| Objective: To investigate the anomalous changes on alternative splicing in glioma cells during the transcription process of GDNF gene and reveal the potential impact of these changes on the migration of glioma cells.Methods: Efficient primers to the cDNA of two highly homologous splice variants of GDNF were used to detect the expression level of mRNA of both GDNF splice variants(α- and β-pro-GDNF), western blot and immunofluorescence were applied to discover the expression level and distribution of α- and β-pro-GDNF in glioma cells respectively 。 Constructed lentivirus and transfected glioma cells U251, so that over-expression and inhibit the expression of α-pro-GDNF protein and then observed the influence of α-pro-GDNF protein on migration of glioma cells through wound scratch assay.Results: Real-time PCR and western blot both indicated the existence of alternative splicing of GDNF gene in glioma cells, the expression levels of mRNA and protein of α- and β-pro-GDNF increased gradually with the raise of the WHO grade, and compared to β-pro-GDNF, the increasing trend of mRNA and protein of α-pro-GDNF is particularly significant, showing predominant expression. The α- and β-pro-GDNF was mainly localized in the cytoplasm of glioma cells, and quantitative analysis of fluorescence intensity reconfirmed more protein in glioma cells than normal cells; after transfection, α-pro-GDNF protein increased in overexpression group and decreased in interference group, α-pro-GDNF protein might be associated with migration of glioma cells by wound scratch assay.Conclusion: We found abnormal alternative splicing of GDNF gene in glioma cells, in which α-pro-GDNF expressed preferentially,it was related to the migration of glioma. This may provide a new therapeutic targets for glioma. |
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