BackgroundHigh frequency of recurrence is the major cause of the poor outcomes for patients with hepatocellular carcinoma (HCC). microRNA (miR)-182-5p emerged as a high-priority miRNA in HCC and was found to be related to HCC metastasis. Whether the expression of miR-182-5p in tumor tissue correlated with early recurrence in HCC patients underwent curative surgery was unknown.MethodsReal-time PCR (RT-PCR) and in situ hybridization (ISH) were conducted to assess the expression of miR-182-5p in HCC cells and tissues. Cell Counting Kit-8 (CCK-8), transwell assays were performed to detected cells proliferation and migration ability. Flow cytometry assays were used to detect cell apoptosis rate, and xenograft model was employed to study miR-182-5p in HCC growth and lung metastasis. The target of miR-182-5p was validated with a dual-luciferase reporter assay and western blotting. Immunohistochemistry, immumoblotting, and immunoprecipitation were performed to test relative protein expression.ResultsWe showed that high expression of miR-182-5p in tumor tissues correlated with poor prognosis as well as early recurrence in HCC patients underwent curative surgery. miR-182-5p enhanced motility and invasive ability of HCC cells both in vitro and in vivo. miR-182-5p directly targets 3′-UTR of FOXO3a and repressed FOXO3a expression, activating AKT/FOXO3a pathway to promote HCC proliferation. Notably, miR-182-5p activated Wnt/β-catenin signaling by inhibiting the degradation of β-catenin and enhancing the interaction between β-catenin and TCF4 which was mediated by repressed FOXO3a.ConclusionsConsistently, miR-182-5p can be a potential predictor of early recurrence for HCC patients underwent curative surgery, and FOXO3a plays a key mediator in miR-182-5p induced HCC progression.

BackgroundOur previous study reported that microRNA-26a (miR-26a) inhibited tumor progression by inhibiting tumor angiogenesis and intratumoral macrophage infiltration in hepatocellular carcinoma (HCC). The direct roles of miR-26a on tumor cell invasion remain poorly understood. In this study, we aim to explore the mechanism of miR-26a in modulating epithelial-mesenchymal transition (EMT) in HCC.MethodsIn vitro cell morphology and cell migration were compared between the hepatoma cell lines HCCLM3 and HepG2, which were established in the previous study. Overexpression and down-regulation of miR-26a were induced in these cell lines, and Western blot and immunofluorescence assays were used to detect the expression of EMT markers. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Immunohistochemical assays were conducted to study the relationships between miR-26a expression and enhancer of zeste homolog 2 (EZH2) and E-cadherin expression in human HCC samples.ResultsDown-regulation of miR-26a in HCCLM3 and HepG2 cells resulted in an EMT-like cell morphology and high motility in vitro and increased in tumor growth and pulmonary metastasis in vivo. Through down-regulation of EZH2 expression and up-regulation of E-cadherin expression, miR-26a inhibited the EMT process in vitro and in vivo. Luciferase reporter assay showed that miR-26a directly interacted with EZH2 messenger RNA (mRNA). Furthermore, the expression of miR-26a was positively correlated with E-cadherin expression and inversely correlated with EZH2 expression in human HCC tissue.ConclusionsmiR-26a inhibited the EMT process in HCC by down-regulating EZH2 expression.

