Biological features and biomarkers in hepatocellular carcinoma

Similar to other cancers, a multistep process of carcinogenesis is observed in hepatocellular carcinoma(HCC). Although the mechanisms underlying the development of HCC have been investigated in terms of oncology, virology, and stem cell biology, the whole picture of hepatocarcinogenesis remains to be elucidated. Recent progress in molecular biology has provided clues to the underlying cause of various diseases. In particular, sequencing technologies, such as whole genome and exome sequencing analyses, have made an impact on genomic research on a variety of cancers including HCC. Comprehensive genomic analyses have detected numerous abnormal genetic alterations, such as mutations and copy number alterations. Based on these findings, signaling pathways and cancer-related genes involved in hepatocarcinogenesis could be analyzed in detail. Simultaneously, a number of novel biomarkers, both from tissue and blood samples, have been recently reported. These biomarkers have been successfully applied to early diagnosis and prognostic prediction of patients with HCC. In this review, we focus on the recent developments in molecular cancer research on HCC and explain the biological features and novel biomarkers.

Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signalregulated kinase phosphorylation

BACKGROUND Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines.The Chinese herbal medicine,Gan Shen Fu Fang(GSFF)is composed of salvianolic acid B and diammonium glycyrrhizinate.In this study,we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment.AIM To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase(ERK)phosphorylation.METHODS Common bile duct-ligated rats were used for in vivo experiments.Hepatic stellate cells-T6(HSC-T6)cells were used for in vitro experiments.Hematoxylin and eosin staining and Masson staining,biochemical assays,hydroxyproline(Hyp)assays,enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis,liver function,the inflammatory response and ERK phosphorylation.The CCK8 assay,immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells.RESULTS GSFF improved liver function and inhibited liver fibrosis in common bile ductligated rats after 3 wk of treatment,as demonstrated by histological changes,hydroxyproline assays and collagen I concentrations.GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines[tumor necrosis factor-α(TNF-α)and interlukin-1β]and NF-κB.In addition,GSFF decreased ERK phosphorylation.In vitro,GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factorβ1(TGF-β1)stimulation and decreased the synthesis of collagen I.GSFF had the greatest effect at a concentration of 0.5μmol/L.GSFF inhibited the expression ofα-smooth muscle actin(α-SMA),a marker of HSC activation,in HSC-T6 cells.Consistent with the in vivo results,GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB.CONCLUSION GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro.These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation.

Expression of PTEN,PPM1A and P-Smad2 in hepatocellular carcinomas and adjacent liver tissues

AIM： To investigate the expressions of PTEN, PPMIA and P-Smad2 in hepatocellular carcinoma （HCC） and their significance.METHODS： The expressions of PTEN, PPMIA and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique. RESULTS： The positive expression （64.52%） and staining intensity （4.19 ± 3.31） of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues （97.37%, 7.88 ± 0.93; 100%, 7.77 ± 0.93, respectively） （P 〈 0.05）, and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades Ⅱ-Ⅲ and above, was also lower than that of grade I and I-Ⅱ. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease （r = -0.339, P = 0.002）; most of PPMIA might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson grade I and I - Ⅱ HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade ≥ Ⅱ, weakly or negatively expressed in the nucleus （P 〈 0.05）, and its location was negatively correlated with the progression of liver disease （r = -0.45, P = 0.0000）. P-Smad2, which was mostly located in the nucleus and cytoplasm of grade I and I -Ⅱ HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade Ⅱ and above （P 〈 0.001）, and its location was positively correlated with the disease progression （r = 0.224, P = 0.016）. Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPMIA （r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively）; and PTEN and PPMIA were positively correlated with HCC carcinogenesis （r = 0.428, P = 0.000）.CONCLUSION： The aberrant location of expression and staining intensity of PTEN, PPMIA and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

Hepatocellular carcinoma staging systems: Hong Kong liver cancer vs Barcelona clinic liver cancer in a Western population

