Hai-Jun Zhou

MicroRNA-26a suppresses tumor growth and metastasis of human hepatocellular carcinoma by targeting interleukin-6-Stat3 pathway†‡

Down‐regulation of microRNA‐26a (miR‐26a) is associated with poor prognosis of hepatocellular carcinoma (HCC), but its functional mechanism in HCC remains unclear. In this study, we investigated the roles of miR‐26a in tumor growth and metastasis of HCC and found that miR‐26a was frequently down‐regulated in HCC tissues. Down‐regulation of miR‐26a correlated with HCC recurrence and metastasis. Through gain‐ and loss‐of‐function studies, miR‐26a was demonstrated to significantly inhibit in vitro cell proliferation, migration, and invasion. In addition, miR‐26a induced G1 arrest and promoted apoptosis of HCC cells. Importantly, miR‐26a suppressed in vivo tumor growth and metastasis in nude mice models bearing human HCC. Interleukin‐6 (IL‐6) was identified as a target of miR‐26a. Knockdown of IL‐6 induced effects on HCC cells similar to those induced by miR‐26a. In contrast, IL‐6 treatment abrogated the effects induced by miR‐26a up‐regulation. Moreover, miR‐26a dramatically suppressed expression of signal transducer and activator of transcription 3 (Stat3) target genes, including Bcl‐2, Mcl‐1, cyclin D1, and MMP2. IL‐6 treatment antagonized this effect, while knockdown of IL‐6 by IL‐6 short hairpin RNA (shIL‐6) induced inhibitory effects on the expression of p‐Stat3 and its main target genes, similar to miR‐26a. The messenger RNA and protein levels of IL‐6 inversely correlated with miR‐26a in HCCs. Patients with high miR‐26a or low IL‐6 in HCC tissues had a better prognosis with longer overall survival (OS) and time to recurrence (TTR). In multivariate analysis, miR‐26a, IL‐6, and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Conclusion: miR‐26a could suppress tumor growth and metastasis of HCC through IL‐6‐Stat3 signaling and is a novel prognostic marker and therapeutic target for HCC. (HEPATOLOGY 2013)

Human mesenchymal stem cells inhibit metastasis of a hepatocellular carcinoma model using the MHCC97-H cell line

The effects of mesenchymal stem cells (MSC) on the growth and metastasis of human malignancies including hepatocellular carcinoma (HCC) are controversial, and the underlying mechanisms are not yet understood. The aim of this study was to explore the role of MSC in the progression of HCC. We investigated the effect of MSC on in vitro proliferation and invasion and in vivo tumor growth and pulmonary metastasis of MHCC97‐H HCC cells with a high metastatic potential. The mRNA and protein levels of transforming growth factor‐beta 1 (TGFβ1) and MMP, and their association with the effects of MSC on HCC cells were also evaluated. Co‐culture of MHCC97‐H cells with MSC conditioned medium significantly enhanced in vitro proliferation but inhibited invasiveness. Following MSC treatment of a nude mouse model bearing human HCC, the MSC were predominantly located in the HCC tissues. Compared with controls, MSC‐treated mice exhibited significantly larger tumors (3080.51 ± 1234.78 mm3vs 2223.75 ± 1000.60 mm3, P = 0.045), but decreased cellular numbers of lung metastases (49.75 ± 18.86 vs 227.22 ± 74.67, P = 0.046). Expression of TGFβ1 and MMP‐2 was significantly downregulated in the MSC‐treated HCC cells. TGFβ siRNA concurrently downregulated expression of TGFβ and MMP‐2 in HCC cells and blocked the MSC‐induced proliferation and invasiveness of MHCC97‐H cells. The MSC enhanced tumor growth but significantly inhibited the invasiveness and metastasis of HCC, possibly through downregulation of TGFβ1. These findings suggest that MSC could be useful in controlling metastatic recurrence of HCC. (Cancer Sci 2010; 101: 2546–2553)

Involvement of protein kinase C β–extracellular signal‐regulating kinase1/2/p38 mitogen‐activated protein kinase–heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion

To understand the molecular mechanism that underlies the role of various prominent signal pathways in hepatocellular carcinoma (HCC) metastasis, a human signal transduction oligonucleotide microarray analysis was carried out in cultured HCC cell models with increasing spontaneous metastatic potential (MHCC97L, MHCC97H, and HCCLM6). The results revealed that the mitogen‐activated protein kinase (MAPK) pathway is the prominently upregulated pathway in HCC metastasis. Further study showed that basal phosphorylated levels of extracellular signal‐regulating kinase (ERK)1/2 and p38 MAPK consecutively increased from MHCC97L to MHCC97H to HCCLM6 cells, but not c‐Jun N‐terminal kinase. The phosphorylation of ERK1/2 and p38 MAPK was regulated by upregulated protein kinase Cβ (PKCβ) in HCC cells through the integrated use of PKCβ RNA interference, the PKCβ specific inhibitor enzastaurin and a PKC activator phorbol‐12‐myristate‐13‐acetate. Heat shock protein 27 (HSP27) was also verified as a downstream common activated protein of PKCβ–ERK1/2 and PKCβ–p38 MAPK. In vitro migration and invasion assay further showed that the depletion of PKCβ or inhibition of PKCβ activation effectively decreased HCC cell motility and invasion. Moreover, the motility and invasion of phorbol‐12‐myristate‐13‐acetate‐stimulated PKCβ‐mediated HCC cells was significantly negated by an ERK inhibitor, 1.4‐diamino‐2.3‐dicyano‐1.4‐bis[2‐aminophenylthio] butadiene, or a p38 MAPK inhibitor, 4‐(4‐Fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole. It also showed that HSP27 is critical in PKCβ‐mediated HCC cell motility and invasion. Taken together, this study reveals the important role of this PKCβ‐ERK1/2/p38MAPK‐HSP27 pathway, which was verified for the first time, in modulating HCC cell motility and invasion. (Cancer Sci 2008; 99: 486–496)

