LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma

Lysyl oxidase‐like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid‐Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Long non‐coding RNA SNAI3‐AS1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the UPF1/Smad7 signalling pathway

Emerging evidence has indicated that deregulation of long non‐coding RNAs (lncRNAs) can contribute to the progression of human cancers, including hepatocellular carcinoma (HCC). However, the role and exact mechanism of most lncRNAs in tumours remains largely unknown. In the current study, we found a novel long non‐coding RNA termed SNAI3‐AS1 which was generally up‐regulated in HCC tissues compared with normal control. Higher expression of SNAI3‐AS1 was significantly correlated with shorter overall survival of HCC patients. Knockdown of SNAI3‐AS1 inhibited the proliferation and metastasis of HCC cells in vitro, whereas overexpression of SNAI3‐AS1 promoted the proliferation and metastasis of HCC cells. Further investigations showed that SNAI3‐AS1 could affect HCC tumorigenesis by binding up‐frameshift protein 1 (UPF1), regulating Smad7 expression and activating TGF‐β/Smad pathway. Functionally, SNAI3‐AS1 promoted HCC growth and metastasis by inducing tumour epithelial to mesenchymal transition (EMT). Taken together, these findings showed that SNAI3‐AS1 promotes the progression of HCC by regulating the UPF1 and activating TGF‐β/Smad pathway.     

Long non‐coding RNA deleted in lymphocytic leukaemia 1 promotes hepatocellular carcinoma progression by sponging miR‐133a to regulate IGF‐1R expression

Long non‐coding RNA (lncRNA) deleted in lymphocytic leukaemia 1 (DLEU1) was reported to be involved in the occurrence and development of multiple cancers. However, the exact expression, biological function and underlying mechanism of DLEU1 in hepatocellular carcinoma (HCC) remain unclear. In this study, real‐time quantitative polymerase chain reaction (qRT‐PCR) in HCC tissues and cell lines revealed that DLEU1 expression was up‐regulated, and the increased DLEU1 was closely associated with advanced tumour‐node‐metastasis stage, vascular metastasis and poor overall survival. Function experiments showed that knockdown of DLEU1 significantly inhibited HCC cell proliferation, colony formation, migration and invasion, and suppressed epithelial to mesenchymal transition (EMT) process via increasing the expression of E‐cadherin and decreasing the expression of N‐cadherin and Vimentin. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay demonstrated that DLEU1 could sponge miR‐133a. Moreover, miR‐133a inhibition significantly reversed the suppression effects of DLEU1 knockdown on HCC cells. Besides, we found that silenced DLEU1 significantly decreased insulin‐like growth factor 1 receptor (IGF‐1R) expression (a target of miR‐133a) and its downstream signal PI3K/AKT pathway in HCC cells, while miR‐133a inhibitor partially reversed this trend. Furthermore, DLEU1 knockdown impaired tumour growth in vivo by regulating miR‐133a/IGF‐1R axis. Collectively, these findings indicate that DLEU1 promoted HCC progression by sponging miR‐133a to regulate IGF‐1R expression. Deleted in lymphocytic leukaemia 1/miR‐133a/IGF‐1R axis may be a novel target for treatment of HCC.     

Integrated bioinformatics analysis revealed the regulation of angiogenesis by tumor cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, metastasis accounts for most of the cases. Angiogenesis plays an important role in cancer metastasis, but how tumor cells affect the function of endothelial cells by dictating their microRNA (miRNA) expression remains largely unknown.Differentially expressed miRNAs (DEMs) were identified through dataset downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. We then used online tools to obtain potential targets of candidate miRNAs and functional enrichment analysis, as well as the protein-protein interaction (PPI). Finally, the function of miR-302c-3p was validated through in vitro assay.In the current study, we found that HCC cells altered miRNA expression profiles of human umbilical vein endothelial cells (HUVECs) and miR-302c-3p was the most down-regulated miRNA in HUVECs when they were co-cultured with HCC-LM3 cells. Functional enrichment analysis of the candidate targets revealed that these genes were involved in epigenetic regulation of gene expression, in particular, cytosine methylation. In addition, PPI network demonstrated distinct roles of genes targeted by miR-302c-3p. Importantly, inhibition of angiogenesis, migration and permeability by the most down-regulated miR-302c-3p in HUVECs was confirmed in vitro. These findings brought us novel insight into the regulation of angiogenesis by HCC cells and provided potential targets for the development of therapeutic strategies.     

Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.     

