Role And Mechanism Of GSK3β In Regulating Glioma Growth And Angiogenesis

[Objective] Glioma accounts for the majority of intracranial primary malignant tumors, glioma is well perfused blood vessel and angiogenesis is required for malignant glioma tumorigenesis and development. Anti-angiogenesis targeted therapy has become a new therapy of glioma, and provided a potential new approach in glioma growth, invasion, and metastasis. GSK3βis a multifunctional Ser/Thr kinase, identified as a critical mediator in the synthesis of glycogen in metabolism. Recent studies have showed that GSK3β not only involved in the regulation of glycogen metabolism, but also plays a central role in a variety of signaling pathways, such as the Wnt/beta-catenin/NF-κB/PI3K/AKT/mTOR signaling pathways in tumor development. The biological effects of GSK3β is correlated with cell mobility, proliferation and survival. However, the role of GSK3P in glioma development is focus on proliferation, invasion and apoptosis for the present. However, the role of GSK3β in glioma development is unclear, and molecular mechanisms underlying GSK3P regulation of neoplastic transformation and tumor development remains to be elucidated. In this study, we simulation the hypoxic environment of glioma, applied in vivo and in vitro experiment to elucidate the specific role of GSK3β in glioma and the underlying mechanism of GSK3β in regulating glioma neoplastic transformation and tumor development.[Methods] (1) Human glioma cell lines U87 and U251 were transduced with the lentiviral-mediating plasmids encoding nonspecific ScRNA- or GSK3β-targeting shRNA. Stable cell lines were obtained through puromycin selection. (2) To investigate the role of GSK3βin cell proliferation and tumorigenesis, we performed the Cell Counting Kit-8 (CCK-8) and colony-forming unit assays in hypoxia.To investigate the role of GSK3β in the angiogenesis, tube formation assay was conducted with HUVECs in hypoxia. (3)We explore the underlying mechanism of GSK3β in regulatingglioma neoplastic transformation and tumor development at the molecular level, and also GSK3βtarget gene levels in both U87 and U251cells exposed to hypoxia. (4) To further clarify the role of GSK3β in tumorigenesis and angiogenesis.Stable transfected U87 and U251cells were injected subcutaneously into flanks of the nude mice.The relative angiogenesis responses andGSK3ptarget gene levels were assayed.[Results] (1) In establishment of GSK3P knockdown lines in glioma cell lines, in comparison to the Con and Sc group, the protein levels of GSK3β were greatly repressedin GSK3βi group. (2) Hypoxia-inducible angiogenesis,cell proliferation and tumorigenesis of both cell lines can be blocked by the knockdown of GSK3β in vitro. (3) Immunohistochemistry analysis indicated that tumor growth and formation of tumor microvessels is significantly lower in GSK3βi group than Sc group. (4) To explore the underlying mechanism of GSK3β in regulatingglioma neoplastic transformation and tumor development at the molecular level, the expression levels of the phosphorylation levels of HIF-la, VEGFand p-p70S6Kl was attenuated in GSK3βi group in both U87 and U251 cells exposed to hypoxia. (5) It wasconfirmed that p-p70S6K1、HIF-1α and VEGF mRNA expression in xenograft tumorswere significantly decreased inGSK3βi group.[Conclusion] We found that GSK3P knockdown significantly inhibited the proliferation, tumorigenesis and angiogenesisexposed to hypoxia. The results further indicated that GSK3pknockdown inhibited tumorigenesis and angiogenesis through targeting p70S6K1downstream molecules such as HIF-1α and VEGF. These findings suggest the potential utility of GSK30 gene therapy to inhibit tumor growth and angiogenesis.