Initial Study On VasohibiiN-1Regulating Angiogenesis In Hepatocellular Carcinoma

Background&aimHepatocellular carcinoma (HCC) is one of the most common solid tumors throughout the world. Though surgical ablation and liver transplantation are the effectiffive measures for treating HCC, the effect is dissatisfactory. Metastasis and recurrence of HCC are the maximal barrier influencing therapeutic effect. As HCC is characterized by hypervascularization, angiogenesis is involved in its invasion and migration the interaction between tumor cells and vascular endothelial cells. It is important to discussion the role of angiogenesis in invasion and migration of Hepatocellular carcinoma.Among the known angiogenic factors, vascular endothelial growth factor (VEGF) plays a central role in mediating tumor angiogenesis, associated with tumor progression, invasion, metastasis, microvessel density (MVD), and poorer survival of patients with HCC. Recently, vasohibin-1, a different type of endogenous inhibitor of angiogenesis, was identified. Its basal expression in quiescent endothelial cells is low, but it is induced in response to angiogenic stimuli, such as VEGFA, and inhibits angiogenesis in an autocrine manner. Previous studies demonstrated that vasohibin-1expression played important roles in the regulation of intratumoral angiogenesis and was associated with neovascularization, malignant potential, and unfavorable prognosis of patients with breast carcinoma. However, no study was conducted to focus on the expression pattern in tumor cells and potential role of vasohibin-1in HCC.Methods1. The expression of vasohibin-1in HCC:Formalin-fixed and paraffin-embedded117HCC tissues and corresponding non-cancerous tissues were retrieved from the Department of Pathology, Shandong Provincial Hospital. The expression of vasohibin-1and VEGF-A in HCC was detected by immunochemistry. The relationship between the expression of vasohibin-1and VEGF-A and the clinicopathologic factors was evaluated. Correlation between vasohibin-1and VEGF-A, MVD, and clinicopathological features was then investigated. Prognostic value of these factors was determined using Kaplan-Meier analysis and a Cox proportional hazards regression model.2. The expression of vasohibin-1in HCC cell lines and L-02:The expression of vasohibin-1in the HCC cell line:HepG2,SMMC-7721,BEL-7402and MHCC-97L was detected by immuncytochemistry, RT-PCR and western blot.3. Silencing of vasohibin-1in hepatocellular carcinoma cell mediated by lentivirus vector:Lentiviral vectors(LV-RNAi), containing U6promoter and green fluorescent protein(GFP), were constructed to deliver small hairpin RNA(shRNA) targeting vasohibin-1into hepG2cell according to the manufacturers'instruction. The control group was the hepG2cell transfected with negative vector. The transfection efficiency was determined by detecting the GFP expression with the fluorescent microscopy. The expression level of vasohibin-1mRNA was examined by Real time quantitative polymerase chain reaction, The expression level of vasohibin-1protein was finally examined by Western blotting for identifing the inhibitory efficiency of RNAi.4. Effects of vasohibin-1gene silence on biological behavior of hepatocellular carcinoma cell:The cell proliferation was detected by MMT assay as described in the manufacture's manual. Motility and migrating velocity of cells was tested by scratch wound healing assay. The rates of apoptosis and cell cycle changes of the hepatoma cell line hepG2were studied by flow cyometry. To investigate the possible mechanism of vasohibin-1involved in the procession of angiogenesis, the expression of VEGF-A and MMP-9in hepG2after vasohibin-1RNAi transfection was tested by Real-time PCR and western blot.Results1. The expression of vasohibin-1in HCC:Cytoplasm high expression of vasohibin-1was detected in38.5%(45/117) of the HCC tissues, which was significantly higher than that in16.2%(19/117) of ANLT (P<0.001). Vasohibin-1was statistically correlated with VEGF-A, MVD, and microvascular invasion in HCC.