The Expression Of URG11 In Tissues And Its Role In Development Of HCC

【Background】Hepatocellular carcinoma (HCC) is one of the most common cancers in our country, which severely did harm to the health of people. Epidemiological and molecular studies revealed that 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), might play an important role in the development of HCC. HBx is important for HBV viral replication. HBx is a trans-activator and could regulate a number of genes which are involved in cancer development and progression. HBx could bind to and functionally inactivate the tumor suppressor, p53. Moreover, HBx regulates many cytoplasmic signal transduction pathways which affect cell proliferation and differentiation. However, the mechanisms mentioned above could only partially explain the role of HBx in HCC. There must be some unknown mechanisms which are involved in HBx-associated hepatocarcinogenesis. To further clarify the role of HBx in HCC development, suppression differential hybridization and RACE-PCR were used to search some new molecules which might contribute to HBx mediated transformation. Upregulated gene 11 (URG11), a new gene which was upregulated by HBx, was successfully cloned. This gene was located on chrosome 11, and contains 13 exons.The code sequence is 2022bps and encodes a 70kD protein. This gene contains a transmembrane helices (281-297), five von Willebrand factor type C (VWFC) domains, a single C-type lectin domain, and a long tyrosines in tail, which has a lot of phosphorylation sites. VWFC domain has been observed in many proteins and is associated with cell/cell recognition and cell/matrix interaction, and C-type lectin domain has been found in some proteins that appear to regulate cell growth, indicating that URG11 might be a functional regulator of cell growth. And preliminary studies have also found that URG11 was overexpressed in some tumor tissues inclucing HCC, and could promote tumor cell growth. Moreover, URG11 upregulated the expression ofβ-catenin, a key molecular of Wnt signal pathway. However, the expression files of URG11 in different normal and tumor tissues remain unknown. The role of URG11 on Wnt signal pathway and other possible mechanisms involved in the contribution of URG11 on HCC development remain unclear.【Aims】1. To examine the expression of URG11 in different normal and tumor tissues.2. To detect the expression and significance of URG11 in HCC tissues and cell lines.3. To investigate the relationship between HBx and URG11 in HCC tissues and cells.4. To study the effects of URG11 on HCC cell growth. 5. To clarify the possible mechanisms of URG11 on HCC cell growth.【Methods】1. Immunohistochemistry was perfomed to examine the expression of URG11 in different human normal and tumor tissues.2. Immunohistochemistry was perfomed to examine the expression of URG11 and HBx in HCC tissues, adjacent noncancer tissues, and norml liver tissues.3. RT-PCR and western blot were performed to examine the mRNA and protein expression of URG11 in HCC cell lines (HHCC, HepG2, SMMC-7721), immortal liver cell line (QZG) and HBx transfectd HCC cells.4. Immunocytochemistry was perfomed to examine the distribution of URG11 in HCC cells.5. Liposome 2000 was used to transfect eukaryotic expression vector and siRNA vector of URG11 into QZG cells and HCC cells, respectively, and G418 was used to select stable individual clones.6. MTT method was used to detect the growth of the transfected cells and control ones.7. FCM was used to analyze cell cycle of the transfectants.8. Colony formation assay was used to investigate the anchor-independent growth ability of transfected cells.9. Soft agar and nude mice were used to detect tumorigenesis ability of the transfected cells in vitro and in vivo.10. RT-PCR and western blot were used to detect the cell cycle related moleculars, including p21waf1, cyclinD1, CDK4, and CDK6.11. Dual-luciferase reporter assay was used to examine the activation of Tcf/Lef signaling by URG11. 12. Western blot was used to examine the expression of GSK3βandβ-catenin.【Results】1. Expression of URG11 in different normal and tumor tissuesCytoplasmic positive staining of URG11 was found in the normal and cancer tissue cells. URG11 positive expression was detected in normal tissues of heart, lung, spleen, kidney, adrenal gland, bladder, testicle, womb, muscle, skin and placenta, while not detected in normal tissues of brain, thymus, lymphnode, ovary and prostate. In tumor tissues, positive expression of URG11 was found in carcinoma of lung, kidney, bladder, thyroid gland, mammary gland, testicle, ovary, and prostate, while not found in carcinoma of brain, womb, and skin. URG11 expressions were upregulated in tumor tissues of thyroid gland, mammary gland, ovary, and prostate, while downregulated in cancer tissues of womb and skin, when compared with those in normal tissues, respectively. The expression of URG11 in gastric cancer tissues, colon cancer tissues, and pancreatic cancer tissues were also higher than those in the corresponding normal tissues respectively.2. Expression of URG11 in HCC tissues, adjacent noncancerous tissues, normal liver tissues and HCC cell linesAmong 85 pairs of tissues, URG11 positive expression was detected in 53 HCC tissues (62.35%) and in 59 adjacent nontumor liver tissues (69.41%), while in 27 normal liver tissues, only 5 (18.5%) tissues were with weak URG11 expression. The positive rates of URG11 expression in HCC tissues and adjacent noncancerous tissues were higher than that in normal liver tissues. And in HCC tissues, the level of URG11 expression in well differentiatied HCC tissues was higher than that in moderately differentiated ones and poorly differentiated ones, respectively. URG11 mRNA and protein expression in HCC cell lines was also higher than that in immortal liver cell line, which was with no URG11 expression.3. Relationship between HBx and URG11 in HCC tissues, adjacent noncancerous tissues, and in HBx transfected HCC cellsHBx was almost exclusively cytoplasmic in the cancer and adjacent noncancer cells, occasionally with nuclear staining. Among 72 pairs of HBV related HCC tissues, HBx positive expression was detected in 59 HCC tissues (81.9%) and in 62 adjacent nontumor liver tissues (86.1%), while 27 normal liver tissues were all neative for HBx expression. Correlation coefficient between HBx and URG11 was 0.41 in HCC tissues (p<0.05) and 0.51 in adjacent nontumor tissues (p<0.05). Western blot and RT-PCR analysis showed that both URG11 mRNA and protein were upregulated in HBx transfected cells, when compared with cells transfected with empty vector.4. Effects of URG11 on HCC cell growth.After the eukaryotic expression vector for URG11 was successfully transfected into QZG cells, URG11 enhanced QZG cell growth and proliferation, and promoted more QZG cells entering into S phase from G1 phase. When siRNA vector of URG11 was transfected into HepG2 cells and HHCC cells, both HepG2 cell and HHCC cell growth were suppressed, and the ability of colony formation, anchor-independent growth and tumorigenesis in nude mice of these two cells were decreased, either. Furhermore, downregulation of URG11 blocked G1-S progression of HepG2 cell and HHCC cell, but with no influence on cell apoptosis of these two kinds of cells.5. Regulation of URG11 on G1/S phase associated moleculesAfter URG11 was upregulated, expressions of cylinD1, CDK4 and CDK6 were increased, while p21waf1 expression was decreased. When URG11 was downregulated, cylinD1 was also decreased.6. Effects of URG11 on Wnt signal pathwayAfter URG11 was upregulated,β-catenin expression was increased, Tcf/Lef signal was activated and cylinD1 expression was upregulated inβ-catenin dependent manner. When URG11 expression was downregulated,β-catenin expression was also decreased. However, the expression of GSK3βwas unchanged, no matter URG11 expression was upregulated or downregulated.【Conclusions】1. URG11 might play different role in different types of tumor.2. URG11 might play an important role in HCC development, and it might be mainly regulated by HBx.3. URG11 affects cell growth, cell cycle, and tumorigenesis ability of HCC cells in vivo and in vitro, which is associated with regulation of cylinD1 inβ-catenin dependent manner.