| Glioma is the most common primary intracranial tumor in adults, which grows infiltratively and is highly invasive. It is solid tumor with a poor prognosis and rich blood vessels that recurs easily. Method in the treatment of glioma includes operation, chemotherapy, gene therapy and other new biological treatment. Right now, despite some achievements to improve the prognosis of glioma is exist, it is still limited. There are many reasons for these difficulties, of which most main results from two aspects:1.glioma cells are invasive growth and dividing line of the tumor tissue, but normal brain tissue is not clear. The tumor tissue removal operation cannot be completed, residual tumor cells become the seed of recurrence; 2. glioma is solid tumor with rich blood vessels. Rich blood supplies to the tumor cells provide sufficient nutrients and the material basis for its rapid proliferation. Therefore, to explore an effective molecular marker for diagnosis and provide targets for inhibiting tumor cell proliferation, invasion and angiogenesis, thereby improving the glioma treatment effect and the prognosis currently become the research hotspot. The Sema4D transmembrane protein receptor Plexin-B1 which was originally regarded as neural guidance molecules has been widely researched. In recent years, with the intensively study, it is found that Plexin-B1 may be involved in a variety of cellular processes, in particular, has played an important role in the process of tumor development and angiogenesis, has been used as a new target for tumor diagnosis and molecular therapy. Some research indicated that, Plexin-B 1 in normal brain tissue express little. With the pathological grades of glioma increased its expression quantity also increases. The new target point can be used as a potential molecular marker of glioma diagnosis and immunotherapy. However, Plexin-B1 plays a role in the development of glioma and its role in the related regulation mechanism is unknown. In order to study the effect of Plexin-B1 on neural invasion of glioma cell migration and angiogenesis and related mechanism, we carried out the relevant researches. First of all, through immunohistochemical staining of glioma cells we make sure that the Sema4D and Plexin-B1 almost have no expression in normal astrocytes whereas in glioma cells expression abnormally increased. Then, we use shRNA to inhibit the expression of Plexin-B1 and application of RT-PCR and Western blot to identify whether transduction is successful. The application of puromycin screened the stably transfected cell lines. Secondly, in order to study the effect of Plexin-B1 on the growth and apoptosis of glioma, we used flow cytometry and mitochondrial membrane potential detection technology to research on the effect of Plexin-B1 on glioma apoptosis. The experimental results show that, when the silence of Plexin-B1 group compared with the control group, the early apoptosis of tumor cell number increased obviously, the mitochondrial membrane potential decreased significantly. By constructing the in vivo tumorigenicity of tumor bearing mice, the results showed that Plexin-B1 silencing group tumor size was significantly smaller than in the control group, silencing of Plexin-B1 can inhibit the growth of glioma cells. Thirdly, in order to study the role of Plexin-B1 in glioma invasion and migration ability, we use the scratch test, the Transwell experiment, and cytoskeleton were studied by staining. We use RT-PCR, western blot detects the expression of ?v?3, RhoA, RhoB, RhoC, SRPK1, and coimmunoprecipitation to detect whether Plexin-B1 affects the movement of glioma cells by regulating cytoskeleton. Results show that, Plexin-B1 can promote the invasion and migration of glioma cells by affecting the cytoskeleton, this effect is mediated by the active of avP3, RhoA, SRPK1. In order to study the relationship between Plexin-B1 and SRPK1, we used SRPK1 shRNA to silence SRPK1 and confirmed the effects of SRPK1, RhoA and Plexin-B1 on ?v?3 are little. Then, we study the influence of Plexin-B1, SRPK1 on glioma angiogenesis, measure tumor microvascular density in the body environment (MVD) and use immunofluorescence staining to detect the expression of CD31. Isolated environment on human umbilical vein endothelial cells were small officer forming experiment, results show that silence of Plexin-B1 and SRPK1 could inhibit angiogenesis. Finally, we explore the effects of Plexin-B1 on apoptosis of glioma cell growth, migration and possible mechanisms of angiogenesis invasion. Through the studies of expression of PI3K, p-PI3K, Akt, p-Akt by RT-PCR and Western blot we have found that Plexin-B1 may play a role through PI3K/Akt pathway. This study reveals that Plexin-B1 promotes invasion and angiogenesis of glioma. And mechanism of inhibiting apoptosis, invasion and angiogenesis is through regulating RhoA/?v?3/PI3K/Akt pathway and SRPK1 in glioma. This experiment provides theoretical basis and molecular evidence for the drug research and development of treating glioma. |
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