Loss‐of‐function of miR‐142 by hypermethylation promotes TGF‐β‐mediated tumour growth and metastasis in hepatocellular carcinoma

Objectives

Hypermethylation‐induced epigenetic silencing of tumour suppressor genes (TSGs) are frequent events during carcinogenesis. MicroRNA‐142 (miR‐142) is found to be dysregulated in cancer patients to participate into tumour growth, metastasis and angiogenesis. However, the tumour suppressive role of miR‐142 and the status of methylation are not fully understood in hepatocellular carcinoma (HCC).

Methods

Hepatocellular carcinoma tissues and corresponding non‐neoplastic tissues were collected. The expression and function of miR‐142 and TGF‐β in two HCC cell lines were determined. The miRNA‐mRNA network of miR‐142 was analysed in HCC cell lines.

Results

We found that the miR‐142 expression was reduced in tumour tissues and two HCC cell lines HepG2 and SMMC7721, which correlated to higher TNM stage, metastasis and differentiation. Moreover, miR‐142 was identified to directly target and inhibit transforming growth factor β (TGF‐β), leading to decreased cell vitality, proliferation, EMT and the ability of pro‐angiogenesis in TGF‐β‐dependent manner. Interestingly, the status of methylation of miR‐142 was analysed and the results found the hypermethylated miR‐142 in tumour patients and cell lines. The treatment of methylation inhibitor 5‐Aza could restore the expression of miR‐142 to suppress the TGF‐β expression, which impaired TGF‐β‐induced tumour growth.

Conclusion

These findings implicated that miR‐142 was a tumour suppressor gene in HCC and often hyermethylated to increase TGF‐β‐induced development of hepatocellular carcinoma.

     

Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin β1

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.     

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of β‐catenin and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH‐GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of β‐catenin and reprogramming genes p‐Oct4, Nanog, Klf4, c‐myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co‐localization with β‐catenin in the cytoplasm, preventing β‐catenin from entering the nucleus. Treatment with the specific Wnt/β‐catenin pathway inhibitor salinomycin was able to reduce the expression of β‐catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA‐GATA5 restored β‐catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of β‐catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/β‐catenin pathway and the reduction of reprogramming gene expression.     

Long non‐coding RNA F11‐AS1 inhibits HBV‐related hepatocellular carcinoma progression by regulating NR1I3 via binding to microRNA‐211‐5p

Long non‐coding RNAs (lncRNAs) could regulate growth and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to investigate the mechanism of lncRNA F11‐AS1 in hepatitis B virus (HBV)–related HCC. The relation of lncRNA F11‐AS1 expression in HBV‐related HCC tissues to prognosis was analysed in silico. Stably HBV‐expressing HepG2.2.15 cells were established to explore the regulation of lncRNA F11‐AS1 by HBx protein, as well as to study the effects of overexpressed lncRNA F11‐AS1 on proliferation, migration, invasion and apoptosis in vitro. Subsequently, the underlying interactions and roles of lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis in HBV‐related HCC were investigated. Additionally, the influence of lncRNA F11‐AS1 and miR‐211‐5p on tumour growth and metastasis capacity of HepG2.2.15 cells were studied on tumour‐bearing nude mice. Poor expression of lncRNA F11‐AS1 was correlated with poor prognosis in patients with HBV‐related HCC, and its down‐regulation was caused by the HBx protein. lncRNA F11‐AS1 was proved to up‐regulate the NR1I3 expression by binding to miR‐211‐5p. Overexpression of lncRNA F11‐AS1 reduced the proliferation, migration and invasion, yet induced apoptosis of HepG2.2.15 cells in vitro, which could be abolished by overexpression of miR‐211‐5p. Additionally, either lncRNA F11‐AS1 overexpression or miR‐211‐5p inhibition attenuated the tumour growth and metastasis capacity of HepG2.2.15 cells in vivo. Collectively, lncRNA F11‐AS1 acted as a modulator of miR‐211‐5p to positively regulate the expression of NR1I3, and the lncRNA F11‐AS1/miR‐211‐5p/NR1I3 axis participated in HBV‐related HCC progression via interference with the cellular physiology of HCC.     

