Gab2-AKT-ARK5 Signaling Pathway Promotes Glioma Invasion

Objective: Glioma is one of common malignant tumors in the central nervous system. Its prognosis is closely related to the degree of tumor invasiveness. This study was to explore whether Gab2-Akt-ARK5 signaling pathway can promote glioma invasion, to provide new therapeutic targets for the effective control of invasive glioma. Methods: 90 samples collected from Affiliated Hospital of Weifang Medical College between 2005 and 2013 were selected for the study. All of those were already diagnosed as gliomas including 42 cases of low-grade glimas(WTO Ⅰ, Ⅱ grade) and 48 cases of high-grade gliomas(WTO Ⅲ, Ⅳ grade). All patients with detail clinical and pathological data did not receive radiotherapy and chemotherapy.1. All tissues specimens were conducted HE staining, light microscope observation and pathological diagnosis.2. The expression of ARK5 and Gab2 in 90 cases of glioma tissues were detected by Immunohistochemistry, SP method and the differences of expression in low-grade gliomas and high- grade gliomas were analyzed.3. Glioma LN-229 cells were selected for the study. Small RNA interference plasmid of ARK5 target fragment 5’-GAAGTTATGCTTTATTCAC-3’, small RNA interference plasmids of Gab2 target fragment 5’-GTGAGAACGATGAGAAATA-3’ and SCR target small RNA interference plasmid were transfected into LN-229 cells, which had gone down to the generation 2 times by cell transfected technology. Transfected cell lines were named the experimental group si ARK5/LN-229 cells(experimental A group), si Gab2/LN-229 cells(experimental G group), si ARK5 4 &si Gab2/LN-229 cells(experimental AG group) and SCR/LN-229 cells(control group). The low expression of ARK5 and Gab2 in experimental group and control group were detected by Western blotting.4. Invasive ability of transfected LN-229 cell lines in experimental group and control group were by Transwell cell invasion assay.5. MMP-2 and MMP-9 activity of experimental group and control group with the stimulation of insulin-like growth factor-1(IGF-1) were detected by gelatin zymography assay.6. Under the stimulation of IGF-1, the phosphorylation of ARK5 and Akt in experimental group and control group were detected by Western blotting.Results:1. Immunohistochemistry showed that the expression of ARK5 protein in high-grade gliomas was significantly higher than in low-grade gliomas, the difference was statistically significant(P = 0.002, P <0.05); the expression of Gab2 protein in high-grade gliomas was significantly higher than in low-grade gliomas, the difference was statistically significant(P=0.000, P<0.05); there was a positive correlation between the expression of ARK5 and Gab2 in 90 cases of glioma tissues(P = 0.000, P <0.05).2. Transwell cell invasion assay showed that compared with the control group, the invasion ability of LN-229 cells in experimental groups and control group was all significantly decreased(P <0.05, P <0.05, P <0.05) and invasion ability of LN-229 cells in experimental AG group was decreased more significantly.3. Zymography results showed that compared with control group, the activity of MMP-2 and MMP-9 in experimental groups was all downregulated.4. Western blot results showed that:4.1 Compared with the control group, only the expression of ARK5 was reduced in experimental A group, only the expression of Gab2 was reduced in in experimental G group, the expression of ARK5 and Gab2 were both reduced in in experimental AG group.4.2 Compared with the control group, the level of phosphorylation of Akt and ARK5 in experimental G group was all decreased with IGF-1 stimulation.SPSS16.0 statistical software was selected to analyze data. Immunohistochemistry was analyzed by χ2 test and Spearman correlation coefficient. All quantitative information and data results are expressed by x±s. Comparisons between two groups were used by two-tailed unpaired t-test. P<0.05 indicated that the difference was significant.Conclusion:1. The expression of ARK5 and Gab2 in gliomas related with the grading of glioma and ARK5 and Gab2 protein expression were positively correlated in gliomas, suggesting ARK5 and Gab2 have certain guiding significance for the assessment of the extent of malignant glioma.2. Invasion ability of LN-229 cells was reduced after the expression of ARK5 or Gab2 had been decreased by si RNA interference as well as the activity of MMP2 and MMP9 was downregulated indicating that ARK5 and Gab2 proteins promoted invasion of glioma, suggesting ARK5 and Gab2 can promote glioma invasion and be used as targets for the treatment of glioma combined biological indicators.3. the phosphorylation of Akt and ARK5 of LN-229 cells was suppressed after the expression of Gab2 had been decreased, indicating that Gab2 was located at ARK5 and Akt ’s upstream in signaling pathway as well as ARK5 and Gab2 can promote glioma invasion through Gab2-Akt-ARK5 signal pathway.