Adenoviral-mediated URG11RNAi Inhibits Proliferation Of Human Liver Cancer Cells

Upregulated gene 11 (URG11) is a newly discovered gene upregulated by hepatitis B virus X antigen (HBx),which encodes a 70kD protein consisting of two von Willebrand factor type C (VWFC) domains and a single C-type lectin domain. VWFC repeats have been identified in 200 extracellular matrix proteins. These repeats, named on the basis of amino acid conservation of 10 cysteine residues, have been shown to bind to members of the transforming growth factor-β(TGF-β) superfamily and are proposed to regulate cell movement,adheresion and signal transduction. C-type lectin domain has been found in some proteins that appear to regulate cell proliferation and metastasis. Previous research indecates that URG11 expresses higher in tumor tissue than in non-tumors.It can promote tumor cell growth and differentiation, enhance tumor metastasis through upregulating β-catenin expression,and additionally increase intracellular calcium level.Therefore, URG11 was predicted to be a very important new gene in proliferation and invasion of tumor cells.Despite for its still unknown mechanisms in human liver cancer development,URG11 may be a promising therapeutic target for adenoviral-mediated gene therapy of human liver cancer.【Objectives】1.To determine the effects of adenoviral transgene expression of URG11 on in vitro and in vivo growth of human liver cancer cells by constructing URG11 and URG11-SiRNA adenoviral vector sysytem; 2.To study the effect of liver cancer gene therapy;3.To screen downstream effectors of URG11.【Methods】1.Adenoviruses expressing URG11 and URG11-siRNA (green fluorescent protein CMV,pi0 as control) were constructed and packaged.Expressing level of URG11 protein was effectively decreased by Ad-URG11-siRNA tested by WB;2. Ad-URG11 and Ad-URG11-siRNA viruses were introduced into HCC and QZG cells separately, and the levers of URG11 protein expression had been tested by WB; Viral mediated in vitro cell proliferation, cell cycle and apoptosis were determined separately by MTT assay and FCM; 3.Effects on tumor growth in vivo were investigated in a nude mouse model;4.2-DE assay had been performed to screen URG11 downstream molecules.【Results】 1. Successfully construction of URG11 and URG11-siRNA adenoviral vector systemAdenoviruses expressing URG11, URG11-siRNA(CMV or pi0 as control) were constructed and expressing level of URG11 protein in HCC cells was effectively decreased by Ad-URG11-siRNA tested by WB; Compared with uninfected cells, URG11 was upregulated significantly in QZG cells by Ad-URG11.2. Ad-URG11-siRNA inhibited in vitro tumor cell proliferationAd-URG11-siRNA and Ad-URG11 were introduced into HCC and QZG cells separately, and the lever of URG11 protein expression had been tested by WB; In vitro cell proliferation, cell cycle progression and apoptosis were determined by MTT assay and FCM. We found that downregulation of URG11 in HCC cells could inhibit cell growth, block cell G1-S phase progression and induce cell apoptosis(P<0.05),while upregulation of URG11 in QZG by Ad-URG11 had reverse effects.3. Ad-URG11-siRNA inhibited in vivo tumor cell growthEffects on tumor growth in vivo were studied in a nude mouse model. Beforehand decreasing of URG11 expression by Ad-URG11-siRNA inhibited tumorigenicity of implanted HCC cells, and significantly decreased tumor size. On the other side,in vivo treatment of pre-established tumors by HCC cells with Ad-URG11-siRNA significantly reduced local tumor size (P<0.05).4.Screening of URG11 downstream effectorsThe maps of MKN28-URG11-siRNA and MKN28-pi0 proteome were successfully acquired through two-dimensional electrophoresis. Melanie3 2D software was used to analyze the two maps. Compared with the map of MKN28-pi0, there are 11 protein spots in MKN28-URG11-siRNA which were strongly stained and 8 spots weakly stained. All the spots were treated by destaining of silver, in-gel digestion and then analyzed by MALDI-TOF-MS software. After MASCOT database searching, six proteins were identified. They were calcium binding protein p22,calpain,renin binding protein,choline acetyltransferase,Fibrinogen and c-type lectin,among which c-type lectin and renin binding protein were up-rgulated by URG11.【Conclusion】In conclusion, there is evidence that URG11 can functionally regulate proliferational potential in vitro and in vivo of human hepatocellular carcinoma cells, which was gained by successfully conctruction of URG11 and URG11-siRNA adenoviral vector system. URG11 may be an important therapeutic target for human liver cancer gene therapy by interacting with a series of calcium related molecules.