SRXN1 stimulates hepatocellular carcinoma tumorigenesis and metastasis through modulating ROS/p65/BTG2 signalling

Sulfiredoxin 1 (SRXN1) is a pivotal regulator of the antioxidant response in eukaryotic cells. However, the role of SRXN1 in hepatocellular carcinoma (HCC) is far from clear. The present study aims to elucidate whether SRXN1 participates in tumorigenesis and metastasis of HCC and to determine the molecular mechanisms. We found that SRXN1 expression was up‐regulated in HCC tissue samples and correlated with poor prognosis in HCC patients. We also observed that SRXN1 knockdown by transient siRNA transfection inhibited HCC cell proliferation, migration and invasion. Overexpression of SRXN1 increased HCC cell migration and invasion. B‐cell translocation gene 2 (BTG2) was identified as a downstream target of SRXN1. Mechanistic studies revealed that SRXN1‐depleted reactive oxygen species (ROS) modulated migration and invasion of HCC cells. In addition, the ROS/p65/BTG2 signalling hub was found to regulate the epithelial‐mesenchymal transition (EMT), which mediates the pro‐metastasis role of SRXN1 in HCC cells. In vivo experiments showed SRXN1 promotes HCC tumour growth and metastasis in mouse subcutaneous xenograft and metastasis models. Collectively, our results revealed a novel pro‐tumorigenic and pro‐metastatic function of SRXN1 in HCC. These findings demonstrate a rationale to exploit SRXN1 as a therapeutic target effectively preventing metastasis of HCC.     

Betulinic acid induces apoptosis and suppresses metastasis in hepatocellular carcinoma cell lines in vitro and in vivo

Hepatocellular carcinoma (HCC) is a high incidence and mortality malignant tumour globally. Betulinic acid (BA) is a pentacyclic triterpenoid with potential pro‐apoptotic activities which widely found in many plants. In this study, we determined the effects of BA on proliferation, apoptosis, invasion, and metastasis in HCC cell lines and on tumour growth and pulmonary metastasis in mice. The results suggested that BA could inhibit cell viability and proliferation of HCC cell lines including HepG2, LM3, and MHCC97H. In addition, BA induced apoptosis of HepG2 cells characterised condensed nuclei and nuclear fragmentation. Moreover, western blot analysis showed that BA‐induced apoptosis associated with increasing of pro‐apoptotic protein Bax and cleaved caspase‐3 and decreasing of anti‐apoptotic protein Bcl‐2. Meanwhile, BA also reduced the reactive oxygen species (ROS) level. Furthermore, BA also significantly inhibited HCC growth in vivo and blocked pulmonary metastasis of HCC by regulating the metastasis‐related proteins including MMP‐2, MMP‐9, and TIMP2 without obvious toxicity. In all, the present study suggested that BA might be a promising anti‐HCC drug candidate by inhibiting proliferation, inducing apoptosis, and blocking metastasis.     

Slug promoted vasculogenic mimicry in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. Epithelial–mesenchymal transition (EMT) regulator slug have been implicated in the tumour invasion and metastasis of human hepatocellular carcinoma (HCC). However, the relationship between slug and VM formation is not clear. In the study, we demonstrated that slug expression was associated with EMT and cancer stem cell (CSCs) phenotype in HCC patients. Importantly, slug showed statistically correlation with VM formation. We consistently demonstrated that an overexpression of slug in HCC cells significantly increased CSCs subpopulation that was obvious by the increased clone forming efficiency in soft agar and by flowcytometry analysis. Meantime, the VM formation and VM mediator overexpression were also induced by slug induction. Finally, slug overexpression lead to the maintenance of CSCs phenotype and VM formation was demonstrated in vivo. Therefore, the results of this study indicate that slug induced the increase and maintenance of CSCs subpopulation and contributed to VM formation eventually. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.     

Disulfiram combined with copper inhibits metastasis and epithelial–mesenchymal transition in hepatocellular carcinoma through the NF‐κB and TGF‐β pathways

Late‐stage hepatocellular carcinoma (HCC) usually has a low survival rate because of the high risk of metastases and the lack of an effective cure. Disulfiram (DSF) has copper (Cu)‐dependent anticancer properties in vitro and in vivo. The present work aims to explore the anti‐metastasis effects and molecular mechanisms of DSF/Cu on HCC cells both in vitro and in vivo. The results showed that DSF inhibited the proliferation, migration and invasion of HCC cells. Cu improved the anti‐metastatic activity of DSF, while Cu alone had no effect. Furthermore, DSF/Cu inhibited both NF‐κB and TGF‐β signalling, including the nuclear translocation of NF‐κB subunits and the expression of Smad4, leading to down‐regulation of Snail and Slug, which contributed to phenotype epithelial–mesenchymal transition (EMT). Finally, DSF/Cu inhibited the lung metastasis of Hep3B cells not only in a subcutaneous tumour model but also in an orthotopic liver metastasis assay. These results indicated that DSF/Cu suppressed the metastasis and EMT of hepatic carcinoma through NF‐κB and TGF‐β signalling. Our study indicates the potential of DSF/Cu for therapeutic use.