Identification of Suitable Reference Genes for qRT-PCR Analysis of Circulating microRNAs in Hepatitis B Virus-Infected Patients

Circulating microRNAs (miRNAs) were found to exist in serum/plasma in a highly stable, cell-free form, and aberrantly expressed in many human diseases. Currently, the expression levels of circulating miRNAs are estimated by quantitative real-time polymerase chain reaction. However, no study has systematically evaluated reference genes for evaluating circulating microRNA expression. This study describes the identification and characterization of an appropriate reference gene for the normalization of circulating miRNA levels in hepatitis B virus (HBV)-infected patients and healthy people. Ten miRNAs that resemble the mean expression of the TaqMan low density array together with U6, RNU6B, and miR-16 were validated with two algorithms, geNorm, and NormFinder, after ensuring their equivalent expression between the two study groups. The combination of miR-26a, miR-221, and miR-22* is recommended as the most stable set of reference genes for circulating miRNA evaluation in HBV patients and healthy people.

Evaluation of midkine as a diagnostic serum biomarker in hepatocellular carcinoma.

Purpose: To evaluate the value of serum midkine (MDK) as a diagnostic biomarker in hepatocellular carcinoma, particularly for those with negative alpha-fetoprotein (AFP) and at an early stage. Experimental Design: MDK expression in tumors was assessed by immunohistochemistry from 105 patients with hepatocellular carcinomas or liver cirrhosis. Serum MDK levels were detected by ELISA in 933 participants including hepatocellular carcinomas and hospital controls from different medical centers. Sensitivities and specificities of serum MDK in diagnosing hepatocellular carcinoma according to AFP level and Barcelona Clinic Liver Cancer (BCLC) stage were analyzed. Results: MDK levels were significantly elevated in hepatocellular carcinoma tissues as well as serum samples. The sensitivity of serum MDK for hepatocellular carcinoma diagnosis was much higher than that of AFP (86.9% vs. 51.9%) with similar specificities (83.9% vs. 86.3%). Notably, serum MDK had an outstanding performance in distinguishing AFP-negative hepatocellular carcinomas from different controls: In those AFP-negative hepatocellular carcinomas, the sensitivity could reach as high as 89.2%. Moreover, receiver operating characteristic (ROC) curve analysis also showed that serum MDK had a better performance compared with AFP in distinguishing early-stage hepatocellular carcinomas as well as small hepatocellular carcinomas. Even in very early-stage hepatocellular carcinomas, MDK showed an obviously higher sensitivity compared with AFP (80% vs. 40%). Furthermore, serum MDK level was significantly decreased in patients with hepatocellular carcinomas after curative resection and re-elevated when tumor relapse occurred. Conclusions: Serum MDK is significantly elevated in most hepatocellular carcinomas, including those with negative AFP and at an early stage, which may serve as a novel diagnostic marker in early diagnosis and postoperative monitoring of hepatocellular carcinomas. Clin Cancer Res; 19(14); 3944–54. ©2013 AACR.

Expression and prognostic significance of osteopontin and caspase-3 in hepatocellular carcinoma patients after curative resection

Osteopontin (OPN) plays an important role in the development, invasion, and metastasis of malignancies. Recently, several studies have reported that OPN enhances chemoresistance in small‐cell lung cancer and breast cancer by blocking caspase‐9 and caspase‐3‐dependent cell apoptosis. The aim of this study was to assess the value of OPN and caspase‐3 for predicting tumor recurrence after curative resection in hepatocellular carcinoma (HCC) patients. We found that OPN expression increased concordantly with increasing metastatic potential in human HCC cell lines, whereas caspase‐3 expression declined. In a tumor tissue microarray immunohistochemical analysis, we found that patients with higher levels of OPN and lower levels of caspase‐3 had a significantly poorer prognosis than patients with lower OPN and higher caspase‐3 levels. The combination of OPN and caspase‐3 expression thus served as an effective prognosticator. These findings suggest that OPN alone or in combination with caspase‐3 may act as an independent indicator for HCC patients after curative resection.

