pshuttle-Egr1-shTRAIL-shES双基因—放射治疗的体外抑瘤效应实验研究
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摘要
放射治疗是目前临床治疗肿瘤的重要手段之一,但是由于肿瘤周边组织的放射损伤及某些肿瘤的辐射抗性问题,使放疗的疗效和应用受到限制。恶性肿瘤基因-放射治疗是近年来肿瘤治疗领域新的研究热点之一,是根据放射治疗和基因治疗的各自特点,将二者联合应用,即将辐射诱导性基因的调控序列与肿瘤杀伤基因相偶联,转染肿瘤细胞,在对肿瘤实施局部放疗的同时诱导肿瘤杀伤基因表达的增强,产生辐射和基因表达产物的协同抑瘤作用。该疗法一方面将放疗与基因治疗有机地结合,发挥协同作用;另一方面,由于辐射具有靶向性和可控性,实现了对杀伤基因表达的时空调控。
利用Egr-1启动子具有辐射诱导特性,即电离辐射诱导下可启动其下游基因的表达,本实验构建了含Egr-1启动子和TRAIL、endostatin双基因的重组质粒pshuttle-Egr1- shTRAIL-shES,研究在电离辐射诱导下,该重组质粒携带的双基因TRAIL和endostatin mRNA及蛋白表达的时效和量效规律,以及观察其联合X射线照射后,分别对人乳腺癌细胞MCF-7和人血管内皮细胞ECV304增殖、周期和凋亡的影响。结果表明,重组质粒pshuttle-Egr1-shTRAIl-shES中TRAIL和endostatin mRNA和蛋白的表达具有辐射诱导双基因共表达特性,且在联合X射线照射后,对MCF-7和ECV304细胞均具有明显的增殖抑制和促凋亡作用,并可改变细胞周期进程。本研究为提高基因-放射治疗效果开辟了新途径,为双基因-放射治疗的临床应用提供理论和实验依据。
At present, the primary ways for tumor therapy are operation, radiotherapy, chemotherapy and biotherapy, and there are also other effective ways, including endocrine therapy, the traditional Chinese medicine treatment, thermotherapy, radiofrequency ablation therapy and so on. Tumor therapy is aimed to eliminate tumor and (or) prevent the tumor growth, meanwhile, minimize the injury to normal tissues, and improve the recovery rate. Nowadays, with the rapid development of molecular biology and genetic engineering, gene-radiotherapy has been a hot spot in the research of tumor therapy, which combines gene therapy and radiotherapy together to cooperate the effects of each therapeutic mean, so would possess good application prospect for the treatment of regional tumor. The discovery of radiation-sensitive early growth response-1 (Egr-1) promoter provides an opportunity for the effective combination of radiotherapy and gene therapy, and establishes the theoretical foundation for malignant tumor gene-radiotherapy. In this study, in order to combine radiotherapy and gene therapy together to cure cancer, the plasmid pshuttle-Egr1-shTRAIL-shES, which could express double genes (TRAIL and endostatin), would be constructed successfully by taking advantage of multiple-gene introduction system. There are three features in this study, one is that the radiation could kill tumor cells and activate transcription of the Egr-1 promoter to induce the expression of the downstream genes, another one is the shTRAIL gene could promote apoptosis of tumor cells, and the third one is the shES gene could inhibit angiogenesis. In this way, it could lower the radiation dose, and alleviate or avoid damage to normal tissues, and combine radiotherapy, promoting-apoptosis and anti-angiogenesis together, thus achieve the purpose of inhibiting tumor growth and killing tumor cells, which would provide a promising way for tumor treatment.
1 Construction of recombinant plasmids
1.1 Acquisition of Egr-1 promoter
The specific PCR primers were designed and synthesized with the following primers according to the sequence of Egr-1 promoter gene: upstream 5'-ggggtaccgacccggaaacgccatataag- 3', including KpnⅠenzyme digestion sites, downstream 5'-ataagaatgcggccgcccaagttctgcgcgctgg g-3', including NotⅠenzyme digestion sites. The Egr-1 promoter gene was cloned and amplified from T-Egr1 as the template, and identified by cleavage of endonucleases and sequencing process. The results of identification confirmed that the sequence of the cloned gene was identical to that published on Genbank.
