pEgr-1-AIF_（△1-480）基因—放射治疗乳腺癌的体外抑瘤效应研究
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摘要
恶性肿瘤严重威胁着人类的健康,一直是人们关注的焦点。肿瘤的常规治疗手段包括手术治疗、放射治疗、化学药物治疗及生物治疗等,但是由于多方面原因肿瘤的治疗效果往往不佳。近年来兴起的基因治疗为肿瘤治疗带来了新的希望,尤其是与放射治疗联合应用的肿瘤基因-放射治疗,将放射治疗和基因治疗的各自优点相结合,即能实现电离辐射对肿瘤细胞的直接杀伤目的,又能实现辐射条件下肿瘤杀伤基因的高效表达,继而实现消除肿瘤的作用,产生辐射和基因表达产物的协同抑瘤效果。另外,利用放射治疗具有靶向性和可操控性的特点,可调节杀伤基因表达的时间和位置。
本研究充分利用早期生长反应基因-1(early growth response gene-1, Egr-1)启动子的辐射诱导表达增强的特性以及凋亡诱导因子(apoptosis inducing factor, AIF)强大的诱导细胞凋亡功能,构建Egr-1介导的截短型AIF真核表达载体pEgr-1-AIF_(△1-480),转染乳腺癌MCF-7细胞,研究其在有和无电离辐射条件下蛋白表达的时程和剂量效应规律,继而将重组质粒与2 Gy X射线联合作用于乳腺癌细胞MCF-7,观察两者联合作用后肿瘤细胞的增殖、侵袭力与凋亡的变化。实验结果表明,重组质粒转染后,细胞中截短型AIF△1-480蛋白的表达具有一定的时程和量效规律,重组质粒对人乳腺癌细胞MCF-7具有明显的抑制增殖、降低侵袭能力及促进凋亡的作用,本研究结果为新的肿瘤基因-放射治疗方案提供了重要的理论基础和实验依据。
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-AIF△1 -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 AIF△1-480 gene, and kill tumor cells.
1. Construction of recombinant plasmids
1.1 Acquisition of AIF_(△1-480) gene
The 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 AIF_(△1-480) gene. The AIF_(△1-480) gene was acquired by RT-PCR through agarose gel electrophoresis. The AIF_(△1-480) 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 promoter
The pMD19T-Egr-1 was digested into the fragments with EcoRⅠand HindⅢenzymes. 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 plasmids
The AIF△1-480 gene was ligated to the pcDNA3.1 vector to construct the pcDNA3.1-AIF_(Δ1-480), and the Egr-1 was ligated to the pcDNA3.1-AIF_(Δ1-480) vector to construct the pcDNA3.1-Egr-1-AIF_(Δ1-480) with the technique of genetic engineering. The pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 detection
There were four groups in the experiment: the control, pcDNA3.1, pcDNA3.1- AIF△1-480 and pcDNA3.1-Egr-AIF_(△1-480). 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 cells
3.1 Protein expressions in MCF-7 cells transfected with recombinant plasmids
The 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 AIF_(Δ1-480) protein expressions detected in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 AIF_(Δ1-480). There was time-course law in the AIF_(Δ1-480) protein expressions.
3.2 Time-course changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after 2.0 Gy X-irradiation
The 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 AIF_(Δ1-480) protein expressions detected in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) group was more than that in the pcDNA3.1-AIF_(Δ1-480) 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 AIF_(Δ1-480). There was time-course law in the AIF_(Δ1-480) protein expressions. The promoter of Egr-1 could be activated by irradiation, and enhance the AIF_(Δ1-480) protein expressions.
3.3 Dose-effect changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after X-irradiation with different doses
The 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 AIF_(Δ1-480) protein expressions were not detected in the control and pcDNA3.1 groups, but increased with the increase of doses in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. At the same dose, the expression in the pcDNA3.1- Egr-1-AIF_(Δ1-480) group was more than that in the pcDNA3.1-AIF_(Δ1-480) 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 AIF_(Δ1-480) protein expressions. The promoter of Egr-1 could be activated by irradiation and enhance the AIF_(Δ1-480) protein expressions.
4 Effects of recombinant plasmids combining with X-irradiation on MCF-7 cells
4.1 Effects of X-irradiation on proliferation of MCF-7 cells transfected with recombinant plasmids
The 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. The results suggest that the AIF_(Δ1-480) protein and radiation, respectively, could inhibit the proliferation of MCF-7 cells, so this could increase with the combination of AIF_(Δ1-480) protein and radiation.
4.2 Effects of X-irradiation on cell cycle progression of MCF-7 cells transfected with recombinant plasmids
The percentages of G_0/G_1 phase cells without irradiation increased significantly in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups as compared with that in the control group, but those of G_2/M phase cells decreased significantly (P < 0.05). The percentages of G_0/G_1 and G_2/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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups (P < 0.05). The results indicate that the AIF_(Δ1-480) 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 AIF_(Δ1-480) protein and ionizing radiation.
4.3 Effects of X-irradiation on apoptosis of MCF-7 cells transfected with recombinant plasmids
The early and late apoptotic percentages of the cells without irradiation increased significantly in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. As compared with those in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 AIF_(Δ1-480) protein and ionizing radiation could lead to the early and late apoptosis. The early, late and total apoptosis increased with the combination of AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups (P < 0.01). The results indicate that the AIF_(Δ1-480) protein and ionizing radiation could reduce the invasive ability, particularly in the combination of AIF_(Δ1-480) protein and ionizing radiation.
