Abstract:

Abstract:Objective To clone human truncated apoptosis inducing factor (AIF) cDNA sequence,and to construct early growth response 1 (Egr1)-mediated recombinant expression vector pcDNA3.1-Egr1-AIFΔ1-480 (pEgr1-AIFΔ1-480),and to observe its regularity induced by radiation in human breast cancer   MCF-7 cells.Methods The total mRNA extracted from human leukemia jurkat cells used  as  template,and the human AIFΔ1-480 was acquired by RT-PCR,and it was linked to pMD18T vector for sequencing.Egr1 fragment was digested from pMD19T-Egr1 by restrictive enzyme,and the  Egr1-mediated expression plasmid pEgr1-AIFΔ1-480 was constructed by gene recombination.There were control group,pcDNA3.1 group,pAIFΔ1-480 group and pEgr1-AIFΔ1-480 group in the experiment. After the  plasmids in various groups were transfected into human breast cancer MCF-7 cells,the AIF and AIFΔ1-480 protein expression time-effect (0,2,4,12,24 and 48 h after 2.0 Gy irradiation) and dose-effect (24 h after 0,0.2,0.5,1.0,2.0 and 5.0 Gy irradiation) regularity were measured by Western blotting method.Results The sequencing results showed that the AIFΔ1-480 acquired by RT-PCR was consistent with the sequence expected，the pEgr-AIFΔ1-480 was confirmed  by PCR and restrictive enzyme digestion.After 0-48 h the MCF-7 cells were irradiated by 2.0 Gy,and the AIF protein expressed in the cells in each group,and it increased significantly from 4 h and the AIF expressions in 4,12,24 and 48 h groups were  higher than that in 0 h group(P<0.05),and it reached to maximum value at 48 h.But the AIFΔ1-480 protein expressed  in the  cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups from 2 h (P<0.05),and it reached to peak value at 24 h.The AIFΔ1-480 expressions in pEgr1-AIFΔ1-480 group were higher than those in pAIFΔ1-480 group at 24 and 48 h (P<0.05).After the  MCF-7 cells were irradiated by 0-5 Gy for 24 h,the AIF protein expressed in the cells in each group,but the AIFΔ1-480 protein expressed merely in the cells in pAIFΔ1-480 and pEgr1-AIFΔ1-480 groups.The AIF and AIFΔ1-480 expressions were increased with the dose increasing,the AIF expressions irradiated with 0.2，0.5，1.0 and 5.0 Gy were  higher than that with 0 Gy irradiation.It reached to maximum value by 5.0 Gy irradiation and the AIFΔ1-480  expression  in pEgr1-AIFΔ1-480 group was  higher than that in  pAIFΔ1-480   group.Conclusion The human truncated AIF expression recombinant vetor pEgr1-AIFΔ1-480 is successfully constructed,and the  AIF and AIFΔ1-480 protein expressions increased with time prolongation and dose increasing after irradiation in MCF-7 cells.

Key words:  early growth response gene 1;truncated AIF;radiation;breast neoplasms;gene recombination