1.2 Acquisition of shTRAIL gene
The specific PCR primers were designed and synthesized with the following primers according to the sequence of shTRAIL gene: upstream 5'-ctagtctagacaccatgagcactgaaagcatgatc- 3', including XbaⅠenzyme digestion sites; downstream 5'-cgggatccctagttagccaactaaaaa-3', including BamHⅠenzyme digestion sites. The shTRAIL gene was amplified from pACCMV-shTRAIL as the template, and ligated to T vector for sequencing. The result of sequencing analysis was in coincidence with the anticipated result.
1.3 Acquisition of shES gene
Firstly, the primers for cloning endostatin gene were designed and synthesized: upstream 5'-ttgatatcatgcacagccaccgcgacttccag-3', including EcoRⅤenzyme digestion sites, downstream 5'-gctctagactacttggaggcagtcatgaa-3', including XbaⅠenzyme digestion sites. The mRNA was extracted from the liver of five-month aborted fetus, and acted as the template of reverse transcription. The endostatin gene was acquired by RT-PCR, and ligated to T vector for sequencing. The result of sequencing analysis was in coincidence with the anticipated result. Then the shES gene was gained by PCR amplification and ligated to T vector for sequencing. The PCR amplification system included the pMD19T-Endostatin plasmid as the template, synthesized IL-2 secretory signal-peptide as the upstream primer, and the downstream primer for cloning endostatin gene as the downstream primer.
1.4 Construction of recombinant plasmids
The Egr-1, shTRAIL and shES genes were ligated to pshuttle vector to construct the single-gene recombinant plasmids, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shES, and the double-gene recombinant plasmids pshuttle-Egr1-shTRAIL-shES by making use of the technique of genetic engineering. In the following experiments, the expression rule of these plasmids in human breast cancer MCF-7 cells and human vascular endothelial ECV304 cells, and their effects on the cellular growth, apoptosis and cell cycle of the two cell lines were detected.
2 Experimental grouping and index detection
The experiment was divided into five groups which were the control, pshuttle, pshuttle-Egr1-shTRAIL, pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups. In the time-course experiment, the selected time points were 2, 4, 8, 12, 24 and 48 h, and the irradiation dose was 2.0 Gy. In the dose-effect experiment, the selected irradiation doses were 0, 0.1, 0.5, 1.0, 2.0 and 5.0 Gy. RT-PCR and ELISA were used to detecte the expressions of shTRAIL and shES on both mRNA and protein levels respectively. MTT assay was used to detect the cell proliferation, and flow cytometry to cell cycle and apoptosis.
3 Radiation-induced expression rule of recombinant plasmids in MCF-7 cells
3.1 Time-course changes of shTRAIL and shES mRNA and protein expressions in MCF-7 cells or the supernatant transfected with recombinant plasmids after 2.0 Gy X-irradiation
The cells and supernatant were harvested in different time after 2.0 Gy X-irradiation. The expressions of shTRAIL and shES mRNA in MCF-7 cells and that of shTRAIL and shES protein in the supernatant were detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups,and that of shES mRNA in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly, and manifested the time-effect relationship to some extent. Otherwise, it had no changes in other corresponding groups basically. As compared with those in the 0 h, except for the shTRAIL protein expression in the pshuttle-Egr1-shTRAIL-shES group at 2 and 4 h which increased slightly but without significance, the shTRAIL and shES protein expressions in the supernatant of MCF-7 cells transfected with plasmids increased significantly at other time-points (P < 0.01). The shTRAIL protein expression in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups increased gradually along with the time-prolongation, and the former up to the peak value at 12 h, then decreased, while the later up to the peak value at 48 h. The shES protein expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased with the time-prolongation, and that at 48 h was the highest.
3.2 Dose-effect changes of shTRAIL and shES mRNA and protein expressions in MCF-7 cells or the supernatant transfected with recombinant plasmids after X-irradiation with different doses
The cells and supernatant were harvested at 12 h after X-irradiation with different doses. The expressions of shTRAIL and shES mRNA in MCF-7 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and that of shES mRNA expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly after X-irradiation, and even the irradiation with very low dose (0.1 Gy) induced the gene expression, while it had no changes in other corresponding groups basically. As compared with those in the 0 Gy, except for the shTRAIL protein expression in the pshuttle-Egr1-shTRAIL plus 0.5 Gy group with slight increase but no significance, the shTRAIL and shES protein expressions in the supernatant of MCF-7 cells transfected with plasmids increased significantly after X-irradiation with different doses (P < 0.05, P < 0.01), and increased along with the increasing of doses. The shTRAIL protein expression in the pshuttle-Egr1-shTRAIL group was up to the peak value after 2.0 Gy irradiation, while that in the pshuttle-Egr1-shTRAIL-shES group peaked after 0.5 Gy irradiation and sustained at a higher level afterward. The shES protein expression in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups sustained at a higher level after 0.1 and 0.5 Gy irradiation respectively.
