Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells

A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty‐seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP‐producing) and liver cancer cell line cells (non‐AFP‐producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down‐regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP‐expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up‐regulating expression of metastasis‐related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients.     

The Suppressive Role and Aberrent Promoter Methylation of BTG3 in the Progression of Hepatocellular Carcinoma

Background

BTG3 (B-cell translocation gene 3) has been identified as a tumor suppressor and hypermethylation contributes to its down-regulation in some tumors, but its role in hepatocellular carcinoma (HCC) remain unknown. This study aimed to detect the expression and methylation status of BTG3 in HCC cell lines or tissues, and determine its function in HCC progression.

Methodology

The expression of BTG3 was detected in HCC cell lines and HCC tissue by real-time RT-PCR, Western blot or immunohistochemistry. The promoter methylation status of BTG3 was measured by using methylation-specific PCR in HCC cell lines. A series of assays were performed to evaluate the effect of BTG3 on proliferation, invasion and cell cycle transition in vitro.

Results

BTG3 expression was lower in HCC cell lines than in hepatocyte cell line LO2 (P<0.05). BTG3 was also down-regulated in HCC tissues. Its expression was positively correlated with differentiation and distant metastasis (P<0.05). Patients with lower BTG3 expression had shorter overall survival time (P=0.029). DNA methylation directed repression of BTG3 mRNA expression in HCC cell lines. BTG3 suppressed proliferation, invasion and induces G1/S cycle arrest of HCC cells in vitro.

Conclusion

Down-regulation of BTG3 due to the promoter hypermethylation is closely associated with proliferation, invasion and cell cycle arrest of HCC cells. It may be a novel prognostic biomarker for HCC patients.     

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.     

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.     

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.     

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.     

Pancreatic cancer cell–derived exosomal microRNA‐27a promotes angiogenesis of human microvascular endothelial cells in pancreatic cancer via BTG2

Pancreatic cancer (PC) remains a primary cause of cancer‐related deaths worldwide. Existing literature has highlighted the oncogenic role of microRNA‐27a (miR‐27a) in multiple cancers. Hence, the current study aimed to clarify the potential therapeutic role of PC cell–derived exosomal miR‐27a in human microvascular endothelial cell (HMVEC) angiogenesis in PC. Initially, differentially expressed genes (DEGs) and miRs related to PC were identified by microarray analysis. Microarray analysis provided data predicting the interaction between miR‐27a and BTG2 in PC, which was further verified by the elevation or depletion of miR‐27a. Next, the expression of miR‐27a and BTG2 in the PC tissues was quantified. HMVECs were exposed to exosomes derived from PC cell line PANC‐1 to investigate the effects associated with PC cell–derived exosomes carrying miR‐27a on HMVEC proliferation, invasion and angiogenesis. Finally, the effect of miR‐27a on tumorigenesis and microvessel density (MVD) was analysed after xenograft tumour inoculation in nude mice. Our results revealed that miR‐27a was highly expressed, while BTG2 was poorly expressed in both PC tissues and cell lines. miR‐27a targeted BTG2. Moreover, miR‐27a silencing inhibited PC cell proliferation and invasion, and promoted apoptosis through the elevation of BTG2. The in vitro assays revealed that PC cell–derived exosomes carrying miR‐27a stimulated HMVEC proliferation, invasion and angiogenesis, while this effect was reversed in the HMVECs cultured with medium containing GW4869‐treated PANC‐1 cells. Furthermore, in vivo experiment revealed that miR‐27a knockdown suppressed tumorigenesis and MVD. Taken together, cell‐derived exosomes carrying miR‐27a promotes HMVEC angiogenesis via BTG2 in PC.     

