The Mechanisms Of BFGF Regulate The Expression Of IL-6 And BFGF-siRNA Enhance Chemosensitivity In Glioma Cells

Background:Glioblastoma multiforme(GBM) is the most prevalent intracranial tumors, which lead to progressive disability and death in most case. Owning to characteristic of migration and invasion of glioma cells, they are difficulty to excise radically by surgery. Besides, complicated tumor microenvironment supply the breeding ground, which enhance the resistibility to chemotherapy or radiotherapy. Because of these features, cells survived from operation combined with chemotherapy or radiotherapy lead to tumor recurrence and treatment failure. With the development of biotechnology, the moleculars relate to the glioma and their mechanism of action are researched deeply. And emerging drugs target specific moleculars. Besides, the new findings of biomarkers are important to grade of glioma and clincial prognosis. The new biomarkers are worth of research interests. So we study the specific biomarker of glioma and intent to supply the new therapeutic strategy for clinical treatment.Objectives: Just as the relationship between fertile soil and seeds, the particular tumor microenvironment lead to the ability of cancer cells to generate, proliferation, migration and angiogenesis more stronger. Combined with our early discovery, b FGF act as high level expression cytokine, it is important for the maintenance of glioma microenvironment. Besides,we also found that b FGF regulated IL-6 expression level, which is closely related to malignancy of glioma. Both of b FGF and IL-6 are important for the malignancy maintenance of glioma, and there are mutual regulation relationship between them, but the specific mechanism is unclear. So we want to explore the specific mechanism ofmutual regulation.Besides, we attempt to weaken the resistibility to chemotherapeutics of tumor cells by destroying the glioma microenvironment.Methods:We divided our research into two parties: Part I: Throw light upon the mechanism of b FGF regulated the expression of IL-6 in glioma microenvironment. To confiremed that b FGF could regulate the expression level of IL-6, glioma cells were transfected with b FGF-si RNA to silence the expression of b FGF and another group cells were stimulated by exogenous b FGF. And then the expression of IL-6 m RNA and protein were measured. To make clear the mechanism of the regulation between b FGF and IL-6, we detected the phosphorylation level of MAPK(including ERK1/2?p38?JNK) signaling pathway after stimulation by recombinant human b FGF. Furtherly, we measured the phosphorylation level of IL-6 transcription factor, including AP-1?CREB?C/EBP? and whether the subunit of NF-?B(p65) enter into nucleus. At last, after pre-blocking the signaling pathway of ERK?p38?JNK sespectivly, the cells were stimulated by b FGF and then the phosphorylation level of IL-6 related transcription factor were detected. According to the results of experiments, we confirmed link between upstream and downstream, and concluded the specific mechanism that b FGF regulated the expression of IL-6. Part II: To explore the chemosensitivity to MTZ after silencing the expression of b FGF in glioma cells. The level of b FGF were down-regulated in glioma microenvironment by using b FGF-si RNA. After 24 hours for transfection by small interference RNA, the glioma cells were treated with TMZ for 48 hours. After that, the proliferation of glioma cells were measure by MTT. The apoptosis were increased significantly and the cell cycle of glioma cells were blocked in G0/G1 measured by flow cytometry. The ability of migration were down regulated measured by Transwell assay. At last, the apoptosis and migration proteins were detected by Western Blot. The results indicated that the anti-apoptotic protein Bcl-2 and migration related MMP-2 and MMP-9 were down-regulated, and the Bax was up-regulated.Results: Part I: 1?After transfected with b FGF-si RNA, the m RNA expression level of IL-6 was down-regulated. After stimulated by b FGF, the m RNA expressionlevel of IL-6 was up-regulated. 2?After transfected with b FGF-si RNA, the protein expression level of IL-6 was down-regulated. After stimulated by b FGF, the protein expression level of IL-6 was up-regulated. 3?After stimulated by b FGF, the phosphorylation level of MAPK signaling pathway was enhanced. The phosphorylation level of IL-6 transcription factor was enhanced. 4?After blocking the signaling pathway of ERK?JNK signaling pathway,the phosphorylation level of AP-1 was inhibited. And blocking the signaling pathway of p39 signaling pathway,the phosphorylation level of NF-?B was inhibited. 5?At last, after pre-blocking the signaling pathway of ERK?p38?JNK sespectivly, the protein expression level of IL-6 was inhibited. Part II: 1?Combined application with b FGF-si RNA and temozolomide can enhance the inhibit effect of proliferation to glioma. 2?Combined application with b FGF-si RNA and temozolomide can promote apoptosis of glioma. 3?Combined application with b FGF-si RNA and temozolomide can block cell cycle in G0/G1 phase. 4?Combined application with b FGF-si RNA and temozolomide can enhance the inhibit effect of migration ability to glioma. 5?b FGF-si RNA enhance the cytotoxicity of temozolomide on glioma cells may relate to MAPK signaling pathway.Conclusion: 1.b FGF activates transcription factors NF-?B and AP-1 through MAPK signaling pathway and then regulates the expression of IL-6. 2.Silencing the expression of b FGF can enhance the chemosensitivity to MTZ in glioma cells.