Mutation And Functional Characterization Of Isocitrate Dehydrogenase 1(IDH1) Gene In Human Glioma

Objective To investigate isocitrate dehydrogenase 1R132(IDH1R132) mutation in human gliomas. Methods 90 tumor samples and matched norma blood lymphocytes samples were collected for DNA extraction. Exon 4 of IDH1 gene was amplified with the use of a polymerase-chain-reaction(PCR) assay and sequenced in DNA from the tumor and lymphocytes from each patient. Results IDH1R132 mutation was more than 55% in WHO gradeⅡandⅢgliomas(patients with mutation had a median age of 38.6±2.2 years,while wild-type 47.5±2.9 years), none in WHO grade,21.1% in in WHO gradeⅣ(patients with mutation had a median age of 41.5±2.3 years, wild-type 53.3±3.6 years). None mutation of IDH1R132 was found in lymphocytes. Conclusion Mutations of the IDH1R132were frequently found in several types of grade II and III gliomas with a younger age compared with wild type.Objective To establish Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1. Methods After Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1 were established, RT-PCR,Western-Blotting were used for confirmation. Results We established Hela and U251 cell line stably expressing IDH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1. Conclusion Hela and U251 cell line stably expressing DH1 wild-pCDNA3.1 and IDH1 R132H-pCDNA3.1 were obtained. Objective To investigate the functional change of IDH1R132H mutation gene. Methods (1)The proliferation ability of transfected cells was examined by MTT assay, and then draw cell growth curve. The change of cell cycle and cell apoptosis were analyzed by flow cytometry; (2)The enzymatic activity of wild and IDH1R132H transfected types was assessed; (3)We compared HIF-la and VEGF expression in mutation and wild tumor samples using immunohistochemistry. Results (1)MTT assay was used to measure the proliferative index. Compared with the empty vector, untransfection cells and IDH132H transfected groups, the expression of wild transfected group could effectively inhibit the proliferation in Hela and U251.While no statistics difference between mutant IDH1R132H and empty vector, untransfected groups.The cell cycle was analyzed by flow cytometry.The percentage in G1 phase of Hela-Vector, Hela-Vector-WT and Hela-Vector-IDHR132H were 57.75±4.15%,61.10±3.22%,47.41±2.17% respectively, and S phase were 30.54±2.11%,29.81±2.92%,38.84±1.92%. Compared with wild type and empty vector, G2+S phase in IDH132H increased. While in U251-Vector, U251-Vector-WT-IDH1, and U251-Vector-IDHR132H, the percentage of G2+S were 34.12±3.56%,36.45±4.7%,43.68±3.16%, respectively. The results showed that the percentage of cells(Hela& U251)-Vector-IDH1R132H in G2+S phase all increased, compared with their matched wild type and empty vector. While compared wild type and empty vector, there was no statistics difference. (2)Compared with wild transfected group type,the enzymatic activity of IDH1R132H transfected group decreased 64.09%(Hela,P<0.05),39.79%(U251, P<0.05) respectively, empty-vector decreased 2.44%(Hela, P<0.05),48.9% (U251 P<0.05),while there was no significance between Vector-IDHR132H and empty-vector. (3)The HIF-1α,VEGF positive staining were brown-yellow.They were located in the cell nucleus and cytoplasm respectively. The both positive expression rates in IDHR132H cases increased significantly compared with wild cases(P<0.01). Conclusion After transfection, IDHR132H-vector group had a more proliferative capability,while a higher enzymatic activity in wild-vector group. Two positive(HIF-1α&VEGF) expression rates in mutations cases increased significantly compared with wild cases.Objective To identify Chemosensitivity of IDH1R132H mutation to Chemotherapy agents in U251 cell line. Methods Different concentration DDP was added into U251 cells, and its inhibitive rate was obtained at different time. DDP were added into three kinds of U251 cells. Cell apoptosis was obtained by FCM, and its Caspase-3 protein was expressed by Western-Blotting. Results Compared with empty vector and wild transfected group, IDH1R132H had a obvious apoptosis. The same as cleaved Caspase-3 protein. Conclusion Chemosensitivity was enhanced after IDH1R132H mutation.