Lectin BS‐I inhibits cell migration and invasion via AKT/GSK‐3β/β‐catenin pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is most common malignant cancer worldwide; however, the mortality rate of HCC remains high due to the invasion and metastasis of HCC. Thus, exploring novel treatments to prevent the invasion of HCC is needed for improving clinical outcome of this fatal disease. In this study, we identified lectin from Bandeiraea simplicifolia seeds (BS‐I) binds to metastasis‐associated HCC cell surface glycans by a lectin microarray and inhibits HCC cell migration and invasion through downregulating the matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and urokinase‐type plasminogen activator (uPA) production. These effects of BS‐I were mediated by inhibiting the activation of AKT/GSK‐3β/β‐catenin pathway and depended on specificity of lectin BS‐I binding to GalNAc. GSK3β inhibitors rescued BS‐I‐mediated inhibition of migration and invasion of HCC cell. Further, we identified that lectin BS‐I interacts with sGrp78, affects membrane localization of sGrp78 and attenuates the binding of sGrp78 and p85 to inhibit the activation of AKT/GSK‐3β/β‐catenin pathway. Overexpression of Grp78 or P85 rescues BS‐I‐mediated inhibition of migration and invasion of HCC cell. These findings demonstrated for the first time that BS‐I can act as a novel potential drug to prevent the invasion of HCC.     

MFN1在肝癌转移中的调控作用研究

目的:探讨线粒体融合蛋白MFN1(mito-fusion 1)在肝癌转移中的作用及其机制。方法:1).采用免疫组化实验检测15对肝癌转移灶组织与原发灶组织中MFN1的表达,以明确肝癌转移时是否伴有MFN1表达的改变。2).采用si RNA (small interference RNA)下调肝癌细胞中MFN1的表达后,提高Transwell迁移实验和Transwell侵袭实验分别检测其迁移和侵袭能力,通过实时荧光定量PCR (Quantitative Real-time PCR,qRT-PCR)和Western blot实验分别检测基质金属蛋白酶1 (matrix metalloproteinase 1,MMP1)、MMP2、MMP7及MMP9的m RNA和蛋白表达。结果:1)肝癌转移灶组织中MFN1表达显著低于原发灶组织(P0.05)。2).下调MFN1表达后,肝癌细胞的迁移和侵袭能力显著升高,MMP7的表达显著增加,而MMP1、MMP2与MMP9的表达无明显变化。结论:线粒体融合蛋白MFN1在肝癌转移组织中表达显著降低,可能通过激活MMP7表达,促进肝癌细胞侵袭和转移。     

miR-186 modulates hepatocellular carcinoma cell proliferation and mobility via targeting MCRS1-mediated Wnt/β-catenin signaling

Previous studies have revealed that miR-186 is involved in the pathogenesis of many malignancies. However, the role of miR-186 in hepatocellular carcinoma (HCC) carcinogenesis and its detailed mechanism are poorly understood. This study was to investigate the function of miR-186 in modulating HCC cell proliferation, cell cycle, migration, and invasion. We found that miR-186 was decreased in HCC tissues and cell lines. Loss-of-function experiments showed that reduction of miR-186 dramatically enhanced tumor cell proliferation and metastasis. Besides, miR-186 also participated in the modulation of the cell cycle. In addition, luciferase reporter assays and Western blot analysis showed that MCRS1 was a novel target of miR-186 in HCC cells. Notably, upregulation of miR-186 suppressed the nuclear β-catenin accumulation and blocked the activation of Wnt/β-catenin signaling in HCC cells. Forced MCRS1 expression abrogated the inhibitory effect of miR-186 on cell growth, metastasis and Wnt/β-catenin signaling in HCC cells. Our findings may provide new insight into the pathogenesis of HCC and miR-186/ MCRS1 might function as new therapeutic targets for HCC.     

EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis

Eye absent homolog 4 (EYA4) has been demonstrated to be down‐regulated in hepatocellular carcinoma (HCC), but its biological function and the mechanism in HCC angiogenesis and metastasis remain largely unknown. Herein, we showed that EYA4 expression was frequently low in HCC tissue samples compared with matched adjacent non‐tumourous tissues. In the analysis of 302 HCC specimens, we revealed that decreased expression of EYA4 correlated with tumour differentiation status. Univariate and multivariate analyses identified EYA4 as an independent risk factor for recurrence‐free survival (RFS) and overall survival (OS) among the 302 patients. Functional assays showed that forced expression of EYA4 suppressed HCC cell migration, invasion and capillary tube formation of endothelial cells in vitro, as well as in vivo tumour angiogenesis and metastasis in a mouse model. Furthermore, mechanism study exhibited that EYA4 could inhibit HCC angiogenesis and metastasis by inhibiting c‐JUN/VEGFA pathway. Together, we provide proof that EYA4 is a novel tumour suppressor in HCC and a new prognostic biomarker and therapeutic target in HCC.     