BACKGROUND Despite being the world’s most widely used system for staging and therapeutic guidance in hepatocellular carcinoma(HCC)treatment,the Barcelona clinic liver cancer(BCLC)system has limitations,especially regarding intermediate-grade(BCLC-B)tumors.The recently proposed Hong Kong liver cancer(HKLC)staging system appears useful but requires validation in Western populations.AIM To evaluate the agreement between BCLC and HKLC staging on the management of HCC in a Western population,estimating the overall patient survival.METHODS This was a retrospective study of HCC patients treated at a university hospital in southern Brazil between 2011 and 2016.Demographic,clinical,and laboratory data were collected.HCC staging was carried out according to the HKLC and BCLC systems to assess treatment agreement.Overall survival was estimated based on the treatment proposed in each system.RESULTS A total of 519 HCC patients were assessed.Of these,178(34.3%)were HKLC-I;95(18.3%)HKLC-IIA;47(9.1%)HKLC-IIB;29(5.6%)HKLC-IIIA;30(5.8%)HKLCIIIB;75(14.4%)HKLC-IV;and 65(12.5%)HKLC-V.According to the BCLC,25(4.9%)were BCLC-0;246(47.4%)BCLC-A;107(20.6%)BCLC-B;76(14.6%)BCLCC;and 65(12.5%)BCLC-D.The general agreement between the two systems was 80.0%-BCLC-0 and HKLC-I(100%);BCLC-A and HKLC-I/HKLC-II(96.7%);BCLC-B and HKLC-III(46.7%);BCLC-C and HKLC-IV(98.7%);BCLC-D and HKLC-V(41.5%).When sub-classifying BCLC-A,HKLC-IIB,HKLC-IIIA and HKLC-IIIB stages according to the up-to-7 in/out criterion,13.4,66.0,100 and 36.7%,respectively,of the cases were classified as up-to-7 out.CONCLUSION In a Western population,the general agreement between the two systems was 80.0%,although in BCLC-B cases the agreement was low,suggesting that some individuals could be candidates for the curative treatment recommended by the HKLC.The authors suggest that the BCLC system should be routinely employed,although for BCLC-B cases it should be associated with the HKLC system.

Intracellular sorting pathways of the amyloid precursor protein provide novel neuroprotective strategies

Alzheimer＇s disease（AD）is the most common cause of senile dementia.It is characterized by the formation of plaques mainly composed of the amyloid-beta peptide（Aβ）.Diverse lines of evidence support the notion that accumulation of Aβis a primary cause of AD pathogenesis（Huang and Mucke,2012）.Amyloid precusor protein（APP）processing is dependent on its subcelluar trafficking pathway：Aβis derived from APP by proteolyric processing.

Late retroperitoneal recurrence of hepatocellular carcinoma 12 years after initial diagnosis

Hepatocellular carcinoma (HCC) is an aggressive tumor with poor long-term prognosis.Here,we present an unusual patient with a solitary recurrence of HCC in the right kidney 12 years after the initial diagnosis.This illustrates the importance of considering late recurrence in patients with a history of HCC and the management of these metastases.

Therapeutic application of mesenchymal stem cells-derived extracellular vesicles in colorectal cancer

Colorectal cancer(CRC)is the third most common cancer and the leading cause of cancer death globally.Resistance to therapy is a challenge for CRC treatment.Mesenchymal stem cells(MSCs)have become one of the furthermost effective approaches for tumor treatment due to their specific feature;however,their therapeutic function is controversial.Recently,extracellular vesicles(EVs)derived from MSCs(MSCs-EVs)have attracted extensive research attention due to their promising role in CRC treatment.EVs are cell-derived vesicles that transfer different biomolecules between cells,contributing to intracellular communication.MSCs-EVs can suppress CRC by delivering therapeutic agents to tumor cells.Several studies indicate that MSCs-EVs can serve as a drug delivery system for the treatment of different cancers.Various methods are used to modify(engineer)MSCs-EVs for loading therapeutic agents.Modified MSCs-EVs have improved specificity,targeting ability,and immunogenicity compared to synthetic carriers.Furthermore,CRC-EVs participate in regulating different cells,such as immune cells,fibroblasts,and endothelial cells,promoting tumorigenesis.MSCs-EVs-based therapy indicates a high potential in the treatment of cancer;however,the majority of studies have been conducted in the pre-clinical,and their clinical applications need further scrutiny.In this review,we describe the biogenesis of EVs,focusing on the effect of MSCs-EVs on CRC cells and CRC-derived EVs on other cells.Furthermore,MSCs-EVs as a drug delivery system for cancers is also reviewed,and perspectives regarding the therapeutic application of MSCs-EVs are discussed.