4 Radiation-induced expression rule of recombinant plasmids in ECV304 cells
4.1 Time-course changes of shTRAIL and shES mRNA and protein expressions in ECV304 cells or the supernatant transfected with recombinant plasmids after 2.0 Gy X-irradiation
The cells and supernatant were harvested in different time after 2.0 Gy X-irradiation. The expressions of shTRAIL and shES mRNA in ECV304 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups,and that of shES mRNA in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly, and manifested the time-effect relationship to some extent. Otherwise, it had no changes in other corresponding groups basically. As compared with those in the 0 h, the shTRAIL and shES protein expressions at other time-points increased significantly (P < 0.01). The shTRAIL protein expression increased gradually in both pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and up to the peak value at 48 and 24 h respectively. The shES protein expression increased gradually and up to the peak value at 48 h in the pshuttle-Egr1-shES group, and up to the peak at 12 h in the pshuttle-Egr1-shTRAIL-shES group.
4.2 Dose-effect change of shTRAIL and shES mRNA and protein expression in ECV304 cells or the supernatant transfected with recombinant plasmids after X-irradiation with different doses
The cells and supernatant were harvested 12 h after X-irradiation with different doses. The expressions of shTRAIL and shES mRNA in ECV304 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and that of shES mRNA expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly after X-irradiation, and even the irradiation with very low dose (0.1 Gy) induced the gene expression, while it had no changes in other corresponding groups basically. As compared with those in the 0 Gy, the shTRAIL and shES protein expressions increased significantly after X-irradiation with different doses (P < 0.05, P < 0.01). The shTRAIL protein expressions in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups increased along with the increasing of doses, and that in 5.0 Gy was the highest. The shES protein expressions in both the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL groups increased along with the increasing of doses, and sustained at a higher level after 0.1 Gy, and up to the peak value after irradiation with 5.0 and 2.0 Gy respectively.
5 Experimental study on effects of MCF-7 cells by combining recombinant plasmids and X-irradiation
5.1 Growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
5.1.1 Time-effect relationship of the growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated by 2.0 Gy at 24 h after transfection, then the cellular proliferation was detected by MTT assay at the different time-points after irradiation. The results showed that the growth rates of the MCF-7 cells transfected with recombinant plasmids declined, especially those of the pshuttle-Egr1-shTRAIL-shES group. Under the same time-point, when compared with those in the control group, the growth rates of the pshuttle-Egr1-shTRAIL-shES group declined significantly from the beginning of 4 h, and the differences were significant at other time-points except for 8 h when compared with those in the pshuttle group, and at 8、12、24 and 36 h when compared with those in the pshuttle-Egr1-shES group, and from the beginning of 4 h when compared with those in pshuttle-Egr1-shTRAIL group (P < 0.05 ~ P < 0.001).
5.1.2 Dose-effect relationship of the growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with different doses at 24 h after transfection, then the cellular proliferation was detected by MTT assay at 24 h after irradiation. The results showed that along with the enlargement of the irradiation dose, the inhibition of cellular growth increased gradually. Under the identical irradiation dose, there was significant difference between that of the control and pshuttle groups only after 2.0 Gy X-irradiation (P < 0.05), while that of the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups was inhibited significantly as compared with that of the control and pshuttle groups respectively (P < 0.05 ~ P < 0.001). Among these, the inhibition of cell growth in the 5.0 Gy + pshuttle-Egr1-shTRAIL-shES group was the strongest.
5.2 Effects on apoptosis of MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 24 h later. Then the cell apoptotic percentages were detected by FCM. The results showed that as compared with that of the control group, the apoptotic percentages of other groups were increased significantly (P < 0.001), especially that of the pshuttle-Egr1-shTRAIL-shES group which was the highest. Furthermore, the necrotic percentage of the pshuttle-Egr1-shTRAIL-shES group increased significantly when compared with that of all other groups respectively (P < 0.01, P < 0.001).
