The Effect And Mechanism Of Overexpressing IDH1^R132H On The Growth Of Glioblastoma Cells

OBJECTIVE: To study the effect of IDH1^R132H on proliferation, invasion and migration in U87 GBM cells, and explore its molecular mechanism, in order to provide more comprehensive laboratory evidence and therapeutic targets for the individual treatment of GBM.METHODS: To construct a stable U87 cell line overexpressing IDH1^R132H protein, and be verified by Western blot. The effect of overexpressing IDH1^R132H on proliferation in U87 cells was determined by CCK-8 assay, the effect of overexpressing IDH1^R132H on migration in U87 cells was determined by Wound healing test, and the effect of overexpressing IDH1^R132H on invasion in U87 cells was determined by Transwell assay. mi R-128 a expression level was analyzed between IDH1^R132H GBM and IDH1 WT GBM from TCGA database, the expression level of miR-128 a in U87 cells overexpressing IDH1^R132H was also detected by Real-time PCR. The expression level of HIF-1α in U87 cells overexpressing IDH1^R132H was determined by Western blot, HIF-1α inhibitor was used to observe its effect on miR-128 a expression level in IDH1^R132H U87 cells. The promoter sequence of mi R-128 a was analyzed, and Chip assay was performed to test the binding level of HIF-1α and miR-128 a in IDH1^R132H U87 cells. Using miR-128 a mimics or mi R-128 a inhibitor in U87 cells, the effect of mi R-128 a on proliferation in IDH1^R132H U87 cells and IDH1 WT U87 cells was determined by CCK-8 assay. Real-time PCR and Western blot were performed to detect the expression level of Bmi-1 in IDH1^R132H U87 cells, miR-128 a mimics transfected U87 cells and miR-128 a inhibitor transfected U87 cells. The binding level of miR-128 a and Bmi-1 mRNA 3’-UTR was determined by Luciferase assay. The protein levels of p-Akt, Total Akt, p-FAK and MMP-9 were detected by Western blot in miR-128 a mimics transfected U87 cells or miR-128 a inhibitor transfected U87 cells. Apoptosis and cell cycle were also analyzed by flow cytometry in miR-128 a mimics transfected U87 cells or mi R-128 a inhibitor transfected U87 cells.RESULTS: A stable U87 GBM cell line overexpressing IDH1^R132H was established. Compared with control group and IDH1 WT group, U87 cells with IDH1^R132H overexpression showed decreased proliferation, migration and invasion（P<0.05）. There was an increased expression level of HIF-1α in the IDH1^R132H U87 cells（P<0.05）, Chip assay proved that HIF-1α could binding to the HRE in the miR-128 a promoter（P<0.01）. Compared with IDH1 WT GBM, IDH1^R132H GBM showed higher expression level of miR-128a（P< 0.01）, and miR-128 a could inhibit cell proliferation in vitro experiments（P< 0.01）, thus we speculated that the decreased proliferation of U87 cells was due to the up-regulation of miR-128 a induced by IDH1^R132H. miR-128 a could directly target Bmi-1 3’-UTR（P<0.05）, and negatively regulate the expression of Bmi-1（P<0.05）. Western blot analysis showed that mi R-128 a could negatively regulate p-Akt, p-FAK and MMP-9 expression. Flow cytometry revealed that miR-128 a could induce apoptosis（P<0.05）, and decrease cell proliferation index（P<0.05）.CONCLUSION: Our research investigated the impact of IDH1^R132H on GBM cell growth, the mechanism of which was mainly through the HIF-1α-miR-128a-Bmi-1 pathway.