Comprehensive analysis of partial epithelial mesenchymal transition-related genes in hepatocellular carcinoma

Increasing evidence has revealed that cancer cells undergoing an intermediate state, partial epithelial mesenchymal transition (p-EMT), tend to metastasize rather than complete EMT. We performed a comprehensive analysis of E-cadherin and 25 p-EMT-related genes in HCC to explore the roles and regulatory mechanisms of them in HCC. We analysed E-cadherin and 25 p-EMT-related genes in HCC and constructed an mRNA-miRNA-lncRNA ceRNA subnetwork containing p-EMT-related genes by bioinformatic approaches. IHC was used to identify the protein expression of key p-EMT-related genes, P4HA2, ITGA5, MMP9, MT1X and SPP1. Complete EMT is not necessary for HCC progression. Overexpression of P4HA2, ITGA5, MMP9, SPP1 and down-regulation of MT1X were found in HCC tissues, which were significantly associated with poor prognosis of HCC patients. By means of stepwise reverse prediction and validation from mRNA to lncRNA, an mRNA-miRNA-lncRNA ceRNA subnetwork correlated with HCC prognosis was identified by expression and survival analysis. This study implied that key p-EMT-related genes P4HA2, ITGA5, MMP9, MT1X, SPP1 could be prognostic biomarkers and potential targets of therapy for HCC patients. We constructed an mRNA-miRNA-lncRNA subnetwork containing p-EMT-related genes successfully, among which each component might be utilized as a prognostic biomarker of HCC.     

miR-214-5p targets KLF5 and suppresses proliferation of human hepatocellular carcinoma cells

MicroRNAs (miRNAs) are small endogenous conserved RNAs regulating genes expression through base pairing with the 3′-untranslated region (3′-UTR) of target messenger RNAs. MiR-214-5p is a newly identified miRNA with its biological role largely unknown. In this study, we explored miR-214-5p expression status in 78 paired tumor and nontumor tissues obtained from patients with hepatocellular carcinoma (HCC) by RT-qPCR. The effects of miR-214-5p expression on HCC cell proliferation, cell cycle progression, and cell migration were measured by CCK-8 assay, flow cytometry, and wound-healing assay. A dual-luciferase activity assay was performed to identify whether KLF5 was a target of miR-214-5p. Kaplan-Meier curve and log-rank test were used to investigate the effects of miR-214-5p and KLF5 on overall survival and disease-free survival of patients with HCC. We found miR-214-5p expression was sharply reduced in HCC tissues and cell lines compared with the normal tissues and cell lines. Functional assay revealed that miR-214-5p overexpression could downregulate cell proliferation, cell migration, and arrested cell cycle at G0/G1 phase. Further, we validated Krüppel-like factor 5 (KLF5) as a direct target of miR-214-5p, and was upregulated in HCC and inversely correlated with the expression of miR-214-5p. Moreover, we found the low expression of miR-214-5p and high expression of KLF5 were correlated with tumor size, tumor stage, and poorer 5-year overall survival and disease-free survival of patients with HCC. In conclusion, our results suggested miR-214-5p functions as a tumor suppressor through targeting KLF5 in HCC. Also, miR-214-5p and KLF5 were identified as potential prognostic markers and might be therapeutic targets in HCC.     

DANCR contributed to hepatocellular carcinoma malignancy via sponging miR-216a-5p and modulating KLF12

Long noncoding RNA (lncRNA) differentiation antagonizing nonprotein coding RNA (DANCR) has been identified as an oncogene in several cancers. However, the biological function and role of DANCR in hepatocellular carcinoma (HCC) remain unclear. Our current study aimed to investigate the detailed mechanism of DANCR in HCC. We found that DANCR was significantly upregulated in HCC cell lines in comparison to LO2 cells. Then, we observed that knockdown of DANCR could greatly inhibit Huh7 and HepG2 cell proliferation. In addition, HCC cell apoptosis was increased by silence of DANCR and meanwhile, cell cycle progression was blocked in G1 phase. Apart from these, downregulation of DANCR repressed HCC cell migration and invasion ability obviously. As predicted by the bioinformatics analysis, microRNA-216a-5p (miR-216a-5p) could serve as a direct target of DANCR. MiR-216a-5p has been reported to be involved in many cancers. Here, the correlation between miR-216a-5p and DANCR was confirmed using dual-luciferase reporter assay and radioimmunoprecipitation assay. Subsequently, Kruppel-like factor 12 (KLF12) exerts an important role in different tumor types. KLF12 can function as a downstream target of miR-216a-5p. Finally, the in vivo experiments were used and the data proved that DANCR also strongly suppressed HCC tumor growth in vivo via targeting miR-216a-5p and KLF12. In conclusion, our study indicated that DANCR might provide a new perspective for HCC treatment.     