5.3 Effects on cell cycle of MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 12 h later. Then the percentage of each phase in cell cycle was detected by FCM. The results showed that as compared with that in the control and pshuttle groups respectively, the percentages of G0/G1 phase decreased, and the percentages of S phase increased significantly in the rest groups (P < 0.001), and the percentages of G2/M phase decreased significantly in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL groups (P < 0.01), but that in the pshuttle-Egr1-shTRAIL-shES groups decreased slightly without significance.
6 Effects of combining recombinant plasmids and X-irradiation on ECV304 cells
6.1 Growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
6.1.1 Time-effect relationship of the growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated by 2.0 Gy X-ray at 24 h after transfection, then the cellular proliferation was detected by MTT assay at the different time-points after irradiation. The results showed that the growth rates in the pshuttle group declined slightly after 2.0 Gy irradiation, but when compared with those of the control group, the differences were no significance. All the growth rates in the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL, and pshuttle-Egr1-shTRAIL -shES groups declined, and when compared with those in the control group, the differences began to become significant from the beginning of 12, 12 and 8 h respectively (P < 0.05 ~ P < 0.001). Moreover, as compared with those in the pshuttle-Egr1-shES group respectively, the growth rates in the pshuttle-Egr1-shTRAIL-shES group declined significantly from the beginning of 24 h (P < 0.05 ~ P < 0.001). Moreover, there was significant difference between the growth rates in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups at 36 h (P < 0.05).
6.1.2 Dose-effect relationship of the growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with different doses at 24 h after transfection, then the cellular proliferation was detected by MTT assay at 24 h after irradiation. The results showed that along with the enlargement of the irradiation dose, the inhibition of cellular growth increased gradually. As compared with that in the control and pshuttle groups, the cell growth in the rest groups were inhibited significantly from the beginning of 0.5 Gy (P < 0.05, P < 0.01). Among these, the inhibitory effect in the 5.0 Gy + pshuttle-Egr1-shTRAIL-shES group was the strongest, but there was no significant difference when compared with that in the 5.0 Gy + pshuttle-Egr1-shES and 2.0 Gy + pshuttle-Egr1-shTRAIL-shES groups respectively.
6.2 Effects on apoptosis of ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 24 h later. Then the cell apoptotic percentages were detected by FCM. The results showed that as compared with that of the control group, the apoptotic percentages of the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups were increased significantly (P < 0.05, P < 0.01). As compared with that of the pshuttle group, the apoptotic percentages of the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups were increased significantly (P < 0.05, P < 0.01). The apoptotic percentage of the pshuttle-Egr1-shTRAIL-shES group was the highest. Moreover, the necrotic percentages of both the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups were increased significant when compared with that of the control group, and only that of the later group had significant difference with that of the pshuttle group (P < 0.05).
6.3 Effects on cell cycle of ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 12 h later. Then the percentage of each phase in cell cycle was detected by FCM. The results showed that as compared with that in the control and pshuttle groups, the percentages of G0/G1 phase decreased, and that of G2/M phase increased significantly in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups (P < 0.05, P < 0.01). The percentage of G0/G1 phase decreased in the pshuttle-Egr1-shTRAIL group when compared with that in the pshuttle group(P < 0.05), and that of G2/M phase increased in the pshuttle-Egr1-shTRAIL group when compared with that in the control group (P < 0.05).
Above all, the double-gene coexpression plasmid pshuttle-Egr1-shTRAIL-shES was constructed successfully for the first time in our study, and the plasmid possessed the characteristics of irradiation inducibility and the augumented double-gene coexpression. The effects of growth inhibition and apoptosis promotion on MCF-7 and ECV304 cells by the pshuttle-Egr1-shTRAIL-shES double-gene-radiotherapy were superior to that of both the pure radiotherapy and pshuttle-Egr1-shES or pshuttle-Egr1-shTRAIL single-gene-radiotherapy, and it also could effect the progression of cell cycle. It may be because of the combining action of the promoting apoptosis effect of shTRAIL, the anti-tumor angiogenesis effect of shES, and the directly killing tumor cells by irradiation. The researchs open up a new way to improve the effects of gene-radiotherapy, and provide the theoretical and experimental bases for the clinical application of double-gene-radiotherapy.
