Clinical Hepatocellular Carcinoma Tissues Research Articles

TMB Signature-Related RCAN2 Promotes Apoptosis by Upregulating EHF/DR5 Pathway in Hepatocellular Carcinoma.

The tumour mutation burden (TMB) is a valuable indicator of the accumulation of somatic mutations, and is thought to be associated with the biological behaviour and prognosis of tumours. However, the related genetic mechanism for these association is still unclear. The aim of the present study was to identify the key gene(s) associated with TMB in hepatocellular carcinoma (HCC) and to investigate its biological functions, downstream transcription factors, and mechanism of action. Patients in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database were classified according to TMB signature-related genes. Key genes related to the TMB signature and tumour prognosis were identified. Immunohistochemistry and Quantitative Real-Time Polymerase Chain Reaction (qPCR) were then used to assess gene expression in clinical HCC tissues and HCC cells. Cells with altered gene expression were evaluated for the effect on cell proliferation and apoptosis, both in vitro and in vivo. Three independent databases and cell sequencing data were used to identify the mechanisms involved and the downstream transcription factors. The mechanism was also studied by altering the expression of downstream transcription factors in vitro. The integrated cluster (IC) 2 group, characterized by 99 TMB signature-related genes, showed a significant different TMB score compared to the IC1 group (p < 0.001), as well as more favourable tumour prognosis (p = 0.031). We identified five key prognostic genes that were differentially expressed between IC2 and IC1 and were associated with overall survival. The expression of one of these key prognostic genes, RCAN2, was negatively correlated with TMB in 18 out of 33 tumour types examined. A high level of RCAN2 was correlated with better overall survival in HCC (p = 0.0009). Overexpression of RCAN2 enhanced apoptosis in vitro and in vivo, whereas knockdown of RCAN2 attenuated apoptosis. The mechanism by which RCAN2 promotes apoptosis may involve upregulation of the expression of ETS homologous factor (EHF) and of death receptor 5 (DR5). Downregulation of RCAN2 expression was found to correlate with elevated TMB in multiple cancer types. RCAN2 was also found to be a biomarker of HCC prognosis, and to promote the apoptosis of HCC cells through the EHF/DR5 pathway. These findings provide a new perspective on systemic treatment for advanced HCC with a high TMB.

Aberrant expression of LGALS3BP drives an unfavorable prognosis and more aggressive in HCC via regulating PI3K/AKT signaling

Lectin galactoside-binding soluble 3-binding protein (LGALS3BP) is associated with cancer metastasis and is a promising prognostic marker in neoplasms. In hepatocellular carcinoma (HCC), the prognostic impact and pro-metastatic function of LGALS3BP remain unclear. This study evaluated the endogenous LGALS3BP expression in HCC tissue and its association with prognosis. LGALS3BP protein levels were significantly elevated in clinical HCC tissues and cell lines. Increased LGALS3BP expression was closely associated with disease progression in HCC patients, and they also exhibited an unfavorable prognosis. Furthermore, the knockdown of LGALS3BP inhibited the growth, migration, and invasion of HCC cells in vitro. In mice xenografts, silencing LGALS3BP significantly inhibited tumor cell growth in vivo. Mechanically, upon LGALS3BP depletion, the tumor-suppressive function was dependent on inactivating Phosphatidylinositol 3-kinase (PI3K)/V-akt murine thymoma viral oncogene homolog (AKT) signaling pathway. Collectively, these findings suggest that LGALS3BP employs a pro-tumorigenic function in HCC and may be a promising HCC prognostic marker.

