The Correlation Between CFL1 And Radioresistance In Glioma U251 Cells

Background and objective:Glioma shows infiltrative growth,postoperative radiation therapy has become an important adjunct to the glioma treatment.For the radiation resistance of glioma,the radiation effect is not ideal.CFL1 plays an important role in tumor invasion and migration.This study was designed to explore the relation between CFL1 and radioresistance of glioma U251 cells and to explain the possible mechanism underlying CFL1-mediated radioresistance in U251 cells,so as to provide potential target spots to improve the sensitivity of gliomas to radiotherapy,thereby improving the prognosis of glioma.Methods:RR-U251 cells,pcDNA3.1-CFL1 induced over-expressed CFL1 U251 and RR-U251 cells,knockout CFL1 U251 and RR-U251 cells were established.U251 and RR-U251 cells with above different treatments received radiation therapy.MTT assay,scratch assay,transwell assay and flow cytometry were used to determine cell relative survival rate,cell migration,cell invasion and cell cycle distribution as well as cell apoptosis,respectively.Western Blot and quantitative PCR were also used to monitor CFL1 level change.Through these indicators,changes in cellular radiosensitivity could be observed.Human glioma xenograft model was established.With methods of vivo electroporation and intratumorally transfected injection,CFL1-siRNA was transfected into tumor.Western blot analysis and immunochemistry were utilized to investigate intratumor CFL1 expression.Glioma xenografts received radiotherapy.Tumor growth curve was drawed to further explore the relation between CFL1 and radiation resistance of glioma in vivo.Y-27632 was used to block RhoA-ROCK Ⅱ-LIMKⅡ signaling pathway;the application of CK-666 blocked Cdc42-NWASP-Arp2/3 and Cdc42-MRCKa-Arp2/3 pathway;application PAK1-shRNA blocked Rac-PAK-LIMKⅡand Cdc42-PAK-LIMKⅡ pathway;and MRCKa-shRNA was utilized to block Cdc42-MRCKa-LIMKⅡ and Cdc42-MRCKa-Arp2/3 pathway.Western blot,MTT,scratch assay,and transwell were used to analyed CFL1 expression level,cell viability,cell migration and cell invasion.Results:The expression of CFL1 increased in RR-U251 cells,which showed significant difference compared with untreated U251 cells(p<0.001);relative survival ratio,cell migration and invasion of RR-U251 cells also enhanced significantly compared with untreated U251 cells(p<0.05).After radiation therapy,the number of cells arrested in G2/M phase in RR-U251 cells was smaller than that of U251 cells.When CFL1 expression was inhibited in U251 cells,RR-U251 cells and CFL1-over-expressed U251 cells,cell relative survival ratio decreased markedly,cell migration and invasion significantly weakened and the number of cells arrested in G2/M phase increased(p<0.05).The radio sensitivity of the cells with CLF1 inhibiton increased.When CFL1 was over expressed in U251 cells,cell survival ratio increased,cell migration and invasion enhanced and the number of cells arrested in G2/M phase decreased(p<0.05).The adiosensitivity of the cells with CFL1 over-expression decreased.Results from western blot and immunochemistry showed that the CFL1 level declined in the CFL1-siRNA-transfected tumor.Intratumoral CFL1-siRNA-transfected injection combined radiotherapy had a significantly inhibiting efficiency on tumor growth.The application of Y-27632 and CK-666 in RR-U251 cells,the relative cell survival ratio and migration and invasion ability all weaken significantly(p<0.05)while CFL1 was decreased.The radiosensitivity was increased.When PAK1 and MRCKa expression were inhibited,the relative cell survival ratio and migration and invasion ability all weaken significantly(p<0.05)while CFL1 had no marked variation.Conclusion:Reduction of CFL1 expression in U251 cells could increase the radiosensitivity of glioma.Regulation of ROCK in RhoA-ROCKⅡ-LIMKⅡ and Arp2/3 in Cdc42-NWASP-Arp2/3 and Cdc42-MRCKα-Arp2/3 could affect the radiosensitivity of U251 cells.It indicated that ROCK and Arp2/3 correalted with radiosensivity via CFL1 sigaling pathways.Regulation of PAK1 in Rac-PAK-LIMKⅡ and Cdc42-PAK-LIMK Ⅱ could affect the radiosensitivity of U251 cells,while the same results were observed in regulation of MRCKa for Cdc42-MRCKa-LIMKⅡ and Cdc42-MRCKa-Arp2/3.It indicated that MRCKa and PAK1 correlated with radiosensitivity via other pathways no CFL1.