression inhibited HCC development via the E2F1/TNNC1 axis. KDM5D inhibits E2F1 expression by suppressing H3K4me3 modification in the E2F1 promoter region, which in turn suppresses the binding of E2F1 to the TNNC1 promoter region, thus leading to the inhibition of HCC development.

RFX6 facilitates aerobic glycolysis-mediated growth and metastasis of hepatocellular carcinoma through targeting PGAM1.

Hepatocellular carcinoma (HCC) cells undergo reprogramming of glucose metabolism to support uncontrolled proliferation, of which the intrinsic mechanism still merits further investigation. Although regulatory factor X6 (RFX6) is aberrantly expressed in different cancers, its precise role in cancer development remains ambiguous. Microarrays of HCC tissues were employed to investigate the expression of RFX6 in tumour and adjacent non-neoplastic tissues. Functional assays were employed to explore the role of RFX6 in HCC development. Chromatin immunoprecipitation, untargeted metabolome profiling and sequencing were performed to identify potential downstream genes and pathways regulated by RFX6. Metabolic assays were employed to investigate the effect of RFX6 on glycolysis in HCC cells. Bioinformatics databases were used to validate the above findings. HCC tissues exhibited elevated expression of RFX6. High RFX6 expression represented as an independent hazard factor correlated to poor prognosis in patients with HCC. RFX6 deficiency inhibited HCC development in vitro and in vivo, while its overexpression exerted opposite functions. Mechanistically, RFX6 bound to the promoter area of phosphoglycerate mutase 1 (PGAM1) and upregulated its expression. The increased PGAM1 protein levels enhanced glycolysis and further promoted the development of HCC. RFX6 acted as a novel driver for HCC development by promoting aerobic glycolysis, disclosing the potential of the RFX6-PGAM1 axis for therapeutic targeting.

LncRNA TCTN2 Promotes the Malignant Development of Hepatocellular Carcinoma via Regulating mIR-1285-3p/ARF6 Axis.

Hepatocellular carcinoma (HCC) is one of the most life-threatening malignant diseases. TCTN2 protein participates in tumorigenesis and development. However, whether lncRNA TCTN2 is associated with HCC pathogenesis remains unclear. The expression of lncRNA, TCTN2, miR-1285-3p, and ARF6 in HCC tissues and cells was detected by a quantitative Real-Time PCR (qRT-PCR) assay. lncRNA TCTN2-specific shRNA was transfected into HCC cells, and a functional investigation was performed. The direct interactions between lncRNA TCTN2 and miR-1285-3p and ARF6 were verified by dualluciferase reporter gene assay. A rescue experiment was performed to confirm the role of miR- 1285-3p/ARF6 in association with lncRNA TCTN2. LncRNA TCTN2 exhibited a high expression in HCC tumor tissues and cell lines. Knockdown of lncRNA TCTN2 suppressed cell proliferation and induced cell cycle arrest and apoptosis through regulating Cyclin D1/p21 and Bax/Bcl-2 signals. Meanwhile, the knockdown of lncRNA TCTN2 inhibited HCC cell migration and invasion through upregulating MMP2/MMP9. Mechanistic investigation revealed that lncRNA TCTN2 upregulated the expression of ARF6 via sponging miR-1285-3p. Rescue experiments indicated that miR-1285-3p inhibitor reversed the antitumor effects of lncRNA TCTN2 and ARF6 knockdown inhibited the progression of HCC. Our results suggested that the knockdown of lncRNA TCTN2 inhibited HCC development by regulating the miR-1285-3p/ARF6 axis, implying that the lncRNA TCTN2 is upregulated in HCC and may serve as a diagnostic biomarker in HCC. Furthermore, it may demonstrate an important value for the clinical treatment of patients with HCC.

