Hematological and neurological expressed 1 (HN1) activates c-Myc signaling by inhibiting ubiquitin-mediated proteasomal degradation of c-Myc in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis due to the usually advanced stage at diagnosis. Sustained activation of the MYC oncogene is implicated in the development of HCC; however, the molecular mechanisms of MYC deregulation in HCC are poorly understood. Here, real-time PCR and western blotting were used to measure the expression of hematological and neurological expressed 1 (HN1) in HCC cells. Expression of HN1 and MYC in clinical specimens was analyzed using immunohistochemistry. The role of HN1 in HCC proliferation, migration, and invasion was explored in vitro and in vivo. MYC expression was measured using real-time PCR and western blotting. MYC transcriptional activity was assessed using a luciferase reporter system. Expression of MYC target genes was quantified using real-time PCR. Protein interaction between MYC and HN1 was assessed using co-immunoprecipitation and western blotting. We identified HN1 as a novel regulatory factor of the glycogen synthase kinase (GSK) 3ß-MYC axis. HN1 expression is elevated in liver tumor tissues and cells, and significantly correlates with poor survival in HCC patients. Upregulation of HN1 promotes, and silencing of HN1 represses, the proliferation and metastasis of liver cancer cells in vitro and in vivo. Moreover, our results demonstrate that HN1 sustains stabilization and persistent activity of MYC via interaction with GSK3ß in HCC. Importantly, the tumor-promoting effects of HN1 on HCC cells were attenuated by suppressing MYC. In conclusion, constitutive activation of MYC by HN1 promotes the progression of HCC; therefore, HN1 might be a novel therapeutic target for HCC.

PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication.

A viable therapy is needed to overcome the deadlock of the incurable chronic hepatitis B (CHB). The prolonged existence of covalently closed circular DNA (cccDNA) and integrated HBV DNA in the nucleus of hepatocytes is the root cause of CHB. As a result, it is critical to successfully suppress HBV DNA replication and eliminate cccDNA. RNA interference has been proven in recent research to silence the expression of target genes and thereby decrease HBV replication. However, siRNA is susceptible to be degraded by RNA enzymes in vivo, making it difficult to deliver successfully and lacking of tissue targeting. To exploit the advantages of siRNA technology while also overcoming its limitations, we designed a new strategy and prepared biomimetic nanoparticles that were directed by PreS/2-21 peptides and precisely loaded HBV siRNA. Experiments on these nanoparticles in vitro and in vivo revealed that they are tiny, stable, safe and highly targetable, with high inhibitory effects on HBV DNA, pgRNA, cccDNA, HBeAg and HBsAg. PreS/2-21-directed nanoparticles loaded with HBV gene therapy drugs are expected to be promising for the treatment of CHB.

MicroRNA-379-5p inhibits tumor invasion and metastasis by targeting FAK/AKT signaling in hepatocellular carcinoma.

Some microRNAs (miRNAs) have been implicated in hepatocellular carcinoma (HCC) development and progression. However, the roles and mechanisms of several miRNAs in HCC remain poorly understood. Here, we report that miR-379-5p, which is down-regulated in HCC tissues and cell lines, is associated with advanced TNM stage and metastasis in HCC. The ectopic overexpression of miR-379-5p inhibited HCC cell migration, invasion, epithelial-to-mesenchymal transition (EMT) and metastasis both in vitro and in vivo. Conversely, miR-379 knockdown increased migration, invasion and EMT in HCC cells. Moreover, miR-379-5p exerted this function by directly targeting focal adhesion kinase (FAK) 3'-UTR and repressing FAK expression, thus leading to suppression of AKT signaling. Furthermore, the tumor suppressive effects of miR-379-5p in HCC cells were reversed by activating AKT signaling or restoring FAK expression. In clinical samples of HCC, miR-379-5p negatively correlated with FAK, which was up-regulated in HCC. Taken together, our findings highlight the important role of miR-379-5p in regulating the EMT and metastasis of HCC by targeting FAK/AKT signaling, suggesting that miR-379-5p may represent a novel potential therapeutic target and prognostic marker for HCC.

Histone deacetylase inhibitor valproic acid (VPA) promotes the epithelial mesenchymal transition of colorectal cancer cells via up regulation of Snail.

