Experimental Studies On The Roles Of Beclin1 In Radiation-induced Autophagy

Autophagy is a process that the cells can autolysis the "wasted structure" of itself through a lysosomal degradation pathway so as to provide raw materials and energy for the cell's metabolism and organelles'update. Beclin1, the first autophagy genes found in mammal animals, plays an important role in the regulation of autophagy. In this experiment, we constructed the overexpression model and RNAi model of Beclin1 gene in the MCF7 amd SKOV3 cells by gene engineering to change the expression of Beclin1 at the gene level, and explored the role of Beclin 1 in the autophagy through detecting the expression of Beclin 1 gene after ionizing radiation in different dosese and differet times, providing theoretical basis for radiation combined gene therapy.1 Autophagy was induced by ionizing radiation in tumor cellsIn this experiment, in order to prove that Autophagy can occur after ionizing radiation in tumor cells, the cell model of MCF7 - PQN - GFP - LC3 was construction. Through the confocal microscopy observation, the cells transfected with plasmid PQN-GFP-LC3 can express the green fluorescent protein,so the transfection was successful .what'more, the green fluorescent protein expression enhanced obviously by ionizing radiation in the doses of 8Gy and 12Gy, Presenting a granular distribution,it proved that Autophagy was induced though ionizing radiation2 The expression of Beclin1 gene in wild type MCF-7 cellsThe cells was treated with the dose of 2,4,8Gy .Experimental results show that, , Beclin1 expression hasn't difference at 4,8,16,32 h after IR with 2Gy, 4Gy compared with the control group 0h; At a higher doses of 8Gy irradiation, the gene expression of Beclin1 showed obvious difference between the experimental groups and control group.However, according to the result of Western Blot, The expression of Beclin1 show obvious time effect and dose effect after IR with 2Gy, so we concluded that the posttranscription regulation plays an important role in the regulation of Beclin1.3 The construction of Beclin1 overexpression and RNA silencing modelsThe gene fragment coding for Beclin1 was obtained from human breast cancer cells using the methods of RT-PCR , and inserted into the eukaryotic exppression vector pcDNA3.l(-) to construct the recombinant Plasmid pcDNA3.1/Beclinl,and then the vector was transfected into SKOV3 cells by lipofectamine 2000, while the empty vector was transfected as the control. The expression level of beclin1 was detected by western blot after transfection. The results show that a higher expression of Beclin1 in the model group compare to the control group.that is, the overexpression model of Beclin1 was established successful.The vector Psuper- Beclin1Ri was conserved by our laboratory. And the vector was transfected though Calcium phosphate cell transfection. The expression level of Beclin1 was detected by western blot after transfection.the results show that a lower expression of Beclin1 in the model group compare to the control group.that is, the Beclin1Ri model of Beclin1 was established successful.4 Effect of Beclin1 gene on cell growth statusTo observe the changges of the growth of model cells after transfection, the cells were cultured in twelve pores plate with the concentration of 104 per well,cell counting was done afer 24,48,72,96,120,144h, and the growing curve was plained. And then take the indexing period cells to calculate the multiplication time according to formula: TD=0.693×(T2-T1)/ ln(N2-N1), N1:the cell number at the time of T1; N2:the cell number at the time of T2. Through calculation, the multiplication time of SKOV3- Beclin1 cell was 21.8 h, SKOV3- pcDNA3.1 cell was 14.9 h, and SKOV3 cell was 15.3h. the overexpression group is longerthan the control groups, the two groups have statistically significant differences, so we think that Beclin1 expression inhibit the growth of cells. While Beclin1Ri model was 10.6 h, SKOV3-PsuperRetro was 16.6 h, the two groups have statistically significant differences, Interference of Beclin1 promotes the growth of cells. That is, the gene Beclin1 has the fuction of inhibiting growth of cells.5 Effect of IR on the expression of Beclin1 in Beclin1 overexpression and silencing modelFirstly, we construct Beclin1 overexpression and interference model through genetic engineering. Then Western Blot was applied to detect the change of Beclin1 at 0,4,8,16,32 hours after IR with the dose of 4Gy, Results showed that the protein expression of Beclin1 in the overexpression group was increased more evidently after iradiation with 4Gy, although the control group also have the increasing trend. On the contrary, the interference groups SKOV3-Beclin1Ri hasn't obviously changed after IR with 4Gy, but the expression of Beclin1 still keep the rising trend in SKOV3-PsuperRetro Cell.