A network pharmacology integrated serum pharmacochemistry strategy for uncovering efficacy of YXC on hepatocellular carcinoma

Ethnopharmacological relevanceThe YangzhengXiaoji capsule (YXC) has a wide range of applications as effective traditional Chinese medicine (TCM) preparation for hepatocellular carcinoma (HCC) in China. However, the potential bioactive components and the mechanisms are yet unclear. Aim of the studyThe treatment mechanism of YXC on HCC using a network pharmacology integrated serum pharmacochemistry strategy to investigate associated targets and pathways. Materials and methodsWe utilised HPLC-Q-TOF-MS/MS technology to identify components of the serum samples from both the model group and the YXC (H) group serum, which were collected from nude mice with orthotopic liver tumours. Following this, we conducted compound-target prediction and identified the overlap between the target genes in the YXC group and the oncogenes associated with HCC. The anticancer mechanisms of YXC were investigated by creating a compound-target-pathway network using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis. The anticancer efficacy was evaluated in vitro and in vivo. Also, potential predictive targets and pathways associated with YXC in HCC treatment were assessed by western blotting. ResultsThe YXC (H) serum had 47 bioactive compounds compared to other models, and identified 173 specific target genes. Using the compound-target-disease network, 141 possible target genes were identified. The KEGG pathway analysis revealed vital enrichment of pathways associated with HCC, including regulating Oncology related pathways of inflammation, immunity, apoptosis, and necrosis biological processes. YXC significantly inhibited HCC cell growth in vitro and in vivo. After YXC treatment, western blotting detected alterations in the p53/Bcl-2/Bax/Caspase-3 and PI3K/Akt pathways. ConclusionsYXC can inhibit HCC development and advancement by a variety of components, targets and pathways, especially apoptosis-induction.

Royal jelly acid suppresses hepatocellular carcinoma tumorigenicity by inhibiting H3 histone lactylation at H3K9la and H3K14la sites

Background and purposeHuman hepatocellular carcinoma (HCC) features include enhanced glycolysis and elevated lactate concentrations. Accumulation of lactate during metabolism provides a precursor for histone lysine modification. This study was designed to determine whether royal jelly acid (RJA) acts against HCC through the lactate modification pathway. Experimental approachThe effects of RJA on Hep3B and HCCLM3 cell invasion, migration, proliferation, and apoptosis were investigated using cell scratching, colony formation assay, flow cytometry, western blotting, and real-time qPCR, gas chromatography, and RNA sequencing to determine the pathways and molecular targets involved. Tumor xenografts were used to evaluate the anti-HCC effects of RJA in vivo. In-cell Western blotting and expression correlation analysis were applied to confirm the associations between H3 histone lactylation and the antitumor effects of RJA. Key resultsRJA has good antitumor effects in vivo and in vitro. Multi-omics analysis with metabolome and transcriptome determined that the glycolytic metabolic pathway provided the principle antitumor effect of RJA. Further mechanistic studies showed that RJA inhibited HCC development by interfering with lactate production and inhibiting H3 histone lactylation at H3K9la and H3K14la sites. Conclusions and implicationsThis study first demonstrated that RJA exerts antitumor effects by affecting the glycolytic pathway. RJA could regulate the lactylation of H3K9la and H3K14la sites on H3 histone using lactate as a clue in the glycolytic pathway. Therefore, the lactylation of H3 histone is vital in exerting the antitumor effect of RJA, providing new evidence for screening and exploring antitumor drug mechanisms in the later stage.

LncRNA CASC2 Regulate Cell Proliferation and Invasion by Targeting miR-155/SOCS1 Axis in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNAs) have been reported to be involved in the development and progression of various human malignancies. However, the role of lncRNA CASC2 in hepatocellular carcinoma (HCC) remains mostly unknown. The aim of this study was to investigate the potential roles and underlying mechanisms of CASC2 in HCC progression. We found that CASC2 expressions were downregulated in HCC tissue samples and cell lines. The clinical assays revealed that lower levels of CASC2 were associated with the TNM stage, lymph node metastasis, and a poorer prognosis specific to HCC patients. Overexpression of CASC2 inhibited the proliferating, migratory, and invasion capacity of HCC cells. Bioinformatics analysis and the luciferase reporter assay revealed that CASC2 worked as a molecular sponge for miR-155. And CASC2 could upregulate SOCS1 expression by inhibiting miR-155 expression in HCC cells. Furthermore, SOCS1 inhibition partially inverses the suppression effect of cell proliferation, migration, and invasion regulated by CASC2 in Huh7 and HepG2 cells. Taken together, our findings identified CASC2 as a tumor suppressor to inhibit HCC development by regulating the miR-155/SOCS1 axis, and CASC2 might be a potential therapeutic target of HCC for future clinical treatment.