Energy stress-induced circDDX21 promotes glycolysis and facilitates hepatocellular carcinogenesis

Cancer cells undergo metabolic reprogramming in response to hostile microenvironments, such as energy stress; however, the underlying mechanisms remain largely unclear. It is also unknown whether energy stress-responsive circular RNA (circRNA) is involved in the regulation of glucose metabolism. Here we report that circDDX21 is upregulated in response to glucose deprivation by the transcription factor c-Myc. Functionally, circDDX21 is shown to promote glycolysis by increasing PGAM1 expression. Mechanistically, circDDX21 interacts with the RNA binding protein PABPC1, disrupting its association with the ubiquitin E3 ligase MKRN3. This disassociation attenuates MKRN3-mediated PABPC1 ubiquitination and enhances the binding of PABPC1 to PGAM1 mRNA, thereby leading to PGAM1 mRNA stabilization. The ability of the circDDX21-PGAM1 axis to promote hepatocellular carcinogenesis is validated in a xenograft mouse model. Additionally, in clinical hepatocellular carcinoma tissues, there is a positive correlation between circDDX21 and PGAM1 expression. These findings establish circDDX21 as an important regulator of glycolysis and suggest circDDX21 as a potential therapeutic target for hepatocellular carcinoma.

Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

Resistance to sorafenib, an effective first-line treatment for advanced hepatocellular carcinoma (HCC), greatly compromised the prognosis of patients. The extracellular matrix is one of the most abundant components of the tumor microenvironment. Beyond acting as a physical barrier, it remains unclear whether cell interactions and signal transduction mediated by the extracellular matrix contribute to sorafenib resistance. With the analysis of primary HCC organoid RNA-seq data combined with in vivo and in vitro experiments validation, we discovered that fibronectin extra domain A (FN-EDA) derived from cancer-associated fibroblasts played a critical role in sorafenib resistance. Mechanistically, FN-EDA stimulates the up-regulation of the key one-carbon metabolism enzyme SHMT1 in HCC cells via the TLR4/NF-κB signaling pathway, thereby countering the oxidative stress induced by sorafenib. Moreover, we reinforced the clinical significance of our discoveries by conducting in vivo assays with an immunodeficiency subcutaneous xenograft tumor model, which was established using primary cancer-associated fibroblasts derived from clinical HCC tissues, and through the analysis of HCC samples obtained from The Cancer Genome Atlas (TCGA) database. Our findings suggest that targeting the FN-EDA/SHMT1 pathway could be a potential strategy to improve sorafenib responsiveness in HCC patients.

CGAS suppresses hepatocellular carcinoma independent of its cGAMP synthase activity.

Cyclic GMP-AMP synthase (cGAS) is a key innate immune sensor that recognizes cytosolic DNA to induce immune responses against invading pathogens. The role of cGAS is conventionally recognized as a nucleotidyltransferase to catalyze the synthesis of cGAMP upon recognition of cytosolic DNA, which leads to the activation of STING and production of type I/III interferon to fight against the pathogen. However, given that hepatocytes are lack of functional STING expression, it is intriguing to define the role of cGAS in hepatocellular carcinoma (HCC), the liver parenchymal cells derived malignancy. In this study, we revealed that cGAS was significantly downregulated in clinical HCC tissues, and its dysregulation contributed to the progression of HCC. We further identified cGAS as an immune tyrosine inhibitory motif (ITIM) containing protein, and demonstrated that cGAS inhibited the progression of HCC and increased the response of HCC to sorafenib treatment by suppressing PI3K/AKT/mTORC1 pathway in cellular and animal models. Mechanistically, cGAS recruits SH2-containing tyrosine phosphatase 1 (SHP1) via ITIM, and dephosphorylates p85 in phosphatidylinositol 3-kinase (PI3K), which leads to the suppression of AKT-mTORC1 pathway. Thus, cGAS is identified as a novel tumor suppressor in HCC via its function independent of its conventional role as cGAMP synthase, which indicates a novel therapeutic strategy for advanced HCC by modulating cGAS signaling.