Histone deacetylase inhibitors (HDACIs) have been shown to have antiproliferative activity through cell-cycle arrest, differentiation, and apoptosis in colorectal cancer (CRC) cells. Our present study revealed that one HDAC inhibitor, valproic acid (VPA), can obviously promote in vitro motility of HCT-116 and SW480 cells. VPA treatment significantly down regulates the expression of epithelial markers E-Cadherin (E-Cad) and Zona occludin-1(ZO-1) while up regulates the mesenchymal markers Vimentin (Vim) and N-cadherin (N-Cad), suggesting that VPA can trigger the epithelial-mesenchymal transition (EMT) of CRC cells. VPA treatment significantly increases the expression and nuclear localization of Snail, the key transcription factors of EMT. Snail knockdown by siRNAs obviously reverses VPA induced EMT of HCT-116 and SW480 cells. Further, VPA can decrease the ubiquitination, increase the acetylation, and then elevate the stabilization of Snail. VPA also increases the phosphorylation of Akt/GSK-3ß. The inhibitor of PI3K/Akt, LY2994002, significantly attenuates VPA induced phosphorylation of Akt and GSK-3ß and up regulation of Snail and Vim. Collectively, our data reveal that VPA can trigger the EMT of CRC cells via up regulation of Snail through AKT/GSK-3ß signals and post-transcriptional modification. It suggests that more attention should be paid when VPA used as a new anticancer drug for CRC patients.

Risk Factors Associated with Intraoperative Major Blood Loss during Resection of Hepatocellular Carcinoma.

BACKGROUND/AIMS: Intraoperative blood loss is an independent predictor of recurrence and survival after resection of hepatocellular carcinoma (HCC). The aim of this study was to identify the risk factors associated with intraoperative major blood loss in patients undergoing liver resection for HCC. METHODOLOGY: Clinicopathologic data and perioperative outcomes of 386 patients who underwent liver resection for HCC were retrospectively reviewed. The patients were divided into high (> 1,000 mL) and low (51,000 mL) blood loss groups according to the intraoperative blood loss. Intraoperative blood loss,more than 1,000 mL was defined as major blood loss. The risk factors associated with intraoperative major blood loss were analyzed by univariate and multivariate analyses. RESULTS: Vascular invasion, major hepatectomy and prolonged operation time were risk factors associated with intraoperative major blood loss during resection of HCC on multivariate analysis. Moreover, HCC patients with intraoperative major blood loss had prolonged hospital stay, higher incidence of postoperative complication and mortality compared with patients' with blood loss 1,000 mL. CONCLUSIONS: Vascular invasion, major hepatectomy and prolonged operation time are independent predictors of intraoperative major blood loss during resection of HCC.

Down-regulation of Gli-1 inhibits hepatocellular carcinoma cell migration and invasion.

Glioma-associated oncogene homolog-1 (Gli-1) is considered a marker of Hedgehog pathway activation and is associated with the progression of several cancers. We have previously reported that Gli-1 was correlated with invasion and metastasis in hepatocellular carcinoma (HCC). However, the exact roles and mechanisms of Gli-1 in HCC invasion are unclear. In this study, we found that small interfering RNA mediated down-regulation of Gli-1 expression significantly suppressed adhesion, motility, migration, and invasion of both SMMC-7721 and SK-Hep1 cells. Furthermore, down-regulation of Gli-1 significantly reduced expressions and activities of both matrix metalloproteinase (MMP)-2 and MMP-9. In addition, we found that down-regulation of Gli-1 resulted in up-regulation of E-cadherin and concomitant down-regulation of Snail and Vimentin, consistent with inhibition of epithelial-mesenchymal transition (EMT). Taken together, our results suggest that down-regulation of Gli-1 suppresses HCC cell migration and invasion likely through inhibiting expressions and activations of MMP-2, 9 and blocking EMT.

Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues.

PURPOSE: A comparative proteomic approach was used to identify and analyze proteins related to metastasis of hepatocellular carcinoma (HCC). METHODS: Proteins extracted from 12 HCC tissue specimens (six with metastases and six without) were separated by two-dimensional gel electrophoresis (2-DE). The protein spots exhibiting statistical alternations between the two groups through computerized image analysis were then identified by mass spectrometry. In addition immunohistochemistry (IHC), Western blotting and RT-PCR were performed to verify the expression of certain candidate proteins. RESULTS: 16 proteins including HSP27, S100A11, CK18 were annotated by mass spectrometry, relevant to chaperone function, cell mobility, cytoskeletal architecture, respectively. Most were previously unconnected with metastasis of HCC. Of these HSP27 was found overexpressed consistently in 2-DE patterns of all metastatic HCC tissues compared with nonmetastatic ones. IHC and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. CONCLUSION: There are various proteins joined together in HCC metastasis. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets unique to the metastatic phenotype of HCC.

[Screening hepatocellular carcinoma autoantibodies by serological proteome analysis].