Hydrogel-based miR-192 delivery inhibits the development of hepatocellular carcinoma by suppressing the GSK3β/Wnt/β-catenin pathway.

Hepatocellular carcinoma (HCC) is a primary liver cancer characterized by high invasiveness, metastasis, and poor prognosis, which lacks effective treatments. Although the role of miR-192 in HCC development has been recognized, the underlying molecular mechanism is still poorly understood. This study aimed to explore the impact of mir-192 on HCC and its potential as a therapeutic strategy. Wound healing assay, Transwell assay, CCK-8 assay, and flow cytometry were performed to detect the impact of miR-192 on HCC cell metastasis, invasion, proliferation, and apoptosis, respectively. q-PCR and western blot were applied to measure the relative mRNA and protein expression of the GSK3β/Wnt/β-catenin pathway in miR-192-overexpressing cell lines. Immunofluorescence was carried out to detect the nuclear translocation of β-catenin. starBase website and dual luciferase reporter assay were used to verify the interaction between miR-192 and the target gene WNT10B 3'-untranslated region (3'-UTR) of the Wnt pathway. In addition, we developed algin/polyethyleneimine@miR-192 (AG/PEI@miR-192) nanohydrogel for in vivo delivery of miR-192-agomir. The results revealed that overexpressed miR-192 reduced the expression of HCC cell surface markers CD90, EpCAM, and CD133. Moreover, miR-192 overexpression inhibited HCC cell metastasis, invasion, and proliferation, promoted cell apoptosis, and reduced GSK3β/Wnt/β-catenin pathway expression. Additionally, AG/PEI@miR-192 exhibited good drug release and tumor inhibition. In conclusion, our study suggested that miR-192 inhibits HCC development by suppressing the GSK3β/Wnt/β-catenin pathway and proposed a promising hydrogel-based miR-192 delivery approach to hinder tumor growth.

Epirubicin may enhance the inhibition of hepatocellular carcinoma induced by iodine-125 seeds through downregulating WNT pathway.

To investigate the role of iodine-125 (125 I)combined with epirubicin (EPI) in inhibiting hepatocellular carcinoma (HCC) growth and promoting apoptosis. Both in vivo and in vitro experiments were conducted. CCK-8 assay was performed to determine the cells viability after EPI treatment. HepG2 and SMMC7721 cells were treated with EPI or 125 I or in combination. Colony formation assays were performed to verify the antiproliferation effect. Annexin V-FITC/PI, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and western blotting were performed to analyze cellular apoptosis. Scratch wound healing assays and transwell assays were used to examine migration following different treatments. An isobaric tag for relative and absolute quantitation analysis was used to detect changes in protein expression after 125 I treatment, identifying the potential mediating protein cathelicidin (LL-37). LL-37 protein and WNT pathway-related proteins were detected by western blotting in SMMC7721 and HepG2 cells. Mice were treated with 125 I and EPI to evaluate whether EPI enhanced the antitumor effect of 125 I. EPI promoted 125 I-induced apoptosis and reduced the proliferation of HepG2 and SMMC7721 cells. EPI also prevented the migration of HepG2 and SMMC7721 cells. EPI combined with 125 I may interfere with the WNT signaling pathway by decreasing LL-37 to inhibit HCC development. The antitumor effects of EPI with 125 I were verified in mice. EPI combined with 125 I induced apoptosis and inhibited the proliferation of HCC cells by LL-37 downregulating the WNT pathway.

Down-regulation of circPTTG1IP induces hepatocellular carcinoma development via miR-16-5p/RNF125/JAK1 axis

