The Expression Of Urg4 In Hcc Tissues And The Effects Of Urg4 On Hcc Cell Proliferation

【Background】Hepatocellular carcinoma (HCC), one of the most common cancers in our country, is severely harmed the health of people. Persistent chronic infection of hepatitis B virus (HBV) is a major cause of HCC. And HBx, which is encoded by the smallest X open reading frames (ORF), plays an important role in the development of HCC. HBx is a trans-activator, which regulates many cell proliferation and cell cycle associated genes. HBx could regulate many cytoplasmic signal transduction pathways. HBx could also affect DNA repair. However, the mechanisms how HBx contributed to hepatocarcinogenesis remain unclear. To further clarify the role of HBx in hepatocarcinogenesis, suppression differential hybridization and RACE-PCR were used to search some new molecules which might contribute to HBx mediated transformation. Upregulated gene 4 (URG4), a new gene which was associated with HBx, was successfully cloned. This gene is located on chrosome 7, and contains 3607bp. This gene contains a transmembrane helices, three nuclear localization signals, and a ATP/GTP binding site, which is 34% similar with caspase recruitment domain family 6. Preliminary studies have found that URG4 was overexpressed in some tumors tissues inclucing HCC, and could promote tumor cell growth. However, the exact roles and possible mechanisms involved in the contribution of URG4 on HCC development remain unclear.【Aims】1. To detect the expression and significance of URG4 in HCC tissues and cell lines. 2. To investigate the relationship between HBx and URG4 in HCC tissues and cells. 3. To clarify the effects and possible mechanisms of URG4 on HCC cell growth.【Methods】1.Immunohistochemistry was perfomed to examine the expression URG4 and HBx in HCC tissues, adjacent noncancer tissues, and norml liver tissues.2. RT-PCR and western blot were performed to examine the protein and mRNA expression of URG4 in HCC cell lines, immortal liver cell line and HBx transfectd HCC cells. 3. Liposome 2000 was used to transfect eukaryotic expression vector of URG4 and siRNA vector of URG4 into QZG cells and HCC cells respectively, and G418 was used to select stable individual clones. 4. MTT method was used to detect the growth of the transfected cells and control cells. 5. FCM was used to analyze cell cycle distribution of the transfectants. 6. Colony formation assay was used to investigate the anchor-independent growth ability of transfected cells. 7. Soft agar and nude mice were used to detect tumorigenesis ability of the transfected cells in vitro and in vivo. 8.RT-PCR and western blot were used to detect the expression of cyclinD1. 【Results】1. Expression of URG4 in HCC tissues, adjacent noncancer tissues, normal liver tissues and HCC cell lines.Among 100 pairs of tissue, URG4 positive expression was detected in 48 HCC tissues (48%) and in 54 adjacent nontumor liver tissues (54%), while in 27 normal liver tissues, only 6 (22.2%) tissues were with weak URG4 expression. The positive rates of URG4 expression in HCC tissues and adjacent noncancere tissues were higher than that in normal liver tissues. And in HCC tissues, the level of URG4 expression in well differentiatied HCC tissues was higher than that in moderately differentiated ones and poorly differentiated ones, respectively. mRNA and protein expression of URG4 was also detected in HCC cell lines, especially high in HepG2 cells, but not in immortal liver cell line.2. Relationship between HBx and URG4 in HCC tissues, adjacent noncancer tissues, and in HBx transfected HCC cells.HBx was almost exclusively cytoplasmic in the cancer and adjacent noncancer cells, occasional with nuclear staining. Among 78 pairs of HBV related HCC tissues, HBx positive expression was detected in 60 HCC tissues (76.9%) and in 65 adjacent nontumor liver tissues (83.3%), while 27 normal liver tissues were all neative for HBx expression. Correlation coefficient between HBx and URG4 was 0.38 in HCC tissues (p<0.05) and 0.45 in adjacent nontumor tissues (p<0.05). Furthermore, URG4 mRNA and protein were upregulated in HBx transfected cells, when compared with cells transfected with empty vector.3. Effects of URG4 on HCC cell growth.After the eukaryotic expression vector for URG4 was successfully transfected into QZG cells, exogenous URG4 promoted QZG cell growth and proliferation, and also promoted more QZG cells entering into S phase from G1 phase. When siRNA vector of URG4 was transfected into HepG2 cells, not cell growth, but also the ability of colony formation, anchor-independent growth and tumorigenesis in nude mice of HepG2 cells were decreased. Furhermore, downregulation of URG4 blocked G1-S progression of HepG2 cells.4. Possible mechanisms by which URG4 affected HCC cell growthAfter URG4 expression was upregulated, cylinD1 expression was increased, while when URG4 expression was downregulated, cylinD1 expression was decreased.【Conclusions】1. URG4 might be a downstream effector of HBx, and might play an important role in HCC development. 2. URG4 affects HCC cell growth, cell cycle, and tumorigenesis ability of HCC cells in vivo and in vitro, which is associated with regulation of cylinD1 expression.