OBJECTIVE: To screen hepatocellular carcinoma (HCC) autoantibodies as diagnostic biomarkers or therapy targets by serologic proteome analysis (SERPA). METHODS: Total proteins extracted from human HCC cell line HCCLM3 were separated by two-dimensional electrophoresis (2-DE) and then transferred onto PVDF membranes, which were subsequently incubated with sera from HCC, hepatitis B virus (HBV) infected patients or healthy volunteers. All immuno-reactive protein spots on blot films were matched to those on 2-DE gel maps by image analysis and identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS/MS). RESULTS: 2-DE gel maps of HCCLM3 and corresponding blot films of good quality and reproducibility were established. The number of spots on HCCLM3 2-DE reference gel totaled 603 and those on HCC, HBV and healthy sera blotted films were 70.75+/-24.25, 68.5+/-23.44 and 41.38+/-15.05, respectively. Blot films of HCC and HBV groups had more spots than those of the healthy group (P < 0.05) while no significance was found between films of HCC and HBV groups. By identification, those HCC autoantibodies could be classified as nuclear proteins, cytoskeleton proteins, heat shock proteins and metabolic enzymes. CONCLUSION: Serological proteome analysis is a high throughput technique for screening tumor autoantibodies. Those newly identified HCC associated tumor antigens and corresponding autoantibodies can be used in the early diagnosis or immuno-therapy of HCC.

Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.

[Differential analysis of tyrosine-phosphorylated proteins in human hepatocellular carcinoma cell lines with different metastasis potentials].

OBJECTIVES: To compare expressions of tyrosine-phosphorylated proteins in different hepatocellular carcinoma cell lines with different metastasis potential and to screen key molecules associated with HCC metastasis and recurrence. METHODS: Using two-dimensional electrophoresis, Western blotting and MALDI-TOF-MS/MS, we analyzed tyrosine-phosphorylated protein profiles of Hep3B, MHCC97L and MHCC97H, HCC cell lines with different metastasis potentials. RESULTS: 10 spots were detected in Hep3B, 19 in MHCC97L and 17 in MHCC97H. Seventeen significantly different phosphotyrosine proteins in gel were identified by MALDI-TOF-MS/MS, including Annexin I. CONCLUSION: The changed expression of tyrosine-phosphorylated proteins is associated with HCC metastasis and recurrence.

[Proteomic analysis on metastasis-associated proteins of hepatocellular carcinoma tissues].

OBJECTIVE: A comparative proteomic approach was used to identify and analyze proteins relevant to metastasis of hepatocellular carcinoma (HCC). METHODS: Proteins extracted from 12 liver tumor tissue specimens (6 with metastases and 6 without) were separated by two-dimensional gel electrophoresis (2-DE). Comparative analyses of 2-DE protein patterns between the two groups were done using computerized image analysis. Selected proteins exhibiting statistically significant alternations were identified by mass spectrometry. Immunohistochemistry, Western blotting and RT-PCR were performed to examine the expressions of the candidate proteins. RESULTS: 16 proteins including HSP27, S100A11, CK18 were identified using mass spectrometry, which were related to cell mobility, signal transduction, and energy metabolism respectively. Of these, HSP27 was found to be uniquely over-expressed in 2-DE maps of all metastatic HCCs when compared to the non-metastatic HCC tissues. Immunohistochemistry and Western blotting of HCC tissues confirmed this difference while RT-PCR did not. CONCLUSION: There are different proteins working together that affect the metastasis of HCCs. The overexpression of HSP27 may serve as a biomarker for early detection and therapeutic targets to the metastatic phenotype of HCC. The role of HSP27 in HCC metastasis warrants further investigation.

Differential proteomic analysis of human hepatocellular carcinoma cell line metastasis-associated proteins.

PURPOSE: The comparative study of differentially expression of protein profiles of hepatocellular carcinoma cell lines with various metastasic potential and screening key molecules related to hepatocellular carcinoma metastasis and recurrence. METHODS: Using two-dimensional electrophoresis and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), we analyzed differentially displayed proteomics of human hepatocellular carcinoma cell lines Hep3B, MHCC97L, MHCC97H with different metastasic potential. RESULTS: Approximate 1,000 protein spots were detected on silver-stained gel by ImageMaster (977+/-113 spots in Hep3B, 1092+/-40 in MHCC97L, and 889+/-14 in MHCC97H). Fifty distinct different protein spots were analyzed with online LC-ESI-MS/MS. Only 26 protein spots had a positive result, including annexin1, S100A4, and so on. In comparison with nonmetastasis Hep3B cell lines, there were 16 proteins overexpressed in MHCC97H and MHCC97L, 10 proteins underexpressed in MHCC97H and MHCC97L. Applying cell immunohistochemistry and RT-PCR, we further validated two interesting and different proteins, annexin1 and S100A4. CONCLUSION: The protein profile of metastatic hepatocellular carcinoma cell lines displayed obvious differences compared with non-metastatic liver cancer cell lines. The results imply that various different proteins may lead to HCC metastasis together.