Circular RNAs are known to regulate the biological processes of hepatocellular carcinoma (HCC), and humans with Down syndrome are at low risk of developing solid tumors due to the amplification of several tumor suppressor genes on human chromosome 21 (HSA21). Here, we aimed to investigate the potential role of circRNAs originating from HSA21 in the progression of HCC. CircRNA-sequencing was performed to analyze differentially expressed circRNAs in 4 HCC and peritumor tissues, and circRNAs originating from HSA21 were further analyzed. Circ_0061984 (circPTTG1IP) was chosen for further study because it showed the lowest expression in HCC tissues, and qRT-PCR was used to confirm the expression of circPTTG1IP in HCC patient tissues. The biological function of circPTTG1IP was detected in HCC cells both in vivo and in vitro. Moreover, luciferase reporter assays, circRNA immunoprecipitation, and fluorescence in situ hybridization (FISH) were used to investigate the potential mechanism of circPTTG1IP. Finally, the possible mechanisms of filgotinib in circPTTG1IP-driven HCC were assessed. CircPTTG1IP expression was decreased in HCC compared to peritumoral tissues. Moreover, low circPTTG1IP expression was revealed to be associated with a poor prognosis of HCC patients. Elevation of circPTTG1IP was revealed to inhibit HCC development both in vitro and in vivo. Mechanistically, circPTTG1IP was shown to function as a competing endogenous RNA (ceRNA) of RNF125 by binding miR-16-5p to increase the level of the E3 ubiquitin ligase RNF125, which further ubiquitinated and degraded JAK1 protein. Finally, we demonstrated that administration of filgotinib, a JAK1 inhibitor, restricted HCC progression induced by low circPTTG1IP expression. Thus, we revealed that circPTTG1IP is a novel tumor suppresser circRNA in HCC and that a low circPTTG1IP level promotes HCC development via the miR-16-5p/RNF125/JAK1 axis. Patients with low circPTTG1IP may benefit from filgotinib treatment.

Extracellular Vesicle-Encapsulated MicroRNA-375 from Bone Marrow-Derived Mesenchymal Stem Cells Inhibits Hepatocellular Carcinoma Progression through Regulating HOXB3-Mediated Wnt/β-Catenin Pathway.

Nowadays, microRNA-375 (miR-375) has been implicated in many types of cancers, including hepatocellular carcinoma (HCC), and the functions of miRNAs encapsulated by extracellular vesicles (EV) in HCC progression have also been extensively investigated. In this research, we aimed to probe into the mechanism of EV-encapsulated miR-375 from bone marrow-derived mesenchymal stem cells (BM-MSCs) in HCC progression. At first, miR-375 expression in HCC tissues and cells was detected using RT-qPCR, and miR-375 was overexpressed to specify the effects of miR-375 on the malignant phenotype of HCC cells. miR-375 was downregulated in HCC, and overexpression of miR-375 suppressed HCC cell growth. Then, BM-MSCs and EV were isolated and identified, and, EV were cocultured with HCC cells for further functional assays. It was found that miR-375 encapsulated by EV could restrict the malignant phenotypes of HCC cells. Furthermore, the downstream genes and signaling cascades involved in HCC growth were investigated. HOXB3 was determined to be a downstream target of miR-375, and upregulation of miR-375 decreased Wnt1 and β-catenin protein expression. Furthermore, HOXB3 blocked the repressive effects of miR-375 on HCC cells and Wnt1 and β-catenin expression. This study highlights that miR-375 encapsulated by EV inhibits HCC development via modulating the HOXB3/Wnt/β-catenin axis.

GFI1-Mediated Upregulation of LINC00675 as a ceRNA Restrains Hepatocellular Carcinoma Metastasis by Sponging miR-942-5p.

Hepatocellular carcinoma (HCC) is a common malignant liver tumor worldwide. Tumor recurrence and metastasis contribute to the bad clinical outcome of HCC patients. Substantial studies have displayed lncRNAs modulate various tumorigenic processes of many cancers. Our current work was aimed to investigate the function of LINC00675 in HCC and to recognize the potential interactions between lncRNAs and microRNAs. GFI1 can exhibit a significant role in the progression of human malignant tumors. Firstly, GFI1 was identified using real-time PCR in HCC tissues and cells. In this work, we indicated GFI1 was remarkably reduced in HCC tissues and cells. Meanwhile, GFI1 specifically interacted with the promoter of LINC00675. Up-regulation of LINC00675 obviously repressed the migration and invasion capacity of SMCC-7721 and QGY-7703 cells in vitro. Moreover, decrease of LINC00675 competitively bound to miR-942-5p that contributed to the miRNA-mediated degradation of GFI1, thus facilitated HCC metastasis. The ceRNA function of LINC00675 in HCC cells was assessed and confirmed using RNA immunoprecipitation assay and RNA pull-down assays in our work. Additionally, we proved overexpression of miR-942-5p promoted HCC progression, which was reversed by the up-regulation of GFI1. In summary, LINC00675 might act as a prognostic marker for HCC, which can inhibit HCC development via regulating miR-942-5p and GFI1.