4.5 Effects of X-irradiation on expression of cytochrome c of MCF-7 cells transfected with recombinant plasmids
The expression of cytochrome c protein of the cells without irradiation increased in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups, particularly in pcDNA3.1-Egr-1-AIF_(Δ1-480) group. The results indicate that the AIF_(Δ1-480) protein and ionizing radiation could increase Cyt c protein expression, particularly in the combination of AIF_(Δ1-480) protein and radiation.
Above all, the gene expression plasmid pcDNA3.1-Egr-1-AIF_(Δ1-480) 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.
本研究充分利用早期生长反应基因-1(early growth response gene-1, Egr-1)启动子的辐射诱导表达增强的特性以及凋亡诱导因子(apoptosis inducing factor, AIF)强大的诱导细胞凋亡功能,构建Egr-1介导的截短型AIF真核表达载体pEgr-1-AIF_(△1-480),转染乳腺癌MCF-7细胞,研究其在有和无电离辐射条件下蛋白表达的时程和剂量效应规律,继而将重组质粒与2 Gy X射线联合作用于乳腺癌细胞MCF-7,观察两者联合作用后肿瘤细胞的增殖、侵袭力与凋亡的变化。实验结果表明,重组质粒转染后,细胞中截短型AIF△1-480蛋白的表达具有一定的时程和量效规律,重组质粒对人乳腺癌细胞MCF-7具有明显的抑制增殖、降低侵袭能力及促进凋亡的作用,本研究结果为新的肿瘤基因-放射治疗方案提供了重要的理论基础和实验依据。
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-AIF△1 -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 AIF△1-480 gene, and kill tumor cells.
1. Construction of recombinant plasmids
1.1 Acquisition of AIF_(△1-480) gene
The 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 AIF_(△1-480) gene. The AIF_(△1-480) gene was acquired by RT-PCR through agarose gel electrophoresis. The AIF_(△1-480) 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 promoter
The pMD19T-Egr-1 was digested into the fragments with EcoRⅠand HindⅢenzymes. 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 plasmids
The AIF△1-480 gene was ligated to the pcDNA3.1 vector to construct the pcDNA3.1-AIF_(Δ1-480), and the Egr-1 was ligated to the pcDNA3.1-AIF_(Δ1-480) vector to construct the pcDNA3.1-Egr-1-AIF_(Δ1-480) with the technique of genetic engineering. The pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 detection
There were four groups in the experiment: the control, pcDNA3.1, pcDNA3.1- AIF△1-480 and pcDNA3.1-Egr-AIF_(△1-480). 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 cells
3.1 Protein expressions in MCF-7 cells transfected with recombinant plasmids
The 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 AIF_(Δ1-480) protein expressions detected in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 AIF_(Δ1-480). There was time-course law in the AIF_(Δ1-480) protein expressions.
3.2 Time-course changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after 2.0 Gy X-irradiation
The 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 AIF_(Δ1-480) protein expressions detected in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) group was more than that in the pcDNA3.1-AIF_(Δ1-480) 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 AIF_(Δ1-480). There was time-course law in the AIF_(Δ1-480) protein expressions. The promoter of Egr-1 could be activated by irradiation, and enhance the AIF_(Δ1-480) protein expressions.
3.3 Dose-effect changes of protein expressions in MCF-7 cells transfected with recombinant plasmids after X-irradiation with different doses
The 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 AIF_(Δ1-480) protein expressions were not detected in the control and pcDNA3.1 groups, but increased with the increase of doses in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. At the same dose, the expression in the pcDNA3.1- Egr-1-AIF_(Δ1-480) group was more than that in the pcDNA3.1-AIF_(Δ1-480) 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 AIF_(Δ1-480) protein expressions. The promoter of Egr-1 could be activated by irradiation and enhance the AIF_(Δ1-480) protein expressions.
4 Effects of recombinant plasmids combining with X-irradiation on MCF-7 cells
4.1 Effects of X-irradiation on proliferation of MCF-7 cells transfected with recombinant plasmids
The 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. The results suggest that the AIF_(Δ1-480) protein and radiation, respectively, could inhibit the proliferation of MCF-7 cells, so this could increase with the combination of AIF_(Δ1-480) protein and radiation.
4.2 Effects of X-irradiation on cell cycle progression of MCF-7 cells transfected with recombinant plasmids
The percentages of G_0/G_1 phase cells without irradiation increased significantly in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups as compared with that in the control group, but those of G_2/M phase cells decreased significantly (P < 0.05). The percentages of G_0/G_1 and G_2/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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups (P < 0.05). The results indicate that the AIF_(Δ1-480) 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 AIF_(Δ1-480) protein and ionizing radiation.
4.3 Effects of X-irradiation on apoptosis of MCF-7 cells transfected with recombinant plasmids
The early and late apoptotic percentages of the cells without irradiation increased significantly in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups. As compared with those in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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 AIF_(Δ1-480) protein and ionizing radiation could lead to the early and late apoptosis. The early, late and total apoptosis increased with the combination of AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups (P < 0.01). The results indicate that the AIF_(Δ1-480) protein and ionizing radiation could reduce the invasive ability, particularly in the combination of AIF_(Δ1-480) protein and ionizing radiation.
4.5 Effects of X-irradiation on expression of cytochrome c of MCF-7 cells transfected with recombinant plasmids
The expression of cytochrome c protein of the cells without irradiation increased in the pcDNA3.1-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) 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-AIF_(Δ1-480) and pcDNA3.1-Egr-1-AIF_(Δ1-480) groups, particularly in pcDNA3.1-Egr-1-AIF_(Δ1-480) group. The results indicate that the AIF_(Δ1-480) protein and ionizing radiation could increase Cyt c protein expression, particularly in the combination of AIF_(Δ1-480) protein and radiation.
Above all, the gene expression plasmid pcDNA3.1-Egr-1-AIF_(Δ1-480) 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.
引文
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