Rtn4b Promote Angiogenesis Identification Of Biological Activities And Integrin Beta < Sub > 3 < / Sub > In The Function Of Liver Cancer

Tumor angiogenesis is fundamentally necessary to tumor growth,invasion and metastasis.To date,it is impossible for us to make any significant progress in understanding tumor biology without considering the regulatory rules of vascular network,so it is with the cancer treatment and prevention.RTN4B was firstly cloned by our lab in 1998.Previous work has found that:1) while RTN4B is universally expressed in all normal tissues except liver,it exhibited remarkable up-regulation in hepatocellular carcinoma（HCC） samples at both mRNA and protein levels;2) over-expression of RTN4B in hepatoma cells promote both tumor growth and tumor angiogenesis in nude mice;3) knockdown of endogenous RTN4B in hepatoma cells could successfully inhibited tumor growth,accompanied by decreased blood vessel density.Therefore,it strongly suggests that RTN4B is probably a novel and functional pro-angiogenic factors during HCC development and progress.On the basis of all these findings,in this work,we first carried out various classical assays to demonstrate the pro-angiogenic activity of RTN4B protein.Mammalian RTN4B could not only secret into culture medium,but also be detected in the extracellular basement membrane.By utilizing isolated primary endothelial cells,we demonstrated RTN4B’s activity in cell adhesion,spreading and migration assays, separately.After replated on RTN4B-coated dishes,endothelial cells initiated actin-based cytoskeleton reorganization and formed focal adhesion complex,during which FAK was specifically phosphorylated and downstream ERK and Akt pathway were subsequently activated.To reveal the underlying mechanism of RTN4B’s pro-angiogenic activity,we attempted to isolate its receptor on endothelial cells. Reverse assay using blocking antibody indicated that RTN4B’s activity was integrinα_νβ₃-dependent.This finding was further confirmed by the observation that CHO cells stably expressing integrinα_νβ₃ could specifically well adhere to RTN4B-coated dishes. Moreover,ELISA assay revealed the direct binding relationship between RTN4B and integrinα_νβ₃.Taken together,integrinα_νβ₃ is likely to be a functional receptor of RTN4B on cell surface. Our research work well described the pro-angiogenic activities of RTN4B, revealing an essential role of RTN4B in regulating tumor development and progress. The successful identification of functional receptor of RTN4B makes it possible to find the effective target for blocking RTN4B function,providing a novel experimental evidence for future HCC treatment. The integrin family not only functions in embryonic development and vascular remodeling,but also plays an essential role in tumor initiation and development. Significant alternations in both the expression level and spatial distribution of integrins were observed during tumor progression,meantime more and more functional evidence emerging to demonstrate that integrins were involved in regulating cell survival,proliferation and migration in tumor.However,current knowledge of integrins in tumor is still very limited.Integrins are likely to play diverse roles in different tumor types.As little knowledge of integrinβ₃ in human hepatocellular carcinoma（HCC） has been reported before,exploration of the expression pattern as well as the physiological function of integrinβ₃ in HCC wound contribute to our comprehensive understanding of HCC progression.First of all,by performing quantitative real-time PCR and Western blot,we found that integrinβ₃ was specifically down-regulated in tumor at both mRNA and protein levels when compared with neighboring pathological normal liver tissues.Little endogenous integrinβ₃ were detected in several hepatoma cell lines,either.And transient expression of integrinβ₃ in these cells（including SMMC-7721,SK-Hep1 and QGY-7703） resulted in an enhanced level of apoptosis and suppression of colony formation.We further established stable SMMC-7721 cell lines expressing low level of integrinβ₃.Despite of small difference in apoptotic level under normal condition, there is no detectable difference in cell growth among stable cell lines,which exhibited similar cell growth ratio.However,cell growth inhibition upon serum/ligand deprival and incidences of anoikis were remarkably increased in integrinβ₃-expressing stable lines compared with vector control transfectants.Caspase 8 was also cleaved in these processes.Furthermore,ligands of integrinβ₃ were found to be the key factor in regulating expression level and physiological function of integrin. Expression of two native ligands for integrinα_νβ₃ in liver,vitronectin（VN） and fibrinogen（FG）,was not only inhibited in HCC tissues,but also statistically correlated with the decreased integrinβ₃ expression.Replenished these ligands to the starved SMMC-7721 stable transfectants effectively restored the pro-apoptotic function of integrinβ₃,resulted in less cleaved Caspase 8,which well demonstrated our hypothesis.Consistent results were also gained from SK-Hep1 stable cell lines.Therefore,down-regulation of integrinβ₃ and its ligands in liver is related to the aggressive growth of HCC.Thus,reconstitution of integrinβ₃ in HCC would provide important knowledge for designing novel therapeutic approach to treat liver cancer.