Long noncoding RNA FOXD2-AS1 aggravates hepatocellular carcinoma tumorigenesis by regulating the miR-206/MAP3K1 axis.

LncRNAs play crucial roles in the development of various cancers including hepatocellular carcinoma (HCC). Nevertheless, the function of the long noncoding RNA (lncRNA) FOXD2‐AS1 in HCC is still poorly understood. In this study, we focused on the role of FOXD2‐AS1 in HCC. We found that FOXD2‐AS1 was significantly upregulated in HCC cells in comparison to normal human liver cells, LO2. In this study, we also demonstrated that miR‐206 expression was greatly reduced in HCC cells. Furthermore, the inhibition of FOXD2‐AS1 repressed HCC cell proliferation, enhanced cell apoptosis, and restrained cell invasion and migration. The knockdown of FOXD2‐AS1 elevated miR‐206 expression, and we validated an interaction between these RNAs. Additionally, miR‐206 mimics inhibited HCC development while miR‐206 mimics had the opposite effect. MAP kinase 1 (MAP3K1) was predicted to be a target of miR‐206. We discovered that FOXD2‐AS1 modulated MAP3K1 expression by sponging miR‐206 in MHCC‐97L and HepG2 cells. Finally, our in vivo experiments validated that the knockdown of FOXD2‐AS1 inhibited HCC progression by modulating the miR‐206/MAP3K1 axis. In conclusion, this work implies FOXD2‐AS1 accelerates HCC progression through sponging miR‐206 and regulating MAP3K1 expression.

Nur77-activated lncRNA WFDC21P attenuates hepatocarcinogenesis via modulating glycolysis

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Orphan nuclear receptor Nur77, which is low expressed in HCC, functions as a tumor suppressor to suppress HCC. However, the detailed mechanism is still not well understood. Here, we demonstrate that Nur77 could inhibit HCC development via transcriptional activation of the lncRNA WAP four-disulfide core domain 21 pseudogene (WFDC21P). Nur77 binds to its response elements on the WFDC21P promoter to directly induce WFDC21P transcription, which inhibits HCC cell proliferation, tumor growth, and tumor metastasis both in vitro and in vivo. In clinical HCC samples, WFDC21P expression positively correlated with that of Nur77, and the loss of WFDC21P is associated with worse prognosis. Mechanistically, WFDC21P could inhibit glycolysis by simultaneously interacting with PFKP and PKM2, two key enzymes in glycolysis. These interactions not only abrogate the tetramer formation of PFKP to impede its catalytic activity but also prevent the nuclear translocation of PKM2 to suppress its function as a transcriptional coactivator. Cytosporone-B (Csn-B), an agonist for Nur77, could stimulate WFDC21P expression and suppress HCC in a WFDC21P-dependent manner. Therefore, our study reveals a new HCC suppressor and connects the glycolytic remodeling of HCC with the Nur77-WFDC21P-PFKP/PKM2 axis.

Nanoparticle Conjugation of Ginsenoside Rg3 Inhibits Hepatocellular Carcinoma Development and Metastasis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. The prognosis of HCC remains very poor; thus, an effective treatment remains urgent. Herein, a type of nanomedicine is developed by conjugating Fe@Fe3 O4 nanoparticles with ginsenoside Rg3 (NpRg3), which achieves an excellent coupling effect. In the dimethylnitrosamine-induced HCC model, NpRg3 application significantly prolongs the survival of HCC mice. Further research indicates that NpRg3 application significantly inhibits HCC development and eliminates HCC metastasis to the lung. Notably, NpRg3 application delays HCC-induced ileocecal morphology and gut microbial alterations more than 12 weeks during HCC progression. NpRg3 administration elevates the abundance of Bacteroidetes and Verrucomicrobia, but decreases Firmicutes. Twenty-nine predicted microbial gene functions are enriched, while seven gene functions are reduced after NpRg3 administration. Moreover, the metabolomics profile presents a significant progression during HCC development, but NpRg3 administration corrects tumor-dominant metabolomics. NpRg3 administration decreases 3-indolepropionic acid and urea, but elevates free fatty acids. Importantly, NpRg3 application remodels the unbalanced correlation networks between gut microbiota and metabolism during HCC therapy. In conclusion, nanoparticle conjugation of ginsenoside Rg3 inhibits HCC development and metastasis via the remodeling of unbalanced gut microbiota and metabolism in vivo, providing an antitumor therapy strategy.