The cellular microenvironment modulates the role of PAI-1 and vitronectin in mediating cell-matrix interactions

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily of proteins, circulates in a complex with vitronectin. Furthermore, these two proteins are co-localized in the extracellular matrix (ECM) in many different pathophysiological conditions. Though PAI-1 is a well-characterized inhibitor of serine proteases, recent emphasis has also focused on its protease-independent functions. Vitronectin, a multi-domain protein that binds a wide variety of ligands and proteins, exists in the circulation in a preferred monomeric state, while in the extracellular matrix it exists as a multimer resulting from an altered conformation. Though the mechanism for the conformational alterations and compartmentalization in tissues is unknown, there are a number of biomolecules including PAI-1 that appear to cause such changes. Experimental analysis has established that PAI-1 induces association of vitronectin to higher-order species in a concentration-dependent fashion [1]. This report extends our investigations into the mechanism of the interaction between vitronectin and PAI-1 to explore the physiological relevance of these higher-order complexes for cellular adhesion and migration. In this study, we evaluate the effects of the pericellular microenvironment on the functions of the multimeric complexes in a variety of relevant biological settings. Our findings underscore the importance of the variability of components within this microenvironment, including different receptors and ECM components, in governing the way in which the vitronectin/PAI-1 complex mediates cell-matrix interactions.

Nuclear Mechanotransduction in Cellular Mechanobiology and Molecular Mechanomedicine

It is known that mechanical forces play critical roles in physiology and diseases but the underlying mechanisms remain largely unknown[1].Most studies on the role of forces focus on cell surface molecules and cytoplasmic proteins.However,increasing evidence suggests that nuclear mechanotransduction impacts nuclear activities and functions.Recently we have revealed that transgene dihydrofolate reductase(DHFR)gene expression is directly upregulated via cell surface forceinduced stretching of chromatin [2].Here we show that endogenous genes are also upregulated directly by force via integrins.We present evidence on an underlying mechanism of how gene transcription is regulated by force.We have developed a technique of elastic round microgels to quantify 3D tractions in vitro and in vivo[3].We report a synthetic small molecule(which has been stiffened structurally)that inhibits malignant tumor repopulating cell growth in a low-stiffness(force)microenvironment and cancer metastasis in mouse models without detectable toxicity[4].These findings suggest that direct nuclear mechanotransduction impacts mechanobiology and mechanomedicine at cellular and molecular levels.

Pharmacological Isolation of Experimental Models of Drug-resistant Hepatocellular Carcinoma Cell Line

Drug resistance is one of the major challenges facing the success of chemotherapy against human hepatocarcinoma (HCC) as well as other types of cancer. Studies with cell lines can serve as initial screening for agents that could modulate drug resistance. Development of a good experimental model of drug-resistant cells is a prerequisite for the success of such cellular studies;but could be laborious and generally time-consuming. Additionally, the high mortality rate associated with advanced HCC calls for a probe into the mechanism of resistance by developing experimental model that mimics clinical method of its treatment. Consequently, we have reported a simplified method of selection of drug-resistant hepatocarcinoma cells from human hepatocellular carcinoma (HEPG2) cell line using pharmacologic agents, cisplatin (CDDP) and 5-fluorouracil (5-FU). HEPG2 cell line was incubated for 24 hours with different concentrations of CDDP (0 - 20 μM) or 5-FU (0 - 100 μM). Cell viability was assayed by CCK-8 (Cell Counting Kit) analysis, and the inhibitory concentrations (IC50) for CDDP and 5-FU were established by dose-dependent cytotoxicity curves. The IC50(s) were confirmed by flow cytometric analysis of cell death due to CDDP or 5-FU. Clinical method of treatment was imitated by treating the parental HEPG2 cell line in pulse, at the optimal concentration of either CDDP or 5-FU for 4 to 6 hours. Induction was repeated 6 times, whilst allowing the cells to attain at least 70% confluence between intervals of induction. The resultant drug-resistant sublines, (HEPG2CR) and (HEPG2FR) were found to be stable after over 3 months of drug withdrawal and maintenance in drug-free medium. This was done with the views of establishing a simple, efficient and direct protocol for the development of good cellular models for the study of drug resistance in liver cancer, with possible application in other cancer types.

Extracellular vesicles in the pathogenesis and treatment of acute lung injury

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are common life-threatening lung diseases associated with acute and severe inflammation.Both have high mortality rates,and despite decades of research on clinical ALI/ARDS,there are no effective therapeutic strategies.Disruption of alveolar-capillary barrier integrity or activation of inflammatory responses leads to lung inflammation and injury.Recently,studies on the role of extracellular vesicles(EVs)in regulating normal and pathophysiologic cell activities,including inflammation and injury responses,have attracted attention.Injured and dysfunctional cells often secrete EVs into serum or bronchoalveolar lavage fluid with altered cargoes,which can be used to diagnose and predict the development of ALI/ARDS.EVs secreted by mesenchymal stem cells can also attenuate inflammatory reactions associated with cell dysfunction and injury to preserve or restore cell function,and thereby promote cell proliferation and tissue regeneration.This review focuses on the roles of EVs in the pathogenesis of pulmonary inflammation,particularly ALI/ARDS.

