Effects of gene silencing of Clusterin on radiosensitivity of human glioma U87 cell line

Abstract:

Objective：To investigate the effects of gene silencing of Clusterin（CLU） by lentiviurs-mediated RNA interference on ra－diosensitivity of human glioma U87 cell-line. Methods：shRNAs targeting CLU were cloned into lentiviral vector plentilox3.7 and scramble control shRNA was used as control. Lentiviruses expressing different shRNAs were packaged into 293T cells. Real-time PCR and Western blot were performed to examine mRNA and protein expression of CLU at 72 h after infection of lentivirus. After exposing to different doses of radiation（0，2，4，6 Gy），MTT assay was used to determine cell survival ratio and apoptotic ratio was also detected by flow cytometry. Changes of apoptotic proteins were examined by Western blot. Results：shRNA targeting CLU specifically inhibited the mRNA and protein level of CLU. MTT results showed that survival ratios of CLU-silencing cells under different doses of ionizing irradiation（4，6 Gy） were significantly lower compared with those of cells infected with virus expressing control shRNA（4 Gy：control shRNA vs. CLU shRNA1，P=0.000，control shRNA vs. CLU shRNA2，P=0.003；6 Gy：control shRNA vs. CLU shRNA1，P=0.000，control shRNA vs. CLU shRNA2，P=0.000）. Flow cytometry results showed that apoptotic ratios of CLU-silencing cells under 4 Gy treatment were increased to （35.54±0.95）%，nearly 2 folds higher than those of control cells（control shRNA vs. CLU shRNA1：P=0.000；control shRNA vs. CLU shRNA2：P=0.000）. Results of Western blot showed that gene silencing of CLU markedly upregu－lated apoptosis-related protein Bax expression. Conclusions：Gene silencing of CLU significantly enhances the radio-sensitivity of glioma U87 cell-line in vitro partially through upregulating apoptotic protein Bax. CLU may be a promising target during glioma therapy.