Circular RNA circRHOT1 promotes hepatocellular carcinoma progression by initiation of NR2F6 expression

BackgroundIncreasing evidence has revealed a close relationship between non-coding RNAs and cancer progression. Circular RNAs (circRNAs), a recently identified new member of non-coding RNAs, are demonstrated to participate in diverse biological processes, such as development, homeostatic maintenance and pathological responses. The functions of circRNAs in cancer have drawn wide attention recently. Until now, the expression patterns and roles of circRNAs in hepatocellular carcinoma (HCC) have remained largely unknown.MethodsBioinformatics method was used to screen differentially expressed novel circRNAs in HCC. Northern blotting, qRT-PCR, in situ hybridization (ISH) and RNA-FISH were utilized to analyzed the expression of circRHOT1 in HCC tisues.CCK8, colony formation, EdU assays were used to analyze proliferation of HCC cells. Transwell assay was utilized to analyze HCC cell migration and invasion. FACS was used for apoptosis analysis. Xenograft experiments were used to analyze tumor growth in vivo. Mass spectrum, RNA pulldown, RIP and EMSA was utilized to test the interaction between circRHOT1 and TIP60. RNA-sequencing method was used to analyze the downstream target gene of circRHOT1.ResultsWe identified circRHOT1 (hsa_circRNA_102034) as a conserved and dramatically upregulated circRNA in HCC tissues. HCC patients displaying high circRHOT1 level possessed poor prognosis. Through in vitro and in vivo experiments, we demonstrated circRHOT1 significantly promoted HCC growth and metastasis. Regarding the mechanism, we conducted a RNA pulldown with a biotin-labeled circRHOT1-specific probe and found that circRHOT1 recruited TIP60 to the NR2F6 promoter and initiated NR2F6 transcription. Moreover, NR2F6 knockout inhibited growth, migration and invasion, whereas rescuing NR2F6 in circRHOT1-knockout HCC cells rescued the proliferation and metastasis abilities of HCC cells.ConclusionTaken together, circRHOT1 inhibits HCC development and progression via recruiting TIP60 to initiate NR2F6 expression, indicating that circRHOT1 and NR2F6 may be potential biomarkers for HCC prognosis.

MiR-206 inhibits cell proliferation, invasion, and migration by down-regulating PTP1B in hepatocellular carcinoma.

Protein tyrosine phosphatase 1B (PTP1B) has been reported as an oncogene in hepatocellular carcinoma (HCC). However, how PTP1B is regulated in HCC remains unclear. MicroRNAs (miRNAs) are a class of small non-coding RNAs involved many biological processes including tumorigenesis. In this study, we investigated whether miRNA participated in the regulation of PTP1B in HCC. We found that miR-206, which was down-regulated during tumorigenesis, inhibited HCC cell proliferation and invasion. Overexpression of miR-206 inhibited proliferation, invasion, and migration of HCC cell lines HepG2 and Huh7. Mechanistically, we demonstrated that miR-206 directly targeted PTP1B by binding to the 3′-UTR of PTP1B mRNA as demonstrated by the luciferase reporter assay. Overexpression miR-206 inhibited PTP1B expression while miR-206 inhibition enhanced PTP1B expression in HepG2 and Huh7 cells. Functionally, the regulatory effect on cell proliferation/migration/invasion of miR-206 was reversed by PTP1B overexpression. Furthermore, tumor inoculation nude mice model was used to explore the function of miR-206 in vivo. Our results showed that overexpression of miR-206 drastically inhibited tumor development. In summary, our data suggest that miR-206 inhibits HCC development by targeting PTP1B.

MiR-139-5p, miR-940 and miR-193a-5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1.

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.

Retracted Article: MiR-206 reduced the malignancy of hepatocellular carcinoma cells in vitro by inhibiting MET and CTNNB1 gene expressions.