INTERACTION OF COMPONENT (E) WITH TUMOR CELLULAR DNA

Some antitumor activities of component (E), extracted from the root of Fagopynum Cymosum (Trev) Meisn (FCTM), have recently been discovered in vivo and in vitro. The component E (CE)'s pattern of action with tumor cellular DNA at the molecular pharmacological level was investigated by macromolecular synthesis experiment (MSE) and human DNA interaction system established in our laboratory. The experiments demonstrated that, in vitro, the agent could markedly inhibit the incorporation of 3H-TdR into the cellular DNA, and the IC50 in P388 leukemia cell and in SGC-7901 cell was 17.86 μg/ml and 110.4 μg/ml, respectively. The agent, at mg/ml level, could produce an intercalation reversion pattern with DNA within a short time (2 hours). But when the interval was prolonged for over 4 hours, the action changed to intercalation irreversible pattern. According to these observations, the authors infer that CE interacts with DNA in two ways - directly and indirectly. The indirect action, especially in low concentrations, probably plays the major role. The authors have also compared the interaction of CE with those of components (CB3 and CD1), extracted from FCTM by the same methods, and found that CE is the most active agent against the DNA of cancer cells among the extracts from FCTM.

Novel biomarkers GEP/ABCB5 regulate response to adjuvant transarterial chemoembolization after curative hepatectomy for hepatocellular carcinoma

Background: Transarterial chemoembolization(TACE) is the most commonly used adjuvant therapy for hepatocellular carcinoma(HCC) after curative resection. Responses to TACE are variable due to tumor and patient heterogeneity. We had previously demonstrated that expression of Granulin-epithelin precursor(GEP) and ATP-dependent binding cassette(ABC)B5 in liver cancer stem cells was associated with chemoresistance. The present study aimed to evaluate the association between GEP/ABCB5 expression and response to adjuvant TACE after curative resection for HCC. Methods: Patients received adjuvant TACE after curative resection for HCC and patients received curative resection alone were identified from a prospectively collected database. Clinical samples were retrieved for biomarker analysis. Patients were categorized into 3 risk groups according to their GEP/ABCB5 status for survival analysis: low(GEP-/ABCB5-), intermediate(either GEP +/ABCB5-or GEP-/ABCB5 +) and high(GEP +/ABCB5 +). Early recurrence(recurrence within 2 years after resection) and disease-free survival were analyzed. Results: Clinical samples from 44 patients who had followed-up for more than 2 years were retrieved for further biomarker analysis. Among them, 18 received adjuvant TACE and 26 received surgery alone. Patients with adjuvant TACE in the intermediate risk group was associated with significantly better overall survival and 2-year disease-free survival than those who had surgery alone( P = 0.036 and P = 0.011, respectively). Adjuvant TACE did not offer any significant differences in the early recurrence rate, 2-year disease-free survival and overall survival for patients in low and high risk groups. Conclusions: Adjuvant TACE can only provide survival benefits for patients in the intermediate risk group(either GEP +/ABCB5-or GEP-/ABCB5 +). A larger clinical study is warranted to confirm its role in patient selection for adjuvant TACE.

Mnk kinase pathway: Cellular functions and biological outcomes

The mitogen-activated protein kinase(MAPK) interacting protein kinases 1 and 2(Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs(p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E(eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4 E. The role of Mnk kinases in inflammation and inflammationinduced malignancies is also discussed.

Alzheimer’s disease, neural stem cells and neurogenesis: cellular phase at single-cell level

Alzheimer’s disease cannot be cured as of yet.Our current understanding on the causes of Alzheimer’s disease is limited.To develop treatments,experimental models that represent a particular cellular phase of the disease and more rigorous scrutiny of the cellular pathological mechanisms are crucial.In recent years,Alzheimer’s disease research underwent a paradigm shift.According to this tendency,Alzheimer’s disease is increasingly being conceived of a disease where not only neurons but also multiple cell types synchronously partake to manifest the pathology.Knowledge on every cell type adds an alternative approach and hope for the efforts towards the treatment.Neural stem cells and their neurogenic ability are making an appearance as a new aspect of the disease manifestation based on the recent findings that neurogenesis reduces dramatically in Alzheimer’s disease patients compared to healthy individuals.Therefore,understanding how neural stem cells can form new neurons in Alzheimer’s disease brains holds an immense potential for clinics.However,this provocative idea requires further evidence and tools for investigation.Recently,single cell sequencing appeared as a revolutionary tool to understand cellular programs in unprecedented resolution and it will undoubtedly facilitate comprehensive investigation of different cell types in Alzheimer’s disease.In this mini-review,we will touch upon recent studies that use single cell sequencing for investigating cellular response in Alzheimer’s disease and some consideration pertaining to the utilization of neural regeneration for Alzheimer’s disease research.

