The Effects And Mechanisms Of IDH1 Mutation On Radiosensitivity Of GSCs

Objective:The presence of glioma stem cells(GSCs)leads to glioma difficult to be cured and easily relapsed;50%-80%of WHO ?,? and secondary malignant gliomas are with mutant Isocitrate dehydrogenase 1(IDH1)gene.In this study,IDH1 mutant GSCs were used as the object to investigate effects of IDH1 gene mutation on the radiosensitivity of GSCs,and the mechanism of action was discussed.Methods:(1)The glioma cells were dedifferentiated under serum-free medium.After the cells were cultured to the twelfth generation,the GSCs surface marker protein CD133 was specifically identified by automated flow cytometry,and then getting GSCs.(2)The plasimid in which IDHI gene mutation overexpressed was constructed and transfected into GSCs by Lipofectamine 3000,and then IDHI mutant GSCs were obtained.(3)The cell clone assay and CCK8 experiments were used to detect the effect of 1DH1 mutation combined with X-ray irradiation on the proliferation and viability of GSCs.(4)Cellular immunofluorescence assay detected the number of ?H2AX foci at different time after X-ray irradiation(5)flow cytometry experiments detected cell cycle distribution of IDHI mutant GSCs exposed to X-ray irradiation;(6)The expression levels of the key protein TIGAR of the pentose phosphate pathway after X-ray irradiation for 8 Gy were detected by Western blot.The ratios of NADP+/NADPH and GSSG/GSH were detected by NADPH and glutathione detection kits respectively.Result:(1)The experiment showed that the cell clonal formation rate in the experimental group after X-ray radiation Z,4,6,8 Gy was 68.14%,42.07%,19.44%,and 13.35%,respectively.Compared to wild-type IDH1 group,there was no statistical difference.(2)The results of CCK8 showed that,compared to the wild-type GSCs,the date of IDH1 mutant GSCs before X-ray irradiation,showed unconspicuous change in the cell proliferation activity,upon X-ray irradiation 4 Gy,8 Gy within 48 hours,the average OD value was 1.1 times that of the control group,and the viability of IDH1 mutant GSCs was not significantly decreased,(3)Cellular immunofluorescence experiments showed that the number of ?H2AX foci formed in the mutant group increased rapidly post-irradiation within 0.5 h,and the ?H2AX foci decreased rapidly within 2 hours.The foci disappeared almost within 4 hours.The number of ?H2AX foci formation in IDH1 mutant GSCs was not signi:ficantly different from that in the wild-type IDH1 group.(4)Flow cytometry experiments showed that after 48 hours of plasmid transfection,the percentage of cell cycle distribution in each phase of the wild-type IDH1 group was 72.54%in the G1 phase,18.14%in the S phase,and 9.32%in the G2/M phase.IDH1 mutant group was 65.33%in G1 phase,25.23%in S phase,and 9.44%in G2/M phase.Upon X-ray irradiation,the percentage distribution of cells in each group was not statistically different.(5)Western blot showed that compared to wild-type group,the GSCs in the mutant group did not change TIGAR protein expression within 48 hours of transfection of the mutant plasmid,and the expression of TIGAR protein in the two groups increased after 2 hours of 8 Gy X-ray irradiation.The difference was statistically significant compared to the unirradiated group,but the difference in the amount of protein increased between the two groups was not significant.(6)The data of redox kits showed reducing substances in the IDH1 mutant group increased compared to the cells in the wild-type group,but the difference between the two groups was not statistically significant.Conclusion:Compared to wild-type group,The mutant IDH1 did not have impact on GSCs.Upon X-ray irradiation,the mutant IDH1 had nonsignificant effect on the cell proliferation,the DNA damage repair ability and the percentage distribution of the cell cycle.The redox level of IDH1 mutant GSCs was in stable phase.The results revealed IDH1 mutation had different regulatory mechanisms in glioma cells and GSCs,increases the radiosensitivity of glioma cells,but has no significant effect on the radiosensitivity of GSCs.This may be one of the reasons why glioma is not sensitive to chemoradiotherapy and is prone to relapse after treatment.This study excluded an important factor affecting the radiobiology sensitivity of GSCs,which has certain theoretical significance for clinical interpretation of the radiation tolerance of glioma.