Prediction of chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma by SELDI-based serum decision tree classification

PurposeTo screen potential serological biomarkers and develop decision tree classifications of chronic hepatitis B, liver cirrhosis (LC) and hepatocellular carcinoma (HCC), respectively, with high prediction score for improving diagnosis of liver diseases.MethodsThe total serum samples were randomly divided into three training sets (41 HBV and 35 health; 36 LC and 35 health; 39 HCC and 35 health) and three testing groups (34 HBV and 38 health; 18 LC and 52 health; 42 HCC and 47 health). Selected WCX2 protein chip capture followed by SELDI-TOF-MS analysis was applied to generate the serum protein profiles. Subsequently serum protein spectra were normalized and aligned by Ciphergen SELDI Software 3.1.1 with Biomarker Wizard including baseline subtraction, mass accuracy calibration, automatic peak detection. Once the intensities of selected significant peaks from the training data set were transferred to further BPS analysis, an optimized classification tree with sequence-decision was established to divide training data set into disease group and control group successfully. A double blind test was employed to determine the clinical sensitivity and clinical specificity of three models.ResultsAfter comparative analysis of SELDI based serum protein profile between the cases of disease and healthy, a HCC decision tree classification with sensitivity of 94.872% and specificity of 94.286%; a LC decision tree classification with sensitivity of 91.667% and specificity of 94.286% and a HBV decision tree classification with sensitivity of 95.122% and specificity of 94.286% were produced by BPS respectively. When three decision tree models were challenged by the double-blind test samples, clinical sensitivity and clinical specificity of these models were predicted in diagnosis of three liver diseases (HCC: 90.48 and 89.36%; cirrhosis: 100 and 86.5%; HBV: 85.29 and 84.21%).ConclusionSELDI-based decision tree classifications showed great advantages over conventional serological biomarkers in the diagnosis of chronic hepatitis B, LC as well as HCC.

Osteopontin promotes epithelial-mesenchymal transition of hepatocellular carcinoma through regulating vimentin.

Our previous studies have found that osteopontin (OPN) is a promoter for hepatocellular carcinoma (HCC) progression. However, the molecular mechanism by which OPN enhances HCC metastasis remains elusive. Epithelial-mesenchymal transition (EMT) of cancer cells plays a pivotal role in promoting metastatic process. In this study, we demonstrated that OPN promotes HCC metastasis by inducing an EMT-like, more aggressive cellular phenotype in vitro and in vivo. Furthermore, OPN was identified to interact with vimentin by reciprocal OPN and vimentin immunoprecipitation as well as co-immunofluorescence examination. By using deletion mutants, we found that the residues between 246 and 406 in vimentin are required for binding to OPN. Importantly, OPN significantly increased vimentin stability through inhibition of its protein degradation. Knockdown of vimentin neutralized the EMT induced by OPN both in vitro and in vivo. Moreover, a significant correlation between OPN and vimentin levels was found in clinical HCC specimens and their combination had a worse prognosis with shorter overall survival (OS) and time to recurrence (TTR). In multivariate analysis, OPN and their combination were demonstrated to be independent prognostic indicators for OS and TTR of HCC patients. Collectively, this study indicates that OPN can induce EMT of HCC cells through increasing vimentin stability, which provides more in-depth understanding about the molecular mechanisms of OPN in promoting HCC metastasis and opens tantalizing therapeutic possibilities in HCC.

A glycoproteome database of normal human liver tissue

PurposeTo extensively investigate the glycoproteins of normal human liver tissue, constructing the glycoprotein profile and database of the normal human liver tissue.MethodsThe total proteins were extracted from the normal human liver tissue and then subjected to two-dimensional electrophoresis (2-DE). Finally, 2-DE gels were stained according to the methods of multiplexed proteomics (MP) technology. Glycoprotein spots were excised from 2-DE gel and then characterized by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS).ResultsThe PDQuest software detected 1,011 glycoprotein spots and 1,923 total protein spots in the 2-DE gels of sample from the normal human liver tissue. Furthermore, 116 species of glycoproteins were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and annotated to our databases. In addition, we also applied bioinformatics softwares to predict N- or O-glycosylation sites of identified glycoproteins.ConclusionThis study demonstrates the feasibility of a novel technological platform to contruct glycoprotein databases. These results lay the foundation for future physiological and pathological studies of the human liver.

Analysing protein–protein interaction networks of human liver cancer cell lines with diverse metastasis potential

PurposeHuman hepatocellular carcinoma (HCC) is one of the most mortal tumor. In a previous study, we had constructed glycoprotein expression profiles and glycoprotein databases of three human liver cancer cell lines with diverse metastasis potential. In order to discover vital glycoproteins related to pathogenesis and metastasis of HCC, in this study we analyzed previous data with bioinformatic approach.MethodsWe took previous data to draw the protein–protein interaction (PPI) networks of liver cell lines by searching IntACT database and then using Pajeck software. Further more, we compared the differences between the three PPI networks by drawing the PPI networks of differential glycoproteins and by naming differential display PPI networks.ResultsLarge numbers of proliferation and apoptosis-relative proteins interact with the differential glycoproteins, and among the differential glycoproteins there are many interactions.ConclusionsWe conclude that neither single nor several proteins cause malignant proliferation of liver cells. “Molecule groups” concept should be introduced into diagnosis and metastasis prediction of the HCC.