The Study Of TPM1/TPM3 And Glioma U251 Radioresitance And The Relationship Between TPM1/TPM3 And CFL1

Background and objective:Glioma is the most common intracranial malignant tumor,accounting for about 45%of intracranial tumor.In recent years,new diagnostic technology appear constantly,but most gliomas show infiltrative growth and their boundary with surrounding brain tissue are not clear;so the treatment is not satisfactory,especially the average lifetime of the level IV glioblastoma is less than 12 months.Currently,the mainly treatment of glioma is surgery,chemotherapy and radiotherapy.We found many affective factors in the process of radiotherapy,thus improve the radiosensitivity of glioma is crucial.TPM1、TPM3 play the most import role in the formation and progress of glioma.CFL1 also plays an important role in tumor invasion and migration.This study was designed to explore the relation among TPM1 or TPM3 with the radioresistance of glioma U251 cells and CFL1,so as to provide the potential target spots to improve the sensitivity of gliomas to radiotherapy,thereby improving the prognosis of glioma.Methods:RR-U251 and CFL1-over-U251 cells were established.TPM1-shRNA(TPM3-shRNA)or pcDNA3.1-TPM1(pcDNA3.1-TPM3 was transfected to U251 cells,RR-U251 cells or CFLl-over-U251 cells with the lipo-2000 respectively.MTT assay,scratch assay,transwell assay and flow cytometry were used to determine the relative survival,cell migration,cell invasion and cell cycle distribution as well as cell apoptosis of all kinds of U251 cells,respectively.Western Blot was uesd to detect the protein expression.Through these indicators,changes in cellular radiosensitivity could be observed.In vivo,human glioma xenograft model were transplanted.pcDNA3.1-TPM1 was transfected into tumors with electroporation trasfection.After transfected with plasmid combined with radiation therapy,the tumor growth and draw group curve were monitored and drawed.Western blot analysis and immunochemistry were used to investigate the TPM1 expression in gliama U251 xenograft,and then exploring the relation among TPMlwith the radioresistance of glioma and CFL1 in vivo.Results:1.The expression of TPM1 and p-CFL1 were decreased by 74.13%and 84.82%;TPM3 and CFL1 were increased by 230%and 290%in RR-U251 cells,which showed significant difference compared with U251 cells(p<0.05);Improving the TPM1 expression in U251 cell can promote CFL1 phosphorylation and make its inactivation;but improving TPM3 expression in U251 cell makes no significant change in CFL1 phosphorylation.2.After radiotherapy,the relative survival of RR-U251 cells enhanced significantly compared with U251 cells(p<0.05).TPM1 can improve the radiation sensitivity of glioma.After radiotherapy,the relative survival rate of over-expressed TPM1 in all kinds of U251 glioma cells dropped significantly(p<0.05).And TPM1 could reversed the proliferation of CFLl-over-U251 cells.After radiotherapy,TPM3 can reduce the radiation sensitivity of glioma significantly(p<0.05);And TPM3 intensified the proliferation of CFL1-over-U251 cells.3.After radiotherapy,the migration abilities of RR-U251 cells enhanced significantly compared with U251 cells(p<0.05).After radiotherapy,TPM1 can improve the radiation sensitivity of glioma,the migration rate dropped significantly in over-expressed TPM1 of all kinds of U251 glioma cells(p<0.05).And TPM1 can reversed the migration of CFL1-over-U251 cells.TPM3 also can improve the radiation sensitivity of glioma.After radiotherapy,the migration rate was dropped significantly in over-expressed TPM3 of all kinds of U251 glioma cells(p<0.05).TPM3 can reversed the migration of CFL1-over-U251 cells as well.4.After radiotherapy,the invasion number of RR-U251 were more than that of U251 cells,which has statistically significant(p<0.05).TPM1 can improve the radiation sensitivity of glioma.After radiotherapy,the invasion rate was dropped significantly in over-expressed TPM1 of all kinds of U251 glioma cells(p<0.05).And TPM1 can reversed the invasion of CFL1-over-U251 cells.TPM3 also can improve the radiation sensitivity of glioma.After radiotherapy,the invasion rate was decreased significantly in over-expressed TPM3 of all kinds of U251 glioma cells(p<0.05).TPM3 can reversed the invasion of CFL1-over-U251 cells as well.5.After radiotherapy,the apoptosis ability of RR-U251 cells were less than U251 cells,there was statistically difference between them(p<0.05).TPM1 can improve the radiation sensitivity of glioma.After radiotherapy,the apoptosis rate was increased significantly in over-expressed TPM1 of all kinds of U251 glioma cells(p<0.05).While TPM1 can reversed the less apoptosis of CFL1-over-U251 cells.TPM3 inhibited the apoptosis of various U251 glioma cell,TPM3 decreased the radiation sensitivity of gliomas;Meanwhile,TPM3 exacerbated the resistance to apoptosis of CFL1-over-U251 cells;Synergistic inhibit the apoptosis of glioma cell.6.Immunohistochemistry and western blot results indicated in the group which transfection with pcDNA3.1-TPM1 combined with radiotherapy,the expression of TPM1 significantly higher than the control group,which has a statistical difference(p<0.05).Transfecting with pcDNA3.1-TPM1 and combining with radiotherapy in U251 and RR-U251 group can obviously inhibit the tumor growth compared with the control group,which has statistically significant(p<0.05),improving the sensitivity to radiotherapy;But there is no significant difference between U251 and RR-U251 group.The level protein of CFL1 was decreased,p-CFL1 was increased when transfected with pcDNA3.1-TPM1 and combining with radiotherapyin RR-U251 group.Conclusion:1.Improving the TPM1 expression in varies U251 cells can inhibit the proliferation and migration and invasion of cells,promote the apoptosis of cells so as to improve the effect of radiation therapy of glioma,that is to say TPM1 can improve the radiosensitivity.2.Improving the TPM3 expression in the glioma U251 cells can inhibit the glioma of migration,invasion,and promote the proliferation of glioma,and inhibit the apoptosis of glioma,which affect the radiotherapy of glioma.3.Improving the TPM1 expression in the U251 cells can promote the phosphorylation of CFL1,make its inactivation,so improve the radiosensitivity of gliomas;Improving the expression of TPM1 in CFL1-over-U251 cells can reverse CFL1-inducing radiation resistance,which indicating the competing relationship between TPM1 and CFL1.TPM1 can improve the radiosentivity by regulation the phosphorylation or dephosphorylation of CFL1.4.In vivo results also show that TPM1 can improve the radiation sensitivity of glioma U251 cells,TPM1 can also promote CFL1 phosphorylation.5.The relationship among TPM3,CFL1 and glioma radiation resistance is complex,which needs further study.