| At present, the malignant tumor is one of the major diseases, which threats seriously to human health. The primary ways for tumor treatment are surgery, radiotherapy, chemotherapy and biotherapy and so on. And the comprehensive treatment is a big trend. The gene-radiation therapy has been a hot spot in the research field of oncotherapy. Radiotherapy combines with gene therapy to educe synergistic effect. The discovery of radiation-sensitive early growth response-1 (Egr-1) promoter provides a new thinking for the effective combination of radiotherapy and gene therapy, which establishes the theoretical foundation for gene-radiation therapy on tumor. In this study, the plasmid pEgr-1-AIFâ–³1 -480 constructed successfully as the gene therapy introduction system combined with radiotherapy, which could activate the transcription of the Egr-1 promoter, promote apoptosis of tumor cells with the AIFâ–³1-480 gene, and kill tumor cells.1. Construction of recombinant plasmids1.1 Acquisition of AIFâ–³1-480 geneThe total RNA was extracted from human leukemia Jurkat cells, which was reversely transcribed into cDNA as the template. The specific PCR primers were designed and synthesized according to the sequence of AIFâ–³1-480 gene. The AIFâ–³1-480 gene was acquired by RT-PCR through agarose gel electrophoresis. The AIFâ–³1-480 gene was amplified from the template, and ligated to pMD18T vector. The result of sequencing analysis including enzyme digestion and sequencing process was in coincidence with the anticipated result.1.2 Acquisition of Egr-1 promoterThe pMD19T-Egr-1 was digested into the fragments with EcoRâ… and Hindâ…¢enzymes. Egr-1 fragment was obtained with agarose gel electrophoresis, and identified by cleavage of endonucleases and sequencing analysis. The results of identification confirmed that the sequence of the cloned gene was identical to that published on Genbank. 1.3 Construction of recombinant plasmidsThe AIFâ–³1-480 gene was ligated to the pcDNA3.1 vector to construct the pcDNA3.1-AIFΔ1-480, and the Egr-1 was ligated to the pcDNA3.1-AIFΔ1-480 vector to construct the pcDNA3.1-Egr-1-AIFΔ1-480 with the technique of genetic engineering. The pcDNA3.1-Egr-1-AIFΔ1-480 was identificated by PCR and cleavage of endonucleases. The result of sequencing analysis was in coincidence with the anticipated result. In the following experiments, the gene protein expression rule, the inhibition effects on the tumor growth, apoptosis and invasion in human breast cancer MCF-7 cells were detected after these plasmids combined with radiation.2. Experimental grouping and index detectionThere were four groups in the experiment: the control, pcDNA3.1, pcDNA3.1- AIFâ–³1-480 and pcDNA3.1-Egr-AIFâ–³1-480. In the time-course experiment, the selected time points were 2, 4, 8, 12, 24 and 48 h, and the irradiation dose was 2 Gy. In the dose-effect experiment, the selected irradiation doses were 0, 0.2, 0.5, 1.0, 2.0 and 5.0 Gy, and the observation was done 24 h after irradiation. Western blot, Transwell, MTT and flow cytometry were used to detect the expressions of protein, cell invasion, cell proliferation, cell cycle progression and apoptosis, respectively.3. Radiation-induced expression rule of recombinant plasmids in MCF-7 cells3.1 Protein expressions in MCF-7 cells transfected with recombinant plasmidsThe cells were harvested at different time after transfection. The AIF protein expression in MCF-7 cells was detected and analyzed by Western blot. There were not differences in intrinsic AIF protein expressions between all groups. The AIFΔ1-480 protein expressions detected in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups increased from 4 h after irradiation, and up to the peak value at 48 h. The results indicate that the intrinsic AIF protein expressions could be stable in MCF-7, and were not influenced by transfection with recombinant plasmids and the expressions of Egr-1 and AIFΔ1-480. There was time-course law in the AIFΔ1-480 protein expressions.3.2 Time-course changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after 2.0 Gy X-irradiationThe cells were harvested at different time after irradiation. The AIF protein expression in MCF-7 cells was detected and analyzed by Western blot. The intrinsic AIF protein expressions increased from 4 h after irradiation in all groups, and up to the peak value at 48 h. The AIFΔ1-480 protein expressions detected in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups increased from 2 h after irradiation, and up to the peak value at 24 h. And the expression in the pcDNA3.1- Egr-1-AIFΔ1-480 group was more than that in the pcDNA3.1-AIFΔ1-480 group. The results indicate that ionizing radiation could promote the release of the intrinsic AIF from mitochondria, and the expressions could increase with time extension, and were not influenced by transfection with recombinant plasmids and the expressions of Egr-1 and AIFΔ1-480. There was time-course law in the AIFΔ1-480 protein expressions. The promoter of Egr-1 could be activated by irradiation, and enhance the AIFΔ1-480 protein expressions.3.3 Dose-effect changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after X-irradiation with different dosesThe cells were harvested 24 h after X-irradiation with different doses. The protein expression in MCF-7 cells was detected and analyzed by Western blot. The results showed that the intrinsic AIF protein expression increased gradually at same group with the increase of doses. The AIFΔ1-480 protein expressions were not detected in the control and pcDNA3.1 groups, but increased with the increase of doses in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups. At the