The anti-cancer role of miR-206 in hepatocellular carcinoma (HCC) cells has been reported, but its mechanism of action remains poorly understood. This research aimed to investigate the anti-HCC mechanism of miR-206. We analyzed 25 pairs of HCC and adjacent tissue specimens from HCC patients. Two patient-derived HCC cell lines were established. MiR-206 levels in tissue specimens and cell lines were detected by qRT-PCR. MiR-206 overexpression was mimicked by miR-206 mimic transfection. MET or CTNNB1 gene was overexpressed by transient transfection. Protein and protein phosphorylation levels of interests were assessed by western blotting. HCC cell malignancy in vitro was evaluated by cell proliferation, apoptosis, colony formation, trans-well invasion assays as well as western blotting assessing the marker proteins of epithelial or mesenchymal phenotype. We found that miR-206 level was significantly lower in HCC tissue specimens in comparison to adjacent counterparts. Two patient-derived HCC cell lines showed lower miR-206 level than L02 human hepatocytes. MiR-206 mimic transfection significantly reduced phosphorylation levels of pan-Akt Ser9, Erk1 Thr202/Tyr204 and Gsk-3beta Ser308 as well as protein levels of beta-catenin and c-Met in primary HCC cells in vitro. Luciferase reported assay and AGO2-RNA co-immunoprecipitation assays results demonstrated that miR-206 reduced MET and CTNNB1 gene expressions in HCC cells by interacting with the 3′ UTR of their mRNAs. Restoring c-Met or beta-catenin protein level by MET or CTNNB1 transient overexpression partially restored the malignancy of HCC cells in vitro. We concluded that miR-206 might inhibit HCC development by targeting MET and CTNNB1 gene expression.

Abstract 4004: The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma

Abstract Introduction: The purpose of this study was to examine whether nizatidine could reduce liver fibrosis and subsequent tumor burden in two rodent models of hepatocellular carcinoma (HCC). Methods: An in silico screen identified nizatidine as a compound that can reverse a previously identified gene signature associated with disease progression and HCC development in human cirrhosis patients. We tested the ability of nizatidine to inhibit HCC development in two rodent models. In the first study, male Wistar rats received weekly intraperitoneal injections of 50 mg/kg diethylnitrosamine (DEN). This model has previously been shown to reliably recapitulate the histologic and molecular features of human HCC development including fibrosis after 8 weeks, cirrhosis after 12 weeks, and HCC by 18 weeks. DEN-injured rats were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) after 8 weeks. In the second study, male C57BL/6J mice received a single intraperitoneal injection of 25 mg/kg DEN at day 15 followed by initiation of a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) at 6 weeks of age. DEN+CDAHFD-injured mice were randomized to receive oral gavage of nizatidine (n=7) or vehicle control (n=9) at 12 weeks of age and were sacrificed at 30 weeks of age after development of HCC in the setting of nonalcoholic steatohepatitis (NASH). Results: As expected, repeated injections of DEN in rats resulted in progressive fibrosis followed by HCC formation. Treatment with nizatidine resulted in a 35% reduction of tumor nodules relative to vehicle controls (p<0.18). Liver sections were stained by picrosirius red to assess fibrosis and nizatidine reduced collagen deposition in DEN-injured rats (collagen proportional area = 5±0.03 vs. 9.2±0.04; p<0.05). This histologic observation was further confirmed by gene expression analysis with reduction in several profibrotic markers, such as alpha-smooth muscle actin and collagen type I, after treatment with nizatidine. All mice receiving DEN+CDAHFD developed HCC. Treatment with nizatidine resulted in a 60% reduction in tumor nodules relative to vehicle controls (p<0.0001). Nizatidine treatment also resulted in a significant reduction in liver to body weight (p<0.01). Nizatidine treatment reduced collagen proportional area (11±0.05 vs. 15±.01; p<0.05) and expression of profibrotic markers. Nizatidine treatment also reduced the expression of several proinflammatory markers including CD68, interferon gamma, and interleukin-6. Conclusion: Our data suggest that the H2 receptor antagonist nizatidine reduces fibrosis and subsequent HCC development. This could be beneficial in patient management given the low cost and ready availability of this agent. Citation Format: Shen Li, Sarani Ghoshal, Gunisha Arora, Derek J. Erstad, Mozhdeh Sojoodi, Michael Lanuti, Yujin Hoshida, Thomas Baumert, Kenneth K. Tanabe, Bryan C. Fuchs. The H2 receptor antagonist nizatidine inhibits carcinogenesis in two rodent models of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4004.