Egr1介导的人截短型凋亡诱导因子表达载体的构建及其在乳腺癌MCF-7细胞中的辐射诱导表达规律

Construction of Egr1-mediated human truncated apoptosis inducing factor expression vector and its expression regularity induced by radiation in breast cancer MCF-7 cells

目的:克隆人截短型凋亡诱导因子(AIF)cDNA序列,并构建早期生长反应因子1(Egr1)介导的重组表达载体pcDNA3.1-Egr1-AIFΔ1-480(pEgr1-AIFΔ1-480),观察其在人乳腺癌MCF-7细胞中的辐射诱导表达规律。方法:以人白血病Jurkat细胞mRNA为模板,RT-PCR法扩增获得人AIFΔ1-480,与pMD18T载体连接后行全自动测序,限制性内切酶切取pMD19T-Egr1中Egr1片段,并利用基因重组技术构建Egr1启动子介导的人AIFΔ1-480表达质粒pEgr1-AIFΔ1-480。实验分为对照组、pcDNA3.1组、pAIFΔ1-480组和pEgr1-AIFΔ1-480组。各组质粒分别转染MCF-7细胞,Western blotting法检测AIF及AIFΔ1-480蛋白的辐射诱导表达时程-效应(2.0Gy照射后0、2、4、12、24和48h)和剂量-效应(0、0.2、0.5、1.0、2.0、5.0Gy照射后24h)规律。结果:经测序证实,RT-PCR获得的人AIFΔ1-480基因与预期一致,pEgr1-AIFΔ1-480经PCR和酶切鉴定完全正确;各组MCF-7细胞经2.0Gy照射后0～48h,AIF蛋白在各组中均有表达,从4h开始显著增加,4、12、24和48h各组AIF表达与0h组比较差异有统计学意义(P<0.05),48h达到最大;而在pAIFΔ1-480和pEgr1-AIFΔ1-480组,AIFΔ1-480从2h开始表达,4、12、28和48h各组AIFΔ1-480表达与2h比较差异有统计学意义(P<0.05),24h达到峰值;而且24和48h时pEgr1-AIFΔ1-480组AIFΔ1-480表达较pAIFΔ1-480组显著增加(P<0.05)。各组MCF-7细胞经0～5.0Gy照射后24h,各组均有AIF蛋白表达,而AIFΔ1-480只在pAIFΔ1-480和pEgr1-AIFΔ1-480组表达,二者均随着剂量增加而增加,0.2、0.5、1.0、2.0和5.0Gy照射后各组AIF表达与0Gy比较差异有统计学意义(P<0.05),在5.0Gy照射时达到最大,相同照射剂量pEgr1-AIFΔ1-480组AIFΔ1-480表达高于pAIFΔ1-480组。结论:成功构建Egr1介导的人截短型AIF重组表达载体pEgr1-AIFΔ1-480,AIF和AIFΔ1-480蛋白在MCF-7细胞中的表达随照射时间延长和照射剂量增加而增加。

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.