Psilostachyin C reduces malignant properties of hepatocellular carcinoma cells by blocking CREBBP-mediated transcription of GATAD2B.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality globally. Many herbal medicines and their bioactive compounds have shown anti-tumor properties. This study was conducted to examine the effect of psilostachyin C (PSC), a sesquiterpenoid lactone isolated from Artemisia vulgaris L., in the malignant properties of HCC cells. CCK-8, flow cytometry, wound healing, and Transwell assays revealed that 25 μM PSC treatment significantly suppressed proliferation, cell cycle progression, migration, and invasion of two HCC cell lines (Hep 3B and Huh7) while promoting cell apoptosis. Bioinformatics prediction suggests CREB binding protein (CREBBP) as a promising target of PSC. CREBBP activated transcription of GATA zinc finger domain containing 2B (GATAD2B) by binding to its promoter. CREBBP and GATAD2B were highly expressed in clinical HCC tissues and the acquired HCC cell lines, but their expression was reduced by PSC. Either upregulation of CREBBP or GATAD2B restored the malignant properties of HCC cells blocked by PSC. Collectively, this evidence demonstrates that PSC pocessess anti-tumor functions in HCC cells by blocking CREBBP-mediated transcription of GATAD2B.

Upregulation of CYR61 by TGF-β and YAP signaling exerts a counter-suppression of hepatocellular carcinoma

TGF-β and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-β target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-β in liver cancer cells. In addition, TGF-β-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-β- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-β- or YAP-induced malignant transformation of HCC cells in vitro, and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-β and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Arachidonic acid metabolism CYP450 pathway is deregulated in hepatocellular carcinoma and associated with microvascular invasion.

Arachidonic acid metabolism plays a crucial role in the development and progression of inflammatory and metabolic liver diseases. However, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression of key genes involved in the arachidonic acid metabolism pathway in HCC using a combination of bioinformatics, proteomics and immunohistochemistry analyses. Through a comprehensive analysis of publicly available datasets, clinical HCC tissues, and tissue microarrays, we compared the expression of hepatic arachidonic acid metabolic genes. We observed significant downregulation of cytochrome P450 (CYP450) pathway genes at both the messenger RNA and protein levels in HCC tissues compared to normal liver tissues. Furthermore, we observed a strong correlation between the deregulation of the arachidonic acid metabolism CYP450 pathway and the pathological features and prognosis of HCC. Specifically, the expression of CYP2C8/9/18/19 was significantly correlated with pathological grade (r = -.484, p < .0001), vascular invasion (r = -.402, p < .0001), aspartate transaminase (r = -.246, p = .025), gamma-glutamyl transpeptidase (r = -.252, p = .022), alkaline phosphatase (r = -.342, p = .002), alpha-fetoprotein (r = -.311, p = .004) and carbohydrate antigen 19-9 (r = -.227, p = .047). Moreover, we discovered a significant association between CYP450 pathway activity and vascular invasion in HCC. Collectively, these data indicate that arachidonic acid CYP450 metabolic pathway deregulation is implicated in HCC progression and may be a potential predictive factor for early recurrence in patients with HCC.

CENPA-driven STMN1 Transcription Inhibits Ferroptosis in Hepatocellular Carcinoma.

The growing knowledge of ferroptosis has suggested the regulatory role of ferroptosis in hepatocellular carcinoma (HCC), but the pertinent molecular mechanisms remain unclear. Herein, this study investigated the mechanistic basis of ferroptosis-related genes (ferrGenes) in the growth of HCC. Differentially expressed human ferrGenes and tumor-related transcription factors (TFs) were obtained from the The Cancer Genome Atlas (TCGA) dataset and the GTEx dataset. Spearman method-based correlation analysis were conducted to construct TF-ferrGene coexpression regulatory network. Key genes associated with prognosis were singled out with Lasso regression and multivariate Cox analysis to construct the prognostic risk model. Then the accuracy and independent prognostic ability of the model were evaluated. Expression of CENPA and STMN1 was determined in clinical HCC tissues and HCC cells, and their binding was analyzed with dual-luciferase and chromatin immunoprecipitation (ChIP) assays. Furthermore, ectopic expression and knockdown assays were performed in HCC cells to assess the effect of CENPA and STMN1 on ferroptosis and malignant phenotypes. The prognostic risk model constructed based on the eight TF-ferrGene regulatory network-related genes accurately predicted the prognosis of HCC patients. It was strongly related to the clinical characteristics of HCC patients. Moreover, CENPA/STMN1 might be a key TF-ferrGene regulatory network in ferroptosis of HCC. CENPA and STMN1 were overexpressed in HCC tissues and cells. Additionally, CENPA facilitated STMN1 transcription by binding to STMN1 promoter, thus facilitating the malignant phenotypes and suppressing the ferroptosis of HCC cells. Taken together, CENPA curbs the ferroptosis of HCC cells by upregulating STMN1 transcription, thereby promoting HCC growth.