BackgroundSorafenib is recognized as a standard treatment for advanced hepatocellular carcinoma (HCC). However, many patients have to adopt dose reduction or terminate the use of sorafenib because of side effects. In addition, a large number of patients are resistant to sorafenib. Thus, it is essential to investigate the underlying mechanisms of the resistance to sorafenib and seek potential strategy to enhance its efficacy.MethodsThe protein expression of hypoxia-inducible factors (HIF)-2α, 30-kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK was detected by Western blot. Cell viability assays were performed to study the influence of metformin and sorafenib on cell proliferation. Annexin V-FITC apoptosis assays were used to detect the influence of metformin and sorafenib on cell apoptosis. The relationship between HIF-2α and TIP30 was studied using gene silencing approach and chromatin immunoprecipitation assay. To investigate the effect of metformin and sorafenib on postoperative recurrence and lung metastasis of HCC in tumor-bearing mice, the mice were orally treated either with metformin or sorafenib once a day for continuous 37 days after the operation to remove the lobe where the tumor was implanted. CD31, Ki67, and TUNEL were examined by immunohistochemistry.ResultsOur study demonstrated that metformin synergized with sorafenib reduced HIF-2α expression as examined by Western blot. Gene silencing approach indicated TIP30 was upregulated after knocking-down of HIF-2α and chromatin immunoprecipitation assay revealed that HIF-2α could bind to TIP30 promoter under hypoxic condition. Cell Counting Kit-8 (CCK8) cell viability assay and Annexin V-FITC apoptosis assay showed that metformin in combination with sorafenib suppressed cell proliferation and promoted cell apoptosis. Besides, combined therapy suppressed epithelial-mesenchymal transition (EMT) process both in vitro and in vivo. Moreover, metformin in combination with sorafenib significantly minimized postoperative recurrence and lung metastasis of HCC in orthotopic mouse model. Combined therapy inhibited CD31 and Ki67 expression but promoted TUNEL expression.ConclusionsMetformin may potentially enhance the effect of sorafenib to inhibit HCC recurrence and metastasis after liver resection by regulating the expression of HIF-2α and TIP30.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0253-6) contains supplementary material, which is available to authorized users.

Most hepatocellular carcinoma (HCC) patients are diagnosed at an advanced stage; however, the effect of systemic therapy on advanced HCC remains undetermined. Therefore, new treatment targets must be identified. We analyzed Gene Expression Omnibus datasets from two HCC patient cohorts and found that NT5DC2 was associated with vascular invasion and poor survival. In two hepatoma cell lines, NT5DC2 overexpression promoted HCC cell proliferation and clone formation in vitro and promoted tumor growth in vivo. Coimmunoprecipitation assays and liquid chromatography with tandem mass spectrometry analysis revealed that NT5DC2 bound directly to epidermal growth factor receptor (EGFR). NT5DC2 upregulated EGFR expression by downregulating EGFR ubiquitination and preventing its degradation via the ubiquitin-proteasome pathway but did not upregulate its transcription. EGFR upregulation activated downstream signal transduction, which played a critical role in the protumor effects of NT5DC2. Erlotinib, a small-molecule inhibitor of EGFR, blocked the effect of NT5DC2 in promoting HCC cell proliferation. In a cohort of 79 patients who underwent curative resection for HCC, NT5DC2 expression in the tumors was associated with larger tumors and microvascular invasion. NT5DC2 expression was also independently associated with recurrence-free survival. The present study demonstrated for the first time that NT5DC2 promotes tumor cell proliferation in HCC and may serve as a potential molecular target for treating HCC. EGFR blockage could be used to treat selected patients with NT5DC2 upregulation.

Overexpression of CUB domain-containing protein 1 (CDCP1), a transmembrane glycoprotein and major substrate of Src family kinases (SFKs), always indicates unfavorable outcomes in various cancers. The characteristics of CDCP1 in hepatocellular carcinoma (HCC) have not been assessed. Most recently, CDCP1 was identified as a specific target gene of HIF-2α in clear cell renal carcinoma (CC-RCC). However, considering the role of HIF-2α in the progression of HCC is highly controversial, it is necessary to figure out whether HIF-2α and CDCP1 play a significant part in the metastasis of HCC. Our results showed that HIF-2α and CDCP1 were both induced by hypoxia, and the activation of CDCP1 was HIF-2α dependent. CDCP1 was governed by HIF-2α at mRNA and protein levels in HCC cell lines. Moreover, knocking down of HIF-2α not only inhibited cell invasion but also impaired the expression of Tyr(311) phosphorylation of protein kinase Cδ (PKCδ) which is a downstream factor of CDCP1 and has been reported to induce malignant migration in various tumors. Analysis of human HCC samples showed a negative correlation of CDCP1 expression with disease-free survival, and CDCP1 was an independent prognostic factors of disease-free survival. Taken together, these data demonstrated that HIF-2α could promote HCC cell migration by regulating CDCP1, and targeting HIF-2α-CDCP1-PKCδ pathway might be effective to inhibit HCC metastasis.

