| Glioma is the most common and incurable intracranial tumor. Studying its pathogenesis and trying to find more effective therapeutic modalities are the major research projects in neurosurgical field. Gliomas, just like tumors in other sites of the body, are involved in the processes of uncontrolled cell proliferation, cell dedifferentiation and dysregulation of cell apoptosis. With the development of molecular biology and molecular genetics, it has been found that some of the important oncogenes and tumor suppressor genes such as EGFR and P53 etc are related to gliomagenesis. However, up to date, it is still not clear which are the primary initiating molecular events and which are the secondary promoting molecular events. In addition, gene products exert their function through signaling pathways and there are links and crosstalks even forming a complex network among different signaling pathways. Accordingly, it is more important to study the abnomal activities of gene transduction pathways or the crosstalks among the different gene transduction pathways than to study a single gene activity.The Wnt signaling pathway regulates cell morphology, proliferation, adhesion, migration and structural remodeling. Specific Wnt ligands bind to their target "frizzled" membrane receptors and interact with the cytoplasmic multi-protein degradation complex, which consists of AXIN, APC and GSK-3 β , result in the accumulatin of β -catenin in cell nuclei and β -catenin activates the transcription of its downstream target genes to promote cell proliferation and inhibition of cell apotosis. Therefore, either abnormal activation of Wnt singnaling or mutation of AXIN, APC and β -catenin may lead to the tumorigenesis. Growing evidence indicates that abnormal activity of Wnt signaling pathway exists in many tumors.However, a few investigations have been conducted on the activity of Wnt signaling pathway in gliomas.Based on our previous studies with Atlas cDNA array and RT-PCR, it was found that some members of wnt signaling pathways were upregulated in medulloblastomas and ependymomas. Additionaly, as shown by realtime PCR and western blot, Wnt5a expression was downregulated after RNAi knocking down EGFR expression in TJ905 glioma cell line . So, there may be abnormal activation of Wnt signaling pathway and interaction between EGFR and Wnt signalling pathway in gliomas that should be further investigated..In the present study, we focused on the abnormal Wnt signaling pathway activity in gliomas and its relationshiop with EGFR pathway. The study was divided into 3 parts.In the first part, the mRNA expression of major Wnt members were studied in 47cases of gliomas and 4 normal brain tissues by RT-PCR method, the overexpression of these members were further demonstrated by immunohistochemistry, and the relationship between Wnt and EGFR signaling pathway was also studied. By PCR and sequencing the 3 rd exon of CTNNB1, mutation of P-catenin phosphorylation site and nearby codons was studied.In the second part, RNAi technology was used to observe its inhibitory effect on the growth of human glioblastoma U251 cells. siRNA targeting Wnt2 and P -catenin was transfected into U251 cells mediated by oligofectamine. Wnt2 and 3 -catenin mRNA expression were detected by realtime PCR after transfection. The expression of Wnt2, 3 -catenin and other main members in Wnt pathway and the activity of important downstream pathway of EGFR, PI3K /AKT pathway, was also studied by Western blotting and immunofluorescence staining after transfection. The phenotypic change of U251 cells including proliferation, apoptosis and invasive ability after RNAi trasfection was studied by MTT assay, annexin V staining, flow cytometry and Matrigel 3D growth experiment.In the third part, subcutaneous U251 glioma model was established in nude mice. Every 4 days the mixture of oligofectamine and siRNA was injected into the tumorsand the tumor volumes were measured. On 32n day after the first injection, tumors were resected. The expression of Wnt2, P -catenin and other major members in Wnt pathway and the downstream PI3K and p-AKT expression of EGFR signaling pathway were studied by immunohistochemistry. Apoptosis in tumors were detected by TUNEL method.. Results: The first part:The mRNA expression of the members of Wnt signaling pathway including Wntl, Wnt2, Wnt3, Wnt4, Wnt5a, WntlOb, Wntl3, frizzled2, frizzled5, P -catenin were determined in 47 cases of gliomas. It was found that Wnt2, Wnt5a and frizzled 2 mRNA expressions were upregulated and protein expression of Wnt2, frizzled2, P -catenin, cyclin Dl, C-myc, PCNA, PI3K and p-AKT was also elevated in gliomas. Wnt2 expression was positively correlated with the expression of other genes. It is noteworthy that there was no difference of P -catenin mRNA expression as compared to the normal brain tissues and among the various grades of gliomas. However, the protein expression of P -catenin is significantly enhanced in gliomas and elevated with the increasing malignant degree of gliomas. Sequencing the 3rd exon of CTNNB1 with the phosphorylated site, mutation at the 42nd codon was demonstrated in 2 cases of glioblastomas. The second partU251 cells were transfected with siRNA targeting wnt2 and P -catenin mediated by oligofectamine in vitro. The transfection efficiency was up to over 70%. Wnt2 mRNA and p1 -catenin mRNA expression were obviously knocked down after transfection with siRNA. Wnt2, frizzled2, 3 -catenin, cyclin Dl, PI3K and p-AKT protein expression were also decreased, but C-myc expression didn't change significantly. U251 cells transfected with siRNA targeting wnt2 and p" -catenin showed lowering proliferation activity, decrease of SPF and arresting cell cycle in the Go/Gl phase. The cell invasive ability was attenuated and cell apoptosis was induced. The third part:Subcutaneous U251 glioma model was established in nude mice for in vivo study. As compared with the control group, the tumors in mice treated with siRNA targeting Wnt2 and ￡ -catenin grew slowly and the difference of tumor volumes became significant since the 20th day after the first time of siRNA therapy until the 32nd day tumors were resected. Meanwhile, cell apoptosis was increased. Wnt2, frizzled 2, ￡ -catenin, cyclin Dl, PI3K and p-AKT's expression were downregulated and C-myc expression didn't change as shown by immunohistochemistry.CONCLUSION1. Wnt2 overexpression or mutation of the 3rd exon of CTNNB1 can lead to Wnt signaling pathway activation and result in cell proliferation and invasion. Meanwhile, the Wnt signaling pathway links and crosstalks with the PI3K/AKT signaling pathway and maybe they are modulated with each other.2. Using RNAi technology, siRNA targeting Wnt2 and P-catenin mediated by oligofectamine efficiently knocks down the expression of Wnt2 and P-catenin in human glioblastoma U251 cells, inhibits activation of both Wnt and PI3K/AKT signaling activity, results in decrease of cell proliferation activity and invasive ability, arrests cell cycle and induces apoptosis.3. The established subcutaneous U251 glioma models in nude mice are treated with siRNA targeting Wnt2 and ￡ -catenin. the tumor growth is inhibited and cell apoptosis is induced. The findings of in vivo study is in accordance with those in vitro study4.Wnt2 and p-catenin can be the candidate genes for gene therapy of malignant gliomas. RNAi is a new gene silencing technology with high efficiency, which will have potential application prospects in clinics.
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