Targeting WDxR motif reprograms immune microenvironment and inhibits hepatocellular carcinoma progression.

The WD-repeat (WDR) family affects carcinogenesis, but its role in the immune microenvironment is poorly characterized. Although functional loss or gain of WDR6 does not markedly change in vitro proliferative and invasive capacity of HCC cells, its deficiency in hepa1-6 cells drastically inhibits the growth and lung metastasis of orthotopically implanted tumors in immune-competent C57BL/6J mice. Mechanistically, WDR6 targets tumor suppressor UVRAG to the CUL4A-DDB1-ROC1 E3 ubiquitin ligase complex through a unique WDxR motif and promotes its degradation. This upregulates chromatin accessibility at the TNFα locus by blocking autophagic degradation of p65, elevates intratumoral myeloid-derived suppressor cell (MDSC) number, and reduces CD8+ T cell infiltration, thereby promoting HCC progression. These immunosuppressive effects are reversed by TNFα blockade. TNFα recruits NF-κB to activate the transcription of WDR6, establishing a WDR6-TNFα loop. Clinically, the WDR6/UVRAG/NF-κB pathway is hyperactivated in HCC, predicting a poor prognosis. Importantly, a WDxR-like peptide disrupts the WDR6/UVRAG complex and enhances the efficiency of anti-PD-L1 against HCC with WDR6 dysregulation.

Gene variation profile and its potential correlation with clinical characteristics in HBV-associated HCC patients of Sichuan Han nationality in China

ObjectiveTo explore the correlation between hepatocellular carcinoma (HCC) gene variation profile and clinical characteristics in Han nationality with HBV infection in Sichuan province. MethodsThe clinical data and HCC tissues were obtained from the enrolled patients. Whole exome sequencing and bioinformatics analysis were performed on formalin-fixed and paraffin-embedded samples from HCC. Tumor mutational burden (TMB) was measured by an algorithm developed in-house. ResultsSixteen high-frequency mutated genes with differential expressions were identified by WES. SMG1 gene variation could be positively correlated with satellite lesions. AMY2B and RGPD4 gene mutation seemed to have a greater chance of vascular invasion. The patients with TATDN1 variation have bigger diameters and greater chances of vascular and microvascular invasion (all P < 0.05). Univariate analysis indicated patients with gene TATDN1 variation had worse prognoses both in disease free survival (DFS) and overall survival (OS). In addition, the enrichment analysis showed many pathways, including the cell cycle pathway, viral oncogene pathway, MAPK pathway, PI3K-AKT pathway, etc., may be associated with HCC. ConclusionThis study explores the gene variation profile of HCC patients with HBV infection in Han nationality of Sichuan Province for the first time, which confirmed the existence of some high-frequency mutated genes and the possibility that the gene variations are involved in the tumorigenesis of HCC through multiple signal pathways. Also, patients with TATDN1 wild type showed a trend of better prognosis both in DFS and OS.

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

CKLF as a Prognostic Biomarker and Its Association with Immune Infiltration in Hepatocellular Carcinoma.

The Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing (CMTM) family, comprising nine members, is involved in the tumorigenesis and progression of various cancers. However, the expression profiles and clinical significance of CMTM family members in hepatocellular carcinoma (HCC) are not fully clarified. In this study, the RNA-sequencing and clinical data were downloaded from The Cancer Genome Atlas (TCGA) databases. The Kaplan-Meier method and the Cox proportional hazards regression analysis were used to evaluate the prognostic significance of CMTM family members. Single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE algorithms were employed to explore the relationship between CMTM family genes and the tumor microenvironment in HCC. Finally, the prognostic CMTM family gene expression was further validated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical (IHC) staining in clinical HCC tissue specimens. The results indicated that, compared with normal tissues, the expression of CKLF, CMTM1, CMTM3, CMTM4, CMTM7, and CMTM8 were significantly upregulated in HCC, while the expression of CMTM2, CMTM5, and CMTM6 were significantly downregulated in HCC. Univariate and multivariate Cox regression analysis demonstrated that CKLF was an independent prognostic biomarker for the overall survival (OS) of HCC patients. In HCC, the expression of CKLF was found to be correlated with immune cell infiltration, immune-related functions, and immune checkpoint genes. The qRT-PCR and IHC confirmed that CKLF was highly expressed in HCC. Overall, this research suggested that CKLF is involved in immune cell infiltration and may serve as a critical prognostic biomarker, which provides new light on the therapeutics for HCC.

SNS-023 sensitizes hepatocellular carcinoma to sorafenib by inducing degradation of cancer drivers SIX1 and RPS16.

Hepatocellular carcinoma (HCC) remains challenging due to the lack of efficient therapy. Promoting degradation of certain cancer drivers has become an innovative therapy. The nuclear transcription factor sine oculis homeobox 1 (SIX1) is a key driver for the progression of HCC. Here, we explored the molecular mechanisms of ubiquitination of SIX1 and whether targeting SIX1 degradation might represent a potential strategy for HCC therapy. Through detecting the ubiquitination level of SIX1 in clinical HCC tissues and analyzing TCGA and GEPIA databases, we found that ubiquitin specific peptidase 1 (USP1), a deubiquitinating enzyme, contributed to the lower ubiquitination and high protein level of SIX1 in HCC tissues. In HepG2 and Hep3B cells, activation of EGFR-AKT signaling pathway promoted the expression of USP1 and the stability of its substrates, including SIX1 and ribosomal protein S16 (RPS16). In contrast, suppression of EGFR with gefitinib or knockdown of USP1 restrained EGF-elevated levels of SIX1 and RPS16. We further revealed that SNS-023 (formerly known as BMS-387032) induced degradation of SIX1 and RPS16, whereas this process was reversed by reactivation of EGFR-AKT pathway or overexpression of USP1. Consequently, inactivation of the EGFR-AKT-USP1 axis with SNS-032 led to cell cycle arrest, apoptosis, and suppression of cell proliferation and migration in HCC. Moreover, we showed that sorafenibcombined with SNS-032 or gefitinib synergistically inhibited the growth of Hep3B xenografts in vivo. Overall, we identify that both SIX1 and RPS16 are crucial substrates for the EGFR-AKT-USP1 axis-driven growth of HCC, suggesting a potential anti-HCC strategy from a novel perspective.

Hepatitis B virus promotes hepatocellular carcinoma development by activating GP73 to repress the innate immune response