(P=0.014, 0.035, and0.002, respectively). Patients with vasohibin-1high expression had significantly poor disease-free survival (DFS) and overall survival (OS) at5years after curative hepatectomy (P<0.001for each). Multivariate analysis confirmed that vasohibin-1high expression was an independent prognosticator for unfavorable DFS (HR=2.554, P<0.001) and OS (HR=2.232, P=0.002), along with VEGF-A and TNM stage.2. The expression of vasohibin-1in HCC cell line:Immuncytochemistry, Real time PCR and western blot showed that vasohibin-1was high expressed in the HCC cell line HepG2,SMMC-7721,BEL-7402and MHCC-97L, but hepG2is the strongest.3. Four RNAi lentivirus expression vectors targeting to four various sites of vasohibin-1gene were produced, and the sequence and correct site of ds oligos inserted were confirmed by PCR and sequencing assay; The lentivirus were packaged in293T cells with high titer; Small hairpin RNA (shRNA) targeting vasohibin-1was transfected into hepG2cells. The efficiency of vasohibin-1RNAi sequence against the expression of vasohibin-1was detected by Real-time PCR and western blot. Real-time PCR showed that the relative mRNA levels in vasohibin-1RNAi-1,2,3,4group was33.1%,56.5%,48.3%and81.6%, compared to the negative control group. All the four RNAi significantly reduced the vasohibin-1mRNA(P<0.05), and the vasohibin-1RNAi1showed strongest inhibition. Western blot showed that the protein level of vasohibin-1was down-regulation in the vasohibin-1RNAi, with strongest down-regulation in the vasohibin-1RNAi1group (P<0.05). Real-time PCR and western blot suggested that vasohibin-1RNAi1showed strongest inhibition of the expression of vasohibin-1compared with vasohibin-1RNAi2,3,4. Vasohibin-1RNAi1was choosed for the following experiments to investigate the role of vasohibin-1in the invasion of HCC cell lines.4. Role of vasohibin-1RNAi on proliferation of hepG2:1) vasohibin-1RNAi sequence could decrease the hepG2cell proliferation. A MMT assay revealed that treatment with vasohibin-1RNAi did change the number of viable cells, compared with the control groups (P<0.05).2) vasohibin-1RNAi sequence could not change the migration of hepG2:Scratch wound healing results showed that the wound distance 24h and48h after scratching in negative control cells, The migrating distances of vasohibin-1RNAi cells was not significantly shorter than those of NC RNAi cells and negative control cells (P>0.05).4) vasohibin-1RNAi sequence change the cell cycle and increased the apoptotic index of hepG2(P<0.05).5) vasohibin-1RNAi sequence down-regulated the expression of VEGF-A:Real-time PCR suggested that the expression of VEGF-A was significant lower in vasohibin-1RNAi group than in the control groups (P<0.05). Western blot results further confirmed that the expression of VEGF-A was down regulated in the vasohibin-1RNAi group, while those of MMP-9were slightly lower in the experimental group than in the two control ones without statistical significance..Conclusion1. Upregulation of vasohibin-1expression is associated with angiogenesis and poor prognosis of HCC. Vasohibin-1and VEGF-A are the most important factors influencing the dismal prognosis based on the modulation of angiogenesis in HCC, which provides a rational approach for treatment in the future.2. Vasohibin-1gene silencing can inhibit the expression of vasohibin-1in HepG2cells, HepG2cells transfected with vasohibin-1RNAi could inhibit cell growth, and be arrested on G2/M phase, increased cell apoptosis. Above all, the expression of VEGF-A downregulation, which suggested that vasohibin-1may be involved in angiogenesis of hepatocellular carcinoma cells by regulating the expression of VEGF-A. Vasohibin-1plays an important role in the regulation of HCC cells in angiogenesis and counld be used as a potential therapeutic target for the treatment of liver cancer.3. Lentiviral vector pGCSIL-GFP-vasohibin-1RNAi can inhibit vasohibin-1stably and high efficiently was identified, and effectively infected the hepG2cell line, satisfying the need for the experiments. And the vector provides an efficient tool for further investigating the biological function of vasohibin-1on the hepG2cell line and potential target genes.