利用Egr-1启动子具有辐射诱导特性,即电离辐射诱导下可启动其下游基因的表达,本实验构建了含Egr-1启动子和TRAIL、endostatin双基因的重组质粒pshuttle-Egr1- shTRAIL-shES,研究在电离辐射诱导下,该重组质粒携带的双基因TRAIL和endostatin mRNA及蛋白表达的时效和量效规律,以及观察其联合X射线照射后,分别对人乳腺癌细胞MCF-7和人血管内皮细胞ECV304增殖、周期和凋亡的影响。结果表明,重组质粒pshuttle-Egr1-shTRAIl-shES中TRAIL和endostatin mRNA和蛋白的表达具有辐射诱导双基因共表达特性,且在联合X射线照射后,对MCF-7和ECV304细胞均具有明显的增殖抑制和促凋亡作用,并可改变细胞周期进程。本研究为提高基因-放射治疗效果开辟了新途径,为双基因-放射治疗的临床应用提供理论和实验依据。
At present, the primary ways for tumor therapy are operation, radiotherapy, chemotherapy and biotherapy, and there are also other effective ways, including endocrine therapy, the traditional Chinese medicine treatment, thermotherapy, radiofrequency ablation therapy and so on. Tumor therapy is aimed to eliminate tumor and (or) prevent the tumor growth, meanwhile, minimize the injury to normal tissues, and improve the recovery rate. Nowadays, with the rapid development of molecular biology and genetic engineering, gene-radiotherapy has been a hot spot in the research of tumor therapy, which combines gene therapy and radiotherapy together to cooperate the effects of each therapeutic mean, so would possess good application prospect for the treatment of regional tumor. The discovery of radiation-sensitive early growth response-1 (Egr-1) promoter provides an opportunity for the effective combination of radiotherapy and gene therapy, and establishes the theoretical foundation for malignant tumor gene-radiotherapy. In this study, in order to combine radiotherapy and gene therapy together to cure cancer, the plasmid pshuttle-Egr1-shTRAIL-shES, which could express double genes (TRAIL and endostatin), would be constructed successfully by taking advantage of multiple-gene introduction system. There are three features in this study, one is that the radiation could kill tumor cells and activate transcription of the Egr-1 promoter to induce the expression of the downstream genes, another one is the shTRAIL gene could promote apoptosis of tumor cells, and the third one is the shES gene could inhibit angiogenesis. In this way, it could lower the radiation dose, and alleviate or avoid damage to normal tissues, and combine radiotherapy, promoting-apoptosis and anti-angiogenesis together, thus achieve the purpose of inhibiting tumor growth and killing tumor cells, which would provide a promising way for tumor treatment.
1 Construction of recombinant plasmids
1.1 Acquisition of Egr-1 promoter
The specific PCR primers were designed and synthesized with the following primers according to the sequence of Egr-1 promoter gene: upstream 5'-ggggtaccgacccggaaacgccatataag- 3', including KpnⅠenzyme digestion sites, downstream 5'-ataagaatgcggccgcccaagttctgcgcgctgg g-3', including NotⅠenzyme digestion sites. The Egr-1 promoter gene was cloned and amplified from T-Egr1 as the template, and identified by cleavage of endonucleases and sequencing process. The results of identification confirmed that the sequence of the cloned gene was identical to that published on Genbank.
1.2 Acquisition of shTRAIL gene
The specific PCR primers were designed and synthesized with the following primers according to the sequence of shTRAIL gene: upstream 5'-ctagtctagacaccatgagcactgaaagcatgatc- 3', including XbaⅠenzyme digestion sites; downstream 5'-cgggatccctagttagccaactaaaaa-3', including BamHⅠenzyme digestion sites. The shTRAIL gene was amplified from pACCMV-shTRAIL as the template, and ligated to T vector for sequencing. The result of sequencing analysis was in coincidence with the anticipated result.
