The Role And Mechanism Of Raf-1 In Gastric Cancer And Its Angiogenesis

[Background]V Raf was separated from pig sarcoma virus in 1980s. It's Oncogene C Raf-1 was found in normal cells and the encoded protein was expressed in many kinds of tissues. Following, A Raf and B Raf were founded, and they only expressed in some special tissues (urinary and nervous system). Among them, Raf-1 protein kinase has been the focus of some researches. Raf-1 is a 70-74kDa protein, which is divided into three functional domains. Raf-1 is involved in the transduction of mitogenic signals from the cell membrane to the nucleus and a part of the ras-dependent signaling pathway from receptors to the nucleus. Recent studies have provided evidence suggesting that Raf-1 is involved in many aspects of tumor development including tumorigenesis, metastasis, cell-cycle control and apoptosis. However, the precise mechanisms underlying the involvement of Raf-1 in one important area of cancer development, tumor angiogenesis, have not yet been defined.A number of recent studies about the Raf-1 involvement in angiogenesis have focused the attention on the role of Raf-1 inendothelial cell regulation. For example, Raf-1 has been shown to influence endothelial cell survival and apoptosis. But to date, the underlying mechanisms of the regulation and the effect of Raf-1 during tumor angiogenesis remain elusive. On one hand, angiogenesis is dependent on endothelial cell survival and apoptosis. On the other hand, angiogenesis is also dependent on tumor cells proliferation and the angiogenesis related factors secreted by them. Now, there have been little available data on the role of raf-1 in tumor cells regulation during tumor angiogenesis.Here we have constructed Raf-1 siRNA, infected gastric cancer cells, and observed the effect of Raf-1 on gastric cancer tumorigenesis and angiogenesis in vitro and in nude mice. We propose that specific Raf-1 targeting may have important therapeutic values for cancer therapy, and that individual Raf-1 may be a useful target in developing angiogenic inhibitors.[Objectives]: l.To investigate the expression of Raf-1 protein in gastric cancer tissue and cells and analyze the relationship between the expression of Raf-1 protein and the clinic pathological characteristics of patients with gastric cancer; 2.To construct raf-1 siRNA and explore the effects of Raf-1 protein on malicious biological behaviours of gastric cancer cells; 3.To elucidate the role and mechanism of Raf-1 in gastric cancer angiogenesis.[Methods]: (1) The expressions of Raf-1 mRNA and protein in gastric cancer cells and immortalized gastric epithelial cells were measured by RT-PCR and Western blot, respectively. (2) The expression of Raf-1 protein in gastric cancer and normal gastric tissues was investigated by immunohistochernistry and the relationship between the Raf-1 expression and clinic pathological characteristicswas statistically analyzed. (3) Raf-1-siRNA vector was constructed and transfected into gastric cancer cells, followed by screening and verifying. (4) Growth curve of gastric cancer cells treated with Raf-1 inhibitor or transfected with siRNA vector was drawn by MTT method. (5) The cell cycle distribution of gastric cancer cells treated with Raf-1 inhibitor or transfected with Raf-1 -siRNA vector was investigated by Flow cytometry. (6) Flow cytometry with Annexin V/PI staining was used to assess apoptosis of gastric cancer cells. (7)ELISA, western blot and MTT were used to assess expression of angiogenesis related factors and effect on proliferation of HUVEC by gastric cancer cells treated with Raf-1 inhibitor or transfected with Raf-1-siRNA vector. (8) The effect of Raf-1-siRNA on tumorigenicity and angiogenesis of gastric cancer cells was revealed by tumorigenesis in nude mice.[Results]: (1) Raf-1 mRNA and protein were highly expressed in four gastric cancer cell lines but lower in immortalized gastric epithelial cell line. (2) Compared with normal gastric tissues, Raf-1 protein was highly expressed in gastric cancer tissues and expression of Raf-1 protein was associated with differentiation and clinic pathological characteristics of patients (PO.05). (3) Both Raf-1 inhibitor and Raf-1-siRNA inhibited proliferation of gastric cancer cells (P<0.05). (4) Raf-1 inhibitor and Raf-1-siRNA inhibited gastric cancer cells entering into S phage from Gl phage and gastric cancer cells transfected with Raf-1-siRNA vector presented lower proliferative index. (5) The results of Flow cytometry showed that both Raf-1 inhibitor and Raf-1-siRNA induced apoptosis in gastric cancer cells (6) The invasiveness and tumorigenicity of gastric cancer cells was reduced when Raf-1 protein was inhibited in nude mice. (P<0.05). (7) Microvasculature density suggested that mean vessel...