Retracted: miR-942 promotes tumor migration,invasion, and angiogenesis by regulating EMT via BARX2 in non-small-cell lung cancer

Epithelial–mesenchymal transition (EMT) has an important function in cancer. Recently, microRNAs have been reported to be involved in EMT by regulating target genes. miR-942 is considered a novel oncogene in esophageal squamous cell carcinoma. However, its role in non-small-cell lung cancer (NSCLC) has not been investigated. In this study, the expression of miR-942 in NSCLC patients tumor and paired adjacent tissues were assessed by quantitative real-time polymerase chain reaction and in situ hybridization. Transwell, wound healing, tube formation, and tail vein xenograft assays were conducted to assess miR-942′s function in NSCLC. Potential miR-942 targets were confirmed using dual-luciferase reporter assays, immunohistochemistry, immunoblot, and rescue experiments. The results showed miR-942 is relatively highly expressed in human NSCLC tissues and cells. In vitro assays demonstrated that overexpression of miR-942 promoted cell migration, invasion, and angiogenesis. Tail vein xenograft assays suggested that miR-942 contributed to NSCLC metastasis in vivo. Three bioinformatics software was searched, and BARX2 was predicted as a downstream target of miR-942. Direct interaction between them was validated by dual-luciferase assays. Rescue experiments further confirmed that BARX2 overexpression could reverse functional changes caused by miR-942. Moreover, miR-942 increased EMT-associated proteins N-cadherin and vimentin by inhibiting BARX2, while E-cadherin expression is reduced. In summary, this study reveals that miR-942 induces EMT-related metastasis by directly targeting BARX2, which may provide a potential therapeutic strategy for NSCLC.     

Overexpression of KiSS1 Induces the Proliferation of Hepatocarcinoma and Increases Metastatic Potential by Increasing Migratory Ability and Angiogenic Capacity

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of β-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.     

CCAT2 enhances autophagy-related invasion and metastasis via regulating miR-4496 and ELAVL1 in hepatocellular carcinoma

Autophagy is thought to contribute to the pathogenesis of many diseases, including cancer. Long non-coding RNA (lncRNA) CCAT2 functions as an oncogene in a variety of tumours. However, it is still unknown whether CCAT2 is involved in autophagy and metastasis of hepatocellular carcinoma (HCC). In our study, we found that lncRNA CCAT2 expression was significantly increased in HCC tissue and was correlated with advanced stage and venous invasion. Further experiments revealed that CCAT2 induced autophagy and promoted migration and invasion in vitro and in vivo. Mechanistic investigations found that CCAT2 involved in HCC by regulating miR-4496/Atg5 in cytoplasm. In nucleus, CCAT2 bound with ELAVL1/HuR to facilitate HCC progression. Our findings suggest that CCAT2 is an oncogenic factor in the progression of HCC with different regulatory mechanisms and may serve as a target for HCC therapy.     

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.     

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.     

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.     

Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment

Every year about 500,000 people in the United States die as a result of cancer. Among them, 90% exhibit systemic disease with metastasis. Considering this high rate of incidence and mortality, it is critical to understand the mechanisms behind metastasis and identify new targets for therapy. In recent years, two broad mechanisms for metastasis have received significant attention: epithelial-to-mesenchymal transition (EMT) and tumor microenvironment interactions. EMT is believed to be a major mechanism by which cancer cells become migratory and invasive. Various cancer cells--both in vivo and in vitro--demonstrate features of epithelial-to-mesenchymal-like transition. In addition, many steps of metastasis are influenced by host contributions from the tumor microenvironment, which help determine the course and severity of metastasis. Here we evaluate the diverse mechanisms of EMT and tumor microenvironment interactions in the progression of cancer, and construct a rational argument for targeting these pathways to control metastasis.     

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.     

肝细胞癌中NF-kBp65,VEGF和MMP-9蛋白的表达及其相关性研究

目的:探讨肝细胞癌(HCC)组织中NF-kBp65,VEGF和MMP-9的表达及其临床意义。方法:应用免疫组织化学(SP法)检测35例HCC组织,15例癌旁肝组织及10例正常肝组织中NF-kBp65,VEGF和MMP-9蛋白的表达并分析其与临床病理因素的关系。结果:NF-kBp65,VEGF和MMP-9蛋白在HCC组织中高表达率(分别为80%,74%,71%)高于对照组(P<0.01)。NF-kBp65的表达与HCC的Edmondson分级有关,即在Edmondson分级Ⅲ-Ⅳ的癌细胞中NF-kBp65的表达强于Edmondson分级Ⅰ-Ⅱ的(P<0.05);有静脉浸润的HCC组织中NF-kBp65,VEGF和MMP-9的表达高于无静脉浸润者,差异有统计学意义(P<0.05)。但NF-kBp65的表达与年龄,性别,肿瘤大小,AFP水平无关(P>0.05);VEGF和MMP-9的表达与年龄,性别,肿瘤大小,AFP水平,HCC的Edmondson分级无关(P>0.05)。NF-kBp65的表达与VEGF和MMP-9呈正相关(rs1=0.579,P1<0.05;rs2=0.406,P2<0.05)。结论:NF-kB...     

Inflammatory microenvironment of fibrotic liver promotes hepatocellular carcinoma growth,metastasis and sorafenib resistance through STAT3 activation

The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl₄ gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.