same dose, the expression in the pcDNA3.1- Egr-1-AIFΔ1-480 group was more than that in the pcDNA3.1-AIFΔ1-480 group. The results indicate that there was the dose-effect law in the intrinsic AIF protein expression; ionizing radiation with the increase of doses could promote the more release of the intrinsic AIF. There was dose-effect law in the AIFΔ1-480 protein expressions. The promoter of Egr-1 could be activated by irradiation and enhance the AIFΔ1-480 protein expressions.4 Effects of recombinant plasmids combining with X-irradiation on MCF-7 cells4.1 Effects of X-irradiation on proliferation of MCF-7 cells transfected with recombinant plasmidsThe proliferation of MCF-7 cells without irradiation was the fastest in the control and pcDNA3.1 groups. The proliferation was significantly inhibited in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups from 12 h after irradiation as compared with that in the control group (P < 0.05). After the cells were irradiated with 2.0 Gy X-ray, the proliferation was significantly slow. In particular, the proliferation was the most slowly and at basically stopping status in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups. The results suggest that the AIFΔ1-480 protein and radiation, respectively, could inhibit the proliferation of MCF-7 cells, so this could increase with the combination of AIFΔ1-480 protein and radiation.4.2 Effects of X-irradiation on cell cycle progression of MCF-7 cells transfected with recombinant plasmidsThe percentages of G0/G1 phase cells without irradiation increased significantly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups as compared with that in the control group, but those of G2/M phase cells decreased significantly (P < 0.05). The percentages of G0/G1 and G2/M phase cells irradiated with 2.0 Gy X-ray in all groups increased as compared with those in the control group, particular significantly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups (P < 0.05). The percentages of S phase cells decreased significantly in all groups as compared with those in the control group, specialy in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups (P < 0.05). The results indicate that the AIFΔ1-480 protein could lead to G1 arrest, and ionizing radiation could lead to G1 and G2 blocking. The G1 and G2 arrests could increase with the combination of AIFΔ1-480 protein and ionizing radiation.4.3 Effects of X-irradiation on apoptosis of MCF-7 cells transfected with recombinant plasmidsThe early and late apoptotic percentages of the cells without irradiation increased significantly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups (P < 0.05), in particular in later. The early apoptotic percentages of the cells irradiated with 2.0 Gy in all groups increased significantly (P < 0.05). The late apoptotic percentages of the cells irradiated with 2.0 Gy in the control and pcDNA3.1groups increased significantly (P < 0.05), but especial done significantly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups. As compared with those in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups without irradiation, the early and late apoptotic percentages of the cells irradiated with 2.0 Gy and transfected with recombinant plasmids were significantly increased (P < 0.05). The results indicate that the AIFΔ1-480 protein and ionizing radiation could lead to the early and late apoptosis. The early, late and total apoptosis increased with the combination of AIFΔ1-480 protein and radiation.4.4 Effects of X-irradiation on invasion of MCF-7 cells transfected with recombinant plasmids The number of cells un-irradiated and crossed membrane decreased in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups. As compared with that in cells without irradiation, the number of cells irradiated with 2.0 Gy and crossed membrane decreased significantly in the control and pcDNA3.1 groups (P < 0.05), particularly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups (P < 0.01). As compared with that in the control and pcDNA3.1 groups, the number of cells irradiated with 2.0 Gy and crossed membrane decreased significantly in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups (P < 0.01). The results indicate that the AIFΔ1-480 protein and ionizing radiation could reduce the invasive ability, particularly in the combination of AIFΔ1-480 protein and ionizing radiation.4.5 Effects of X-irradiation on expression of cytochrome c of MCF-7 cells transfected with recombinant plasmidsThe expression of cytochrome c protein of the cells without irradiation increased in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups. As compared with that in the control and pcDNA3.1 groups, the expression of cytochrome c protein of the cells irradiated with 2.0 Gy increased in the pcDNA3.1-AIFΔ1-480 and pcDNA3.1-Egr-1-AIFΔ1-480 groups, particularly in pcDNA3.1-Egr-1-AIFΔ1-480 group. The results indicate that the AIFΔ1-480 protein and ionizing radiation could increase Cyt c protein expression, particularly in the combination of AIFΔ1-480 protein and radiation.Above all, the gene expression plasmid pcDNA3.1-Egr-1-AIFΔ1-480 was constructed successfully in present study. The plasmid has the characteristics of irradiation inducibility, and combines with ionizing radiation could induce cell G1 and G2 phase arrests, increase Cyt c protein expression, inhibit the proliferation, promote the apoptosis and reduce the invasive ability in MCF-7 cells. The researches open up a new way to improve the effects of gene-radiation therapy, and provide the theoretical and experimental bases for the clinical application. |
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