Effects of microRNA-30a on migration,invasion and prognosis of hepatocellular carcinoma

The role of microRNA-30a (miR-30a) deregulation in tumor progression and its downstream signaling pathways remain unknown. Here we confirmed significant downregulation of miR-30a in hepatocellular carcinoma (HCC) tissues and cell lines compared with non-tumor counterparts. MiR-30a downregulation was significantly associated with worse disease-free survival (DFS) of HCC patients. Gain- and loss-of-function studies revealed that downregulation of miR-30a facilitated tumor cell migration, invasion and epithelial–mesenchymal transition (EMT). We identified SNAI1 as a direct target of miR-30a and demonstrated miR-30a as a novel regulator of EMT by targeting SNAI1, indicating its potential therapeutic value for reducing invasion and metastasis of HCC.     

Autophagy inhibition suppresses pulmonary metastasis of HCC in mice via impairing anoikis resistance and colonization of HCC cells

Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma (HCC), which has been linked to cell-death resistance. Autophagy is an important survival mechanism under conditions of cell stress. We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect. Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes. Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition. The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo. Further invasion, migration, detachment, lung colonization, and epithelial-mesenchymal transition (EMT) assays indicated that autophagy inhibition did not affect cell invasiveness, migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells. Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling. As autophagy inhibition was shown to be able to suppress HCC metastasis, an autophagy-based HCC tissue-specific target therapy system (AFP-Cre/LoxP-shRNA) was constructed. In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner. Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis. Together, our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells. Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.     

Decreased expression of SERPINB1 correlates with tumor invasion and poor prognosis in hepatocellular carcinoma

SERPINB1 (serine protease inhibitor, clade B, member1) is a member of the SERPINB family. Recent studies suggested that SERPINB1 may suppress the migration and invasion of lung and breast cancers. In this study, we investigated a possible involvement of SERPINB1 in the regulation of hepatocellular carcinoma metastasis (HCC). The expression of SERPINB1 was evaluated using western blot analysis in 8 paired fresh HCC specimens and immunohistochemistrical assay on 67 paraffin-embedded HCC slices. SERPINB1 was downregulated in HCC specimens and correlatively related with two clinicopathologic features of HCC, metastasis (P = 0.000) and vein invasion (P = 0.006). Univariate and multivariate survival analyses showed a lower level of SERPINB1 expression is associated with poor prognosis and clinical outcome (P = 0.001). In addition, small interfering RNA targeting SERPINB1 was used to knock down the expression of SERPINB1 in Huh7 and BEL-7404 cells. We showed that interference of SERPINB1 promoted migration and invasion of HCC cells, while cell proliferation was not affected. Finally, we observed an apparent increase in the level of active matrix metalloproteinase-2 (MMP2) after SERPINB1 knockdown, implying that SERPINB1 might participate in the regulation of HCC metastasis through modulating the activation of matrix metalloproteinases. Overall, our results suggested an inhibitory role of SERPINB1 in the migration and invasion of HCC, implying that SERPINB1 might be a potential prognostic indicator of HCC metastasis.     

miR-9 inhibits the metastatic ability of hepatocellular carcinoma via targeting beta galactoside alpha-2,6-sialyltransferase 1

