In The Process Of Development Of Liver Cancer. Rtn4b Role

Hepatocellular carcinoma(HCC) is one of the most common malignancies with the fourth highest incidence rate in China,especially in the region of southeast coast. At present,surgical resection is the only effective way for clinical treatment of HCC. However,even by resection,5-year survival prognosis is rare.Our lab has been working on disclosing the mechanism of HCC development and progression through cancer molecular biology research,in searching for novel strategies for effective anti-tumor therapies.Our lab cloned RTN4B gene by virtual cloning in 1998 for the first time.Both immunohistochemistry and RT-PCR experiments showed that RTN4B was significantly up-regulated in HCC tissues.Meanwhile,we were surprised to find that RTN4B was widely expressed in 15 human normal tissues except liver.Therefore, what's the role of RTN4B in HCC? What's the internal relationship between its high expression level and HCC development and progression?As we know,liver is an organ with sufficient blood supply and the cloning hepatoma cells are usually capsuled in HCC.So there is difference in the blood and oxygen supply between normal hepatic cells and HCC cells outside and inside the capsule separately.In 2004.4,Lisette Acevedo et al.reported that RTN4B could promote vascular endothelial cells' adhesion and migration in vitro and it was also involved in the regulation of vascular remodeling in vivo after injury.Based on these implications,we hypothesized that over-expressed RTN4B protein in HCC might be involved in tumor angiogenesis.RTN4B probably promotes tumor growth through regulation of tumor vascular development.If this hypothesis was demonstrated,RTN4B was probably a novel and effective target in drug screen for anti-tumor therapies. Moreover,exploring the biological function of RTN4B in tumor angiogenesis will bring insights into the research work on the mechanism and rules of HCC development and progression.So we selected RTN4B as our study object and have carried out extensive research on it. We first successfully established two RTN4B stably expressing liver cancer cell lines,L02 and QGY.Through comparison,we found no significant difference on cell growth rate between ceils stably transfected with RTN4B and control cells. Conversely,cells expressing RTN4B could evidently promote tumor growth when subcutaneously injected into nude mice.Immunohistochemical staining showed that the vessel density of the tumor formed by cells expressing RTN4B was much higher that that of control cells.These results suggest that the over-expressed RTN4B in HCC might be involved in tumor angiogenesis.Both immunofluorescent cell staining and FACS experiments showed that RTN4B was localized to cell membrane in cancer cells.Further research work found RTN4B could be secreted into extracellular matrix,probably through exosome.By performing western blotting of cell lyses and supernatants,we also demonstrated that RTN4B was widely expressed in nearly all the liver cancer cell lines and it could also be secreted into corresponding cells' culture medium.Recombinant RTN4B protein was successfully prepared through yeast expression system and protein affinity purification technique.We carried out Boyden assay in vitro,finding this protein was able to enhance the chemotaxis of human umbilicus vascular endothelial cells(HUVECs).While in vivo models,recombinant RTN4B protein could also promote angiogenesis in the classic angiogenesis experiment,mouse corneal pockets assay and Chick Embryo Model,which provided a further support for the biological activity of RTN4B in the organism level.Furthermore,we confirmed endogenous RTN4B function with RNA interference (RNAi) technique.SiRNA effectively down-regulating RTN4B was transfected into SMMC-7721 cell line by lentivirus-derived vector and nude mice were subsequently injected.Tumor sizes in the group transfected with RTN4B siRNA were evidently smaller than those transfected with siRNA control.Further immunohistochemistry results showed the vascular density of tumors in the group transfected with RTN4B siRNA were also remarkably lower than those transfected with siRNA control.Taken together,RNAi experiment demonstrated the involvement of RTN4B in tumor angiogenesis in a reverse way:down regulation of RTN4B could inhibit tumor growth through influencing on tumor angiogenesis.By utilizing rat hepatic artery ligation(HAL) model,we found the artificial hypoxia environment could induce the expression of RTN4B.So the hypoxia microenvironment formed in HCC development may be the reason of the expression of RTN4B in HCC.Taken together,our results are mainly consistent with our former hypothesis.The hypoxia microenvironment formed in HCC development induces the expression and secretion of RTN4B. Extracellular RTN4B is then activate tumor vascular endothelial ceils and promote tumor angiogenesis,thus realizing its regulatory function in HCC development and progression. Down-regulation of RTN4B could inhibit tumor angiogenesis and thereby prevent tumor growth. The specific pro-angiogenic function of RTN4B in HCC presents us a new therapeutic target in HCC treatment and also provides us new theoretical evidences and implications for improved strategies.