Tissue cell differentiation and multicellular evolution via cytoskeletal stiffening in mechanically stressed microenvironments

Evolution of eukaryotes from simple cells to complex multicellular organisms remains a mystery. Our postulate is that cytoskeletal stiffening is a necessary condition for evolution of complex multicellular organisms from early simple eukaryotes. Recent findings show that embryonic stem cells are as soft as primitive eukaryotes-amoebae and that differentiated tissue cells can be two orders of magnitude stiffer than embryonic stem cells. Soft embryonic stem cells become stiff as they differentiate into tissue cells of the complex multicellular organisms to match their microenvironment stiffness. We perhaps see in differentiation of embryonic stem cells (derived from inner cell mass cells) the echo of those early evolutionary events. Early soft unicellular organisms might have evolved to stiffen their cytoskeleton to protect their structural integrity from external mechanical stresses while being able to maintain form, to change shape, and to move.

Management of hepatocellular carcinoma with portal vein tumor thrombosis: Review and update at 2016

Portal vein tumor thrombosis(PVTT) is a common phenomenon in hepatocellular carcinoma(HCC). Compared to HCC without PVTT, HCC with PVTT is characterized by an aggressive disease course, worse hepatic function, a higher chance of complications related to portal hypertension and poorer tolerance to treatment. Conventionally, HCC with PVTT is grouped together with metastatic HCC during the planning of its management, and most patients are offered palliative treatment with sorafenib or other systemic agents. As a result, most data on the management of HCC with PVTT comes from subgroup analyses or retrospective series. In the past few years, there have been several updates on management of HCC with PVTT. First, it is evident that HCC with PVTT consists of heterogeneous subgroups with different prognoses. Different classifications have been proposed to stage the degree of portal vein invasion/thrombosis, suggesting that different treatment modalities may be individualized to patients with different risks. Second, more studies indicate that more aggressive treatment, including surgical resection or locoregional treatment, may benefit select HCC patients with PVTT. In this review, we aim to discussthe recent conceptual changes and summarize the data on the management of HCC with PVTT.

Cellular and molecular basis of chronic constipation: Taking the functional/idiopathic label out

In recent years, the improvement of technology and the increase in knowledge have shifted several strongly held paradigms. This is particularly true in gastroenterology, and specifically in the field of the so-called "functional" or "idiopathic" disease, where conditions thought for decades to be based mainly on alterations of visceral perception or aberrant psychosomatic mechanisms have, in fact, be reconducted to an organic basis (or, at the very least, have shown one or more demonstrable abnormalities). This is particularly true, for instance, for irritable bowel syndrome, the prototype entity of "functional" gastrointestinal disorders, where low-grade inflammation of both mucosa and myenteric plexus has been repeatedly demonstrated. Thus, researchers have also investigated other functional/idiopathic gastrointestinal disorders, and found that some organic ground is present, such as abnormal neurotransmission and myenteric plexitis in esophageal achalasia and mucosal immune activation and mild eosinophilia in functional dyspepsia. Here we show evidence, based on our own and other authors' work, that chronic constipation has several abnormalities reconductable to alterations in the enteric nervous system, abnormalities mainly characterized by a constant decrease of enteric glial cells and interstitial cells of Cajal (and, sometimes, of enteric neurons). Thus, we feel that (at least some forms of) chronic constipation should no more be considered as a functional/idiopathic gastrointestinal disorder, but instead as a true enteric neuropathic abnormality.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts

AIM: To study the effectiveness and mechanisms of anti-human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis,oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. METHODS: The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7721 and,subsequently,polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively,the growth of cells in nude mice and angiogenesis were observed. RESULTS: VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7721 cells and xenografted tumor. Compared to the control group,the transfected cells grew slower in cell cultures and xenografts,and the xenograft formation was delayed as well. In addition,the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstratedby microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. CONCLUSION: Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation,differentiation and tumori-genesis in hepatocarcinoma.