Glycosylation of cell surface proteins regulates critical cellular functions, including invasion and metastasis in cancer cells. Emerging evidence has shown that microRNAs (miRNAs) are involved in regulating both the glycosylation modifications on cell surface and the progression of cancer. In this study, we investigated the role of miR-9 in α-2,6-linked sialylation and the metastasis of mouse hepatocellular carcinoma (HCC). According to array-based miRNA expression profiling data of HCC cell lines Hepa1–6, Hca-P, and Hca-F with different lymphatic metastatic capacities, reverse correlation was found between miR-9 expression levels and the metastatic potential in these HCC cells. Additionally, β-galactoside α-2,6-sialyltransferase 1 (St6gal1) expression level is associated negatively with miR-9 and positively with metastatic potential. Bioinformatics analysis indicated that miR-9 could target St6gal1, which was verified by luciferase reporter assays. miR-9 overexpression reduced expression of St6gal1, which subsequently suppressed HCC cells metastatic potential. Moreover, upregulation of miR-9 could inhibit integrin-β1/FAK-mediated cell motility and migration signaling in mouse HCC cells. Together, our results suggest that miR-9 could act as a tumor suppressor and regulate mouse HCC cells migration and invasion by inhibiting the α-2,6-linked sialylation. This finding may provide insight into the relationship between abnormal miRNA expression and aberrant cell surface glycosylation during tumor lymphatic metastasis.     

MicroRNA-122 Triggers Mesenchymal-Epithelial Transition and Suppresses Hepatocellular Carcinoma Cell Motility and Invasion by Targeting RhoA

The loss of microRNA-122 (miR-122) expression is strongly associated with increased invasion and metastasis, and poor prognosis of hepatocellular carcinoma (HCC), however, the underlying mechanisms remain poorly understood. In the present study, we observed that miR-122 over-expression in HCC cell lines Sk-hep-1 and Bel-7402 triggered the mesenchymal-epithelial transition (MET), as demonstrated by epithelial-like morphological changes, up-regulated epithelial proteins (E-cadherin, ZO-1, α-catenin, occludin, BVES, and MST4), and down-regulated mesenchymal proteins (vimentin and fibronectin). The over-expression of miRNA-122 also caused cytoskeleton disruption, RhoA/Rock pathway inactivation, enhanced cell adhesion, and suppression of migration and invasion of Sk-hep-1 and Bel-7402 cells, whereas, these effects could be reversed through miR-122 inhibition. Additional studies demonstrated that the inhibition of wild-type RhoA function induced MET and inhibited cell migration and invasion, while RhoA over-expression reversed miR-122-induced MET and inhibition of migration and invasion of HCC cells, suggesting that miR-122 induced MET and suppressed the migration and invasion of HCC cells by targeting RhoA. Moreover, our results demonstrated that HNF4α up-regulated its target gene miR-122 that subsequently induced MET and inhibited cell migration and invasion, whereas miR-122 inhibition reversed these HNF4α-induced phenotypes. These results revealed functional and mechanistic links among the tumor suppressors HNF4α, miR-122, and RhoA in EMT and invasive and metastatic phenotypes of HCC. Taken together, our study provides the first evidence that the HNF4α/miR-122/RhoA axis negatively regulates EMT and the migration and invasion of HCC cells.     

The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines

High mobility group protein box1 (HMGB1) and its receptor—receptor for advanced glycation end products (RAGE) are pivotal factors in the development and progression of many types of tumor, but the role of HMGB1-RAGE axis in hepatocellular carcinoma (HCC) especially its effects on metastasis and recurrence remains obscure. Here, we report the role of HMGB1-RAGE axis in the biological behaviors of HCC cell lines and the underlying molecular mechanism. We show that the expressions of HMGB1, RAGE, and extracellular HMGB1 increase consistently according to cell metastasis potentials, while the concentration of soluble form of RAGE (sRAGE) is inversely related to metastasis potential of HCC cells. Furthermore, our data show that rhHMGB1 promotes cellular proliferation, migration, and invasion, and increases the level of nuclear factor kappa B (NF-κB), while administrations of HMGB1-siRNA, RAGE-siRNA, anti-HMGB1 neutralizing antibody, anti-RAGE neutralizing antibody, and sRAGE inhibit cellular proliferation, migration, and invasion. Moreover, we also demonstrate that the expression of NF-кB is inhibited by knockdown of HMGB1 or RAGE. Collectively, these data demonstrate that HMGB1 activates RAGE signaling pathways and induces NF-кB activation to promote cellular proliferation, invasion, and metastasis, in HCC cell lines. Taken together, HMGB1-RAGE axis may become a potential target in HCC therapy.