ABSTRACT
Objective:To explore the effect of ionizing radiation on apoptosis of lung cancer H460 cells after ATRX was silenced by RNAi and its mechanism.Methods:The lentivirus expression vectors targeting ATRX were transfected into the 293T cells,and the lung cancer H460 cells were infected with lentivirus twice,and the ATRX silenced cell model was obtained after puromycin positive screening,then they were named as sh-ATRX1-H460,sh-ATRX2-H460,and sh-ATRX3-H460 cells;the sh-control-H460 cells were regarded as control cells.The cells were divided into sh-control-H460 group and sh-ATRX3-H460 group,accroding to the silencing results and were irradiated by 0,2 and 8 Gy X-rays.The expression levels of ATRX,poly(ADP-ribose) polymerase 1(PARP1),and caspase-3 proteins were measured by Western blotting method;the apoptotic rate was measured by flow cytometry and AnnexinⅤ-FITC/PI kits.Results:The lung cancer cell model of sh-ATRX3-H460 silenced by ATRX was obtained successfully.After 2 and 8 Gy X-ray irradiation,compared with before irradiation,the expression level of ATRX protein in sh-control-H460 group was increased,while there was no expression of ATRX protein in sh-control-H460 group;compared with before irradiation,the apoptotic rates of cells in two groups were increased(P<0.05 or P<0.01);the apoptotic rate in sh-ATRX3-H460 group was significantly higher than that in sh-control-H460 group after 8 Gy X-ray irradiation (P<0.01).The expressions of cleaved PARP1 in the cells in both two groups after 2 Gy and 8 Gy X-ray irradiation were increased and showed similar rule.The expression level of procaspase-3 protein in sh-control-H460 group had little change,and it was increased significantly in sh-ATRX3-H460 group after 8 Gy X-ray irradiation.Conclusion:ATRX silencing can be achived by RNAi,then the silencing could increase the apoptosis induced by irradiation and its mechanism may be related to the PARP1-caspase-3 pathway.
ABSTRACT
Objective To establish the MCF-7 cell models of Beclin 1 over-and low-expressions,and to detect the autophagic and apoptotic changes after 4 Gy irradiation,and to explore their molecular regulation mechanisms. Methods MCF-7,MCF-7 + 4Gy,MCF-7-Beclin 1 + 4Gy and MCF-7-Belcin 1 RNAi+ 4Gy groups were set up. Molecular biology method was used to construct Beclin 1 over-expression vector pcDNA3.1-Beclin 1,and to estabilish the Beclin 1 over- and low-expression cell models.After the cells were irradiated with 4 Gy, the autopahgic cell percentages were measured by fluorescence microscope with MDC staining, the apoptotic cell percentages were measured by FCM with AnnexinⅤ-FITC and PI staining,and the expressions of Beclin1,P53, Bcl-2 and Bax proteins were measured by Western blotting method.Results Compared with MCF-7 group,the autophagic and apoptotic cell percentages in MCF-7+4 Gy,MCF-7 Beclin 1 +4 Gy and MCF-7-Beclin 1 RNAi+4 Gy groups were significantly increased (P <0.05 or P <0.001 ),especially in MCF-7 Beclin 1+4 Gy group which was significantly higher than those in MCF-7 + 4 Gy (P < 0.05);while there was significant difference in the necrotic cell percentages between various groups. After 4 Gy irradiation, compared with MCF-7 group, the expression levels of Beclin 1,P53 and Bax proteins in MCF-7 + 4 Gy and MCF-7-Beclin 1 + 4 Gy groups were increased,but the expression levels of Bcl-2 protein were decreased,especially in MCF-7-Beclin 1 + 4 Gy group. Conclusion The MCF-7 cell models of Beclin 1 over-and low-expressions are successfully established,and ionizing radiation could induce the autophagy and apoptosis of MCF-7 cells,which is more obvious in Beclin 1 over-expression MCF-7 cells.Beclin 1 can activate P53,inhibit Bcl-2 and activate Bax,which forms the regulation of autophagy and apoptosis by P53 .