BackgroundHepatitis B virus (HBV) causes acute and chronic infection in the clinic. Hepatocellular carcinoma (HCC) is closely linked to HBV infection. Serum Golgi protein 73 (GP73) increases during HBV infection. However, the role of GP73 during HBV infection and the occurrence of HBV-related HCC is still poorly understood.MethodsThe underlying role of HBV-induced GP73 in regulating HCC development was investigated in this study. GP73 expression in HBV-related clinical HCC tissues and in HBV-infected hepatoma cells and primary human hepatocytes was evaluated by immunohistochemistry, ELISAs, Western blotting and quantitative real-time PCR (qRT-PCR) analysis. Tumorigenicity of GP73 overexpressed cells was detected by flow cytometry, qRT-PCR, xenograft nude mouse analyses and sphere formation assays. The effects of GP73 and HBV infection on host innate immune responses in hepatocytes were further investigated by Western blotting and qRT-PCR analysis.ResultsInitially, we confirmed that HBV-positive HCC tissues had significantly higher expression of GP73. Ectopic expression of the HBV gene could induce GP73 expression in primary human hepatocytes and hepatoma cells in vitro. In addition, we discovered that GP73 promotes HCC in both normal liver cells and hepatoma cells. We also found that ectopic expression of HBV genes increases GP73 expression, suppressing the host's innate immune responses in hepatocytes.ConclusionsOur results demonstrate that HBV facilitates HCC development by activating GP73 to repress the host's innate immune response. This study adds to our understanding of the pathogenesis of HBV infection-induced HCC. The findings also provide preclinical support for GP73 as a potential HCC prevention or treatment target.

RING finger and WD repeat domain 3 regulates proliferation and metastasis through the Wnt/β-catenin signalling pathways in hepatocellular carcinoma

BACKGROUNDHepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIMTo investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTSCompared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage (P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells (P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway.CONCLUSIONWe provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.

Screening and identification of key genes ATP1B3 and ENAH in the progression of hepatocellular carcinoma: based on data mining and clinical validation

To explore the marker genes correlated with the prognosis, progression and clinical diagnosis of hepatocellular carcinoma (HCC) based on bioinformatics methods. The TCGA-LIHC, GSE84432, GSE143233 and GSE63898 datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were analyzed. The differentially expressed genes (DEGs) shared by different disease types were obtained using GEO2R and edge R packages, and Gene Ontology (GO) and Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analyses of the DEGs were performed. The expression levels of these DEGs in normal and cancerous tissues were verified in TCGA-LIHC to identify the upregulated genes in HCC. Survival analysis, receiver-operating characteristic (ROC) curve analysis, and correlation analysis between the key genes and the clinical features of the patients were carried out using the R language. The differential expressions of 15 key genes were verified in clinical samples of HCC and adjacent tissues using RT-qPCR. A total of 118 common DEGs were obtained in the database, and among them two genes, namely ATPase Na +/K + transport subunit beta 3 (ATP1B3) and actin regulator (ENAH), showed increased expressions with disease progression. Survival analysis combined with the TCGA-LIHC dataset suggested that high expressions of ATP1B3 and ENAH were both significantly correlated with a poor prognosis of HCC patients (P < 0.05), and their AUC values were 0.821 and 0.933, respectively. A high expression of ATP1B3 was correlated with T stage, pathological stage and pathological grade of the tumors (P < 0.05), while that of ENAH was associated only with an advanced tumor grade (P < 0.05). The results of RT-qPCR showed that ATP1B3 and ENAH were both significantly upregulated in clinical HCC tissues (P < 0.05). ATPIB3 and ENAH are both upregulated in HCC, and their high expressions may serve as biomarkers of progression of liver diseases and a poor prognosis of HCC.

Enhancer of zeste homolog 2 promotes hepatocellular cancer progression and chemoresistance by enhancing protein kinase B activation through microRNA-381-mediated SET domain bifurcated 1

