Experimental Study Of The Effects Of Abi1 On Biological Behavior Of Glioma

Part 1 The expression of Abil in glioma and breast cancer tissuesObjective To investigate the expression and significance of Abil protein in glioma. And to explore the expression of Abil, c-Abl and WAVE2 proteins in breast cancer and their correlation.Methods The expression of Abil was examined in 38 glioma tissues and 7 normal brain tissues by immunohistochemical staining. The expression of Abil, c-Abl and WAVE2 were examined in 66 breast cancer specimens and 24 normal mammary tissues by immunohistochemical staining.Results 1. The strong positive rate of Abil was significantly lower in glioma group than in normal control group. The strong positive rate of Abil was negatively correlated with histological grade of glioma.2. The strong positive rate of Abil and WAVE2 were significantly lower in breast cancer group than in normal control group. The positive rate of c-Abl was not significantly lower in breast cancer group than in normal control group, but its location changed.3. In breast cancer, the strong positive rate of Abil was negatively correlated with tumor size, histological grade, lymph node metastasis and clinical stage, but not with age. The positive rate of c-Abl and the strong positive rate of WAVE2 were negatively correlated with histological grade, lymph node metastasis and clinical stage, but not with age and tumor size.4. In breast cancer, positive correlation was found between the expression of Abil protein and that of c-Abl protein, and between the expression of Abil protein and that of WAVE2 protein.Conclusion 1. The reduction of Abil expression is correlated with poor prognosis of glioma and breast cancer patients.2. The reduction of Abil expression may influence the location of c-Abl protein and the expression of WAVE2 protein. So it is hypothesized that Abil may have a key role in Abl/Abil/WAVE2 passway. Part 2 The construction of glioma cell line stably transfected with Abil geneObjective Our prior studies indicated that the expression of Abil in glioma tissues was relatively low. To explore the relationship between Abil and tumorigenesis and development of glioma, glioma cell line stably transfected with Abil gene was constructed.Methods The expression of Abil in 4 glioma cell lines (U251 MG, H4, U-87 MG and C6) were detected by Western blot. pEGFP-C3/Abil plasmid stably transfected glioma cell line in which Abil expression is lowest among four glioma cell lines by Lipofectamine 2000. Then the cells were cultured in DMEM containing G418. After two weeks, cells were seeded into 96 wells plates. So, different clones were got, which were amplified from a single cell. Abil expression in different monoclones were tested by Western blot.Results 1. Abil expression in U251 MG cells was lowest among four glioma cell lines.2. A glioma cell line which stably expressing Abil at high level was obtained by 8-week G418 selection and Western blot identification.Conclusion Glioma cell line U251 MG stably transfected with Abil gene was successfully constructed.Part 3 The effects of Abil on biological behavior of gliomaObjective To explore the effects of Abil on biological behavior of glioma.Methods To evaluate the effect of Abil on cell morpholoy, U251 MG stably transfected with Abil gene were observed by converted microscope with or without Giemza staining. The cell cycle distribution was analyzed by flow cytometry. Growth assay, soft agar colony formation assay and Ki67 staining were used to analyse cell proliferation. For evaluation of in vitro motility of glioma cells, a monolayer wounding(scratch) assay and transwell migration assay were performed. Matrigel invasion assay was performed to evaluate invasive capability in vitro.Results 1. Upregulation of Abil expression changed morphology of U251 MG cells. 2. Abi1 decreased the proportion of multinucleate cells in U251 MG cells.3. Abi1 downregulated the growth rate of U251 MG cells, decreased cloning efficiency and Ki67 expression.4. Abi1 increased migration of U251 MG cells.Conclusion Abi1 could inhibit malignant transformation of glioma. Abi1 suppressed cell proliferaion of glioma, and increase migration.