1.3 Acquisition of shES gene
Firstly, the primers for cloning endostatin gene were designed and synthesized: upstream 5'-ttgatatcatgcacagccaccgcgacttccag-3', including EcoRⅤenzyme digestion sites, downstream 5'-gctctagactacttggaggcagtcatgaa-3', including XbaⅠenzyme digestion sites. The mRNA was extracted from the liver of five-month aborted fetus, and acted as the template of reverse transcription. The endostatin gene was acquired by RT-PCR, and ligated to T vector for sequencing. The result of sequencing analysis was in coincidence with the anticipated result. Then the shES gene was gained by PCR amplification and ligated to T vector for sequencing. The PCR amplification system included the pMD19T-Endostatin plasmid as the template, synthesized IL-2 secretory signal-peptide as the upstream primer, and the downstream primer for cloning endostatin gene as the downstream primer.
1.4 Construction of recombinant plasmids
The Egr-1, shTRAIL and shES genes were ligated to pshuttle vector to construct the single-gene recombinant plasmids, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shES, and the double-gene recombinant plasmids pshuttle-Egr1-shTRAIL-shES by making use of the technique of genetic engineering. In the following experiments, the expression rule of these plasmids in human breast cancer MCF-7 cells and human vascular endothelial ECV304 cells, and their effects on the cellular growth, apoptosis and cell cycle of the two cell lines were detected.
2 Experimental grouping and index detection
The experiment was divided into five groups which were the control, pshuttle, pshuttle-Egr1-shTRAIL, pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups. In the time-course experiment, the selected time points were 2, 4, 8, 12, 24 and 48 h, and the irradiation dose was 2.0 Gy. In the dose-effect experiment, the selected irradiation doses were 0, 0.1, 0.5, 1.0, 2.0 and 5.0 Gy. RT-PCR and ELISA were used to detecte the expressions of shTRAIL and shES on both mRNA and protein levels respectively. MTT assay was used to detect the cell proliferation, and flow cytometry to cell cycle and apoptosis.
3 Radiation-induced expression rule of recombinant plasmids in MCF-7 cells
3.1 Time-course changes of shTRAIL and shES mRNA and protein expressions in MCF-7 cells or the supernatant transfected with recombinant plasmids after 2.0 Gy X-irradiation
The cells and supernatant were harvested in different time after 2.0 Gy X-irradiation. The expressions of shTRAIL and shES mRNA in MCF-7 cells and that of shTRAIL and shES protein in the supernatant were detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups,and that of shES mRNA in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly, and manifested the time-effect relationship to some extent. Otherwise, it had no changes in other corresponding groups basically. As compared with those in the 0 h, except for the shTRAIL protein expression in the pshuttle-Egr1-shTRAIL-shES group at 2 and 4 h which increased slightly but without significance, the shTRAIL and shES protein expressions in the supernatant of MCF-7 cells transfected with plasmids increased significantly at other time-points (P < 0.01). The shTRAIL protein expression in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups increased gradually along with the time-prolongation, and the former up to the peak value at 12 h, then decreased, while the later up to the peak value at 48 h. The shES protein expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased with the time-prolongation, and that at 48 h was the highest.
3.2 Dose-effect changes of shTRAIL and shES mRNA and protein expressions in MCF-7 cells or the supernatant transfected with recombinant plasmids after X-irradiation with different doses
The cells and supernatant were harvested at 12 h after X-irradiation with different doses. The expressions of shTRAIL and shES mRNA in MCF-7 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and that of shES mRNA expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly after X-irradiation, and even the irradiation with very low dose (0.1 Gy) induced the gene expression, while it had no changes in other corresponding groups basically. As compared with those in the 0 Gy, except for the shTRAIL protein expression in the pshuttle-Egr1-shTRAIL plus 0.5 Gy group with slight increase but no significance, the shTRAIL and shES protein expressions in the supernatant of MCF-7 cells transfected with plasmids increased significantly after X-irradiation with different doses (P < 0.05, P < 0.01), and increased along with the increasing of doses. The shTRAIL protein expression in the pshuttle-Egr1-shTRAIL group was up to the peak value after 2.0 Gy irradiation, while that in the pshuttle-Egr1-shTRAIL-shES group peaked after 0.5 Gy irradiation and sustained at a higher level afterward. The shES protein expression in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups sustained at a higher level after 0.1 and 0.5 Gy irradiation respectively.