ABSTRACT Metastasis and chemoresistance are the leading causes of death in patients with hepatocellular carcinoma (HCC). microRNAs (miRNAs or miRs) may be useful as diagnostic, therapeutic and prognostic markers for HCC. In this study, we set out to investigate the possible role of miR-381 in HCC development and chemoresistance along with the related mechanism. Microarray-based gene expression profiling was carried out to analyze the expression of SET domain bifurcated 1 (SETDB1) and histone methyltransferase enhancer of zeste homolog 2 (EZH2) followed by validation in clinical HCC tissues and cells. The potential binding between miR-381 and SETDB1 was found and verified. Then, the role of SETDB1 in HCC in relation to miR-381 and protein kinase B (AKT) pathway was explored through gain- and loss-of-function approaches. After expression determination of EZH2, SETDB1, miR-381, and AKT pathway-related factors, their reactions were analyzed and their functional roles in HCC progression and chemoresistance were investigated in vitro and in vivo. SETDB1 was aberrantly upregulated in clinical HCC tissues and cells. This upregulation activated AKT pathway by promoting its tri-methylation on K64. SETDB1 promoted the proliferation, migration and chemoresistance through the AKT pathway in HCC cells. In a xenograft mouse model, SETDB1 promoted HCC cell tumorigenesis in vivo by activating the AKT pathway. Furthermore, EZH2 suppressed miR-381 by catalyzing the activity of H3K27me3 on its promoter region. In conclusion, EZH2 suppressed miR-381 expression by promoting H3K27me3 activity on its promoter region to facilitate SETDB1 expression, thereby activating the AKT pathway to promote hepatocarcinogenesis and chemoresistance.

MiR-660-5p-loaded M2 macrophages-derived exosomes augment hepatocellular carcinoma development through regulating KLF3

ObjectiveM2 macrophages (M2) can affect tumor development by secreting various cytokines, including exosomes (Exo). Herein, we intended to explore how microRNA (miR)-660-5p-modified M2-Exo affected hepatocellular carcinoma (HCC) development through regulating Kruppel-like factor 3 (KLF3). MethodsmiR-660-5p and KLF3 levels were first measured in clinical HCC tissues. A miR-targeted relation was explored between miR-660-5p and KLF3. M2-Exo were modified by miR-660-5p-related oligonucleotides and co-cultured with HepG2 cells to determine their effects on cell proliferation, colony formation, invasion, migration, apoptosis and epithelial-mesenchymal transition (EMT). Xenografted tumors were collected from mice to further verify the in vitro results. ResultsHigher miR-660-5p and lower KLF3 levels were examined in HCC. KLF3 was targeted by miR-660-5p. Up-regulated miR-660-5p-modified M2-Exo boosted the grwoth and EMT of HepG2 cells, but this effect was impaired by overexpression of KLF3. miR-660-5p-loaded M2-Exo enhanced tumorigenic ability of HCC cells in mice. On the contrary, down-regulated miR-660-5p reduced M2-Exo-mediated promotion of growth of HCC cells in vitro and in vivo. ConclusionOur study summarizes that miR-660-5p-loaded M2-Exo augment HCC development through down-regulating KLF3.

KAT6A is associated with sorafenib resistance and contributes to progression of hepatocellular carcinoma by targeting YAP

Hepatocellular carcinoma (HCC) is a prevalent solid cancer worldwide and sorafenib is a common treatment. Nevertheless, sorafenib resistance is a severe clinical problem. In the present study, we identified that epigenetic regulator, KAT6A, was overexpressed in clinical HCC tissues and sorafenib-resistant HCC samples. The depletion of KAT6A repressed the cell viability and Edu-positive cell numbers of HCC cells. The IC50 value of sorafenib was increased in sorafenib-resistant HCC cells. In addition, the expression of KAT6A was induced in sorafenib-resistant HCC cells. The depletion of KAT6A suppressed the IC50 of sorafenib. Mechanically, YAP was decreased by the depletion of KAT6A. KAT6A was able to enrich in the promoter of YAP. The silencing of KAT6A reduced the enrichment of histone H3 lysine 23 acetylation (H3K23ac) and RNA polymerase II (RNA pol II) on the promoter of YAP in sorafenib-resistant HCC cells. KAT6A inhibitor WM-1119 repressed the cell proliferation of sorafenib-resistant HCC cells, while overexpression of KAT6A or YAP could reverse the effect in the cells. Meanwhile, the treatment of sorafenib inhibited the viability of sorafenib-resistant HCC cells, while the co-treatment of WM-1119 could improve the effect of sorafenib. Collectively, KAT6A was associated with sorafenib resistance and contributes to progression of HCC by targeting YAP. Targeting KAT6A may be served as a promising therapeutic approach for HCC.