Metastasis is a serious risk that may occur during the treatment of hepatocellular carcinoma (HCC), preventing many patients from being surgical candidates and contributing to poor prognosis. Hypoxia has been proved an important factor of metastasis through the epithelial–mesenchymal transition (EMT) pathway. Acetyl‐CoA synthase 2 (ACSS2) provides an acetyl group for the acetylation of hypoxia‐inducible factor (HIF)‐2α, and this epigenetic modification affects the activity of HIF‐2α and the subsequent EMT process. Here, we showed that ACSS2 expression was negatively correlated with HCC malignancy. Knockdown of ACSS2 increased the invasion and migration ability of HCC cells and promoted EMT without increasing the total protein level of HIF‐2α, even in hypoxic conditions. The immunoprecipitation assay revealed downregulated acetylation levels of HIF‐2α after ACSS2 knockdown in hypoxic conditions, which resulted in enhanced HIF‐2α activity. Finally, decreased expression of ACSS2 was found to be related to advanced stage and poor overall survival and disease‐free survival rates in a cohort of patients with HCC. In conclusion, ACSS2 plays an important role in the acetylation process of HIF‐2α, which effectively modifies the activity of HIF‐2α under hypoxic conditions and greatly impacts on the prognosis of patients with HCC.

We previously found that a low dose of sorafenib had a prometastatic effect on hepatocellular carcinoma (HCC), which was caused by downregulation of TIP30 expression. More recently, metformin has been shown to have potential as a preventive and therapeutic agent for different cancers, including HCC. This study evaluated whether the combination of sorafenib and metformin is sufficient to revert the expression of TIP30, thereby simultaneously reducing lung metastasis and improving survival. Our data show that the combination of sorafenib and metformin inhibits proliferation and invasion in vitro, prolongs median survival, and reduces lung metastasis of HCC in vivo. This effect is closely associated with the upregulation of TIP30, partly through activating AMP‐activated protein kinase. Thioredoxin, a prometastasis factor, is negatively regulated by TIP30 and plays an essential role during the process of HCC metastasis. Overall, our results suggest that metformin might be a potent enhancer for the treatment of HCC by using sorafenib.

Our previous studies demonstrated that traditional Chinese herbal medicine 'Songyou Yin' inhibited the growth and invasion of hepatocellular carcinoma (HCC) cells, and altered epithelial‑mesenchymal transition (EMT) markers in oxaliplatin‑treated HCC tissues and cell lines. In the present study, we aimed to explore whether astragaloside IV (AS-IV), a component of 'Songyou Yin', can affect the growth and invasion of HCC cells and the underlying mechanism involved. Human HCC cell lines Huh7 and MHCC97-H, with low and high metastatic potential, respectively, were treated with increasing doses of AS-IV. The Cell Counting Kit-8 (CCK-8), plate clone formation, Transwell, wound healing and immunofluorescence assays were used to investigate the effects of AS-IV on HCC cell proliferation, migration and invasion. The protein expression levels were analyzed by western blotting and immunofluorescence assay. The CCK-8 and plate clone formation assays showed that AS-IV had little effect on the proliferation of HCC cells in vitro. However, the Transwell and wound healing assays demonstrated that AS-IV inhibited the migration and invasion of HCC cells in a dose-dependent manner and the morphology of HCC cells was altered from spindle into oval shaped in the AS-IV pretreated groups. The upregulation of E-cadherin and downregulation of N-cadherin, vimentin, α-SMA and Slug were also observed in the AS-IV pretreated groups. Additionally, AS-IV treatment resulted in a profound decrease in the phosphorylated forms of Akt and GSK-3β, which in turn inhibited the expression of β-catenin. Thus, we conclude that AS-IV attenuates the invasive and migratory abilities of HCC cells through the inhibition of EMT by targeting the Akt/GSK-3β/β-catenin pathway.