4 Radiation-induced expression rule of recombinant plasmids in ECV304 cells
4.1 Time-course changes of shTRAIL and shES mRNA and protein expressions in ECV304 cells or the supernatant transfected with recombinant plasmids after 2.0 Gy X-irradiation
The cells and supernatant were harvested in different time after 2.0 Gy X-irradiation. The expressions of shTRAIL and shES mRNA in ECV304 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups,and that of shES mRNA in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly, and manifested the time-effect relationship to some extent. Otherwise, it had no changes in other corresponding groups basically. As compared with those in the 0 h, the shTRAIL and shES protein expressions at other time-points increased significantly (P < 0.01). The shTRAIL protein expression increased gradually in both pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and up to the peak value at 48 and 24 h respectively. The shES protein expression increased gradually and up to the peak value at 48 h in the pshuttle-Egr1-shES group, and up to the peak at 12 h in the pshuttle-Egr1-shTRAIL-shES group.
4.2 Dose-effect change of shTRAIL and shES mRNA and protein expression in ECV304 cells or the supernatant transfected with recombinant plasmids after X-irradiation with different doses
The cells and supernatant were harvested 12 h after X-irradiation with different doses. The expressions of shTRAIL and shES mRNA in ECV304 cells and that of shTRAIL and shES protein in the supernatant was detected by RT-PCR and ELISA respectively. The results showed that the expressions of shTRAIL mRNA in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups, and that of shES mRNA expressions in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups increased significantly after X-irradiation, and even the irradiation with very low dose (0.1 Gy) induced the gene expression, while it had no changes in other corresponding groups basically. As compared with those in the 0 Gy, the shTRAIL and shES protein expressions increased significantly after X-irradiation with different doses (P < 0.05, P < 0.01). The shTRAIL protein expressions in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups increased along with the increasing of doses, and that in 5.0 Gy was the highest. The shES protein expressions in both the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL groups increased along with the increasing of doses, and sustained at a higher level after 0.1 Gy, and up to the peak value after irradiation with 5.0 and 2.0 Gy respectively.
5 Experimental study on effects of MCF-7 cells by combining recombinant plasmids and X-irradiation
5.1 Growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
5.1.1 Time-effect relationship of the growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated by 2.0 Gy at 24 h after transfection, then the cellular proliferation was detected by MTT assay at the different time-points after irradiation. The results showed that the growth rates of the MCF-7 cells transfected with recombinant plasmids declined, especially those of the pshuttle-Egr1-shTRAIL-shES group. Under the same time-point, when compared with those in the control group, the growth rates of the pshuttle-Egr1-shTRAIL-shES group declined significantly from the beginning of 4 h, and the differences were significant at other time-points except for 8 h when compared with those in the pshuttle group, and at 8、12、24 and 36 h when compared with those in the pshuttle-Egr1-shES group, and from the beginning of 4 h when compared with those in pshuttle-Egr1-shTRAIL group (P < 0.05 ~ P < 0.001).
5.1.2 Dose-effect relationship of the growth inhibitory effects on MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with different doses at 24 h after transfection, then the cellular proliferation was detected by MTT assay at 24 h after irradiation. The results showed that along with the enlargement of the irradiation dose, the inhibition of cellular growth increased gradually. Under the identical irradiation dose, there was significant difference between that of the control and pshuttle groups only after 2.0 Gy X-irradiation (P < 0.05), while that of the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups was inhibited significantly as compared with that of the control and pshuttle groups respectively (P < 0.05 ~ P < 0.001). Among these, the inhibition of cell growth in the 5.0 Gy + pshuttle-Egr1-shTRAIL-shES group was the strongest.
5.2 Effects on apoptosis of MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 24 h later. Then the cell apoptotic percentages were detected by FCM. The results showed that as compared with that of the control group, the apoptotic percentages of other groups were increased significantly (P < 0.001), especially that of the pshuttle-Egr1-shTRAIL-shES group which was the highest. Furthermore, the necrotic percentage of the pshuttle-Egr1-shTRAIL-shES group increased significantly when compared with that of all other groups respectively (P < 0.01, P < 0.001).
5.3 Effects on cell cycle of MCF-7 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 12 h later. Then the percentage of each phase in cell cycle was detected by FCM. The results showed that as compared with that in the control and pshuttle groups respectively, the percentages of G0/G1 phase decreased, and the percentages of S phase increased significantly in the rest groups (P < 0.001), and the percentages of G2/M phase decreased significantly in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL groups (P < 0.01), but that in the pshuttle-Egr1-shTRAIL-shES groups decreased slightly without significance.
6 Effects of combining recombinant plasmids and X-irradiation on ECV304 cells
6.1 Growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
6.1.1 Time-effect relationship of the growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated by 2.0 Gy X-ray at 24 h after transfection, then the cellular proliferation was detected by MTT assay at the different time-points after irradiation. The results showed that the growth rates in the pshuttle group declined slightly after 2.0 Gy irradiation, but when compared with those of the control group, the differences were no significance. All the growth rates in the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL, and pshuttle-Egr1-shTRAIL -shES groups declined, and when compared with those in the control group, the differences began to become significant from the beginning of 12, 12 and 8 h respectively (P < 0.05 ~ P < 0.001). Moreover, as compared with those in the pshuttle-Egr1-shES group respectively, the growth rates in the pshuttle-Egr1-shTRAIL-shES group declined significantly from the beginning of 24 h (P < 0.05 ~ P < 0.001). Moreover, there was significant difference between the growth rates in the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups at 36 h (P < 0.05).
6.1.2 Dose-effect relationship of the growth inhibitory effects on ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with different doses at 24 h after transfection, then the cellular proliferation was detected by MTT assay at 24 h after irradiation. The results showed that along with the enlargement of the irradiation dose, the inhibition of cellular growth increased gradually. As compared with that in the control and pshuttle groups, the cell growth in the rest groups were inhibited significantly from the beginning of 0.5 Gy (P < 0.05, P < 0.01). Among these, the inhibitory effect in the 5.0 Gy + pshuttle-Egr1-shTRAIL-shES group was the strongest, but there was no significant difference when compared with that in the 5.0 Gy + pshuttle-Egr1-shES and 2.0 Gy + pshuttle-Egr1-shTRAIL-shES groups respectively.
6.2 Effects on apoptosis of ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 24 h later. Then the cell apoptotic percentages were detected by FCM. The results showed that as compared with that of the control group, the apoptotic percentages of the pshuttle-Egr1-shES, pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups were increased significantly (P < 0.05, P < 0.01). As compared with that of the pshuttle group, the apoptotic percentages of the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups were increased significantly (P < 0.05, P < 0.01). The apoptotic percentage of the pshuttle-Egr1-shTRAIL-shES group was the highest. Moreover, the necrotic percentages of both the pshuttle-Egr1-shTRAIL and pshuttle-Egr1-shTRAIL-shES groups were increased significant when compared with that of the control group, and only that of the later group had significant difference with that of the pshuttle group (P < 0.05).
6.3 Effects on cell cycle of ECV304 cells by combining recombinant plasmids and X-irradiation
The cells were irradiated with 2.0 Gy X-ray at 24 h after transfected with plasmids, and harvested 12 h later. Then the percentage of each phase in cell cycle was detected by FCM. The results showed that as compared with that in the control and pshuttle groups, the percentages of G0/G1 phase decreased, and that of G2/M phase increased significantly in the pshuttle-Egr1-shES and pshuttle-Egr1-shTRAIL-shES groups (P < 0.05, P < 0.01). The percentage of G0/G1 phase decreased in the pshuttle-Egr1-shTRAIL group when compared with that in the pshuttle group(P < 0.05), and that of G2/M phase increased in the pshuttle-Egr1-shTRAIL group when compared with that in the control group (P < 0.05).
Above all, the double-gene coexpression plasmid pshuttle-Egr1-shTRAIL-shES was constructed successfully for the first time in our study, and the plasmid possessed the characteristics of irradiation inducibility and the augumented double-gene coexpression. The effects of growth inhibition and apoptosis promotion on MCF-7 and ECV304 cells by the pshuttle-Egr1-shTRAIL-shES double-gene-radiotherapy were superior to that of both the pure radiotherapy and pshuttle-Egr1-shES or pshuttle-Egr1-shTRAIL single-gene-radiotherapy, and it also could effect the progression of cell cycle. It may be because of the combining action of the promoting apoptosis effect of shTRAIL, the anti-tumor angiogenesis effect of shES, and the directly killing tumor cells by irradiation. The researchs open up a new way to improve the effects of gene-radiotherapy, and provide the theoretical and experimental bases for the clinical application of double-gene-radiotherapy.
引文
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