Research On Anti-tumor Effect Of Hypoxia/radiation Dual-sensitive Promoter-mediated ShTRAIL Combined With Radiation On Lung Cancer Cells In Vitro

Research on anti-tumor effect of Hypoxia/radiation dual-sensitive promoter-mediated shTRAIL combined with radiation on lung cancer cells in vitroCancer therapy usually applys operation, radiotherapy and chemotherapy, but they have limitation. Gene therapy provides a new way for cancer therapy, some researchers attempt to combine gene therapy with operation, radiotherapy and chemotherapy in one or more of them which can improve the therapy effctiveness. Gene therapy aims at the activation of oncogenes and inactivation of tumor suppressor genes, using known gene fragments introduction, transfer and recombination techniques to eliminate the proliferation of activated oncogenes and tumor suppressor gene function, thereby changes the cell biological character to enhance cell sensitivity to drugs and other factors, and enhances the host anti-tumor properties, holds tumor growth.. Because of the lack of oxygen in the tumor area, tumor cells have strong radiaton resistance, radiotherapy regimen is limited. In recent years, gene therapy was combined with radiotherapy to apply cancer gene-radiotherapy. It mainly utilize radiatoin sensitive promoter to mediate downstream genes high expression under the radiation conditions, both achiving radiation breakdown effect, in generally, radiation sensitive promoter early growth response genel(Egrl) is applied commonly. In order to improve the radiation inducing effects enhancement of Egrl under hypoxic conditions, hypoxia-responsive elements (hypoxia response elenments, HREs) is consider to construct chimeric promoter with Egrl, to increase radiation inducing effects of Egrl under hypoxic conditions. In the study, using radiation enhancing effect property of Egrl promoter, the enhancing downstream gene expression character of HREs promoter under hypoxic condition and strongly inducing cancer cell apoptosis of TRAIL gene, while normal cells are not sensitive to its characteristics, to combine these three together and to construct dual-sensitive promoter mediated expression vector, and to combine the ionizing radiation to approach the inhibition effect on lung adenocarcinoma A549 cells in vitro, The results provide new ideas for cancer treatment.1 Construction and identification of reconminant plasmidsIn this experiment, five plasmids were constructed, they are pcDNA3.1-Egr1-EGFP,pcDNA3.1-HRE/Egr-EGFP, pcDNA3.1-shTRAIL, pcDNA3.1-Egr1-shTRAIL and pcDNA3.1-HRE/Egr1-shTRAIL, respectively. pcDNA3.1, pshuttle-EGFP and pMD19T-Egr1 plasmids were endowed by Li Yanbo doctor, they were digested by different restriction enzymes, then pcDNA3.1 vector, EGFP fragement and Egr1 fragement were gotten respectively, ligating them in order to construct pcDNA3.1-Egr1-EGFP plasmid, call it pcEE, they were identificated by PCR, restriction enzyme digestion and sequencing; HRE sequences synthesized by Shanghai bio-engineering company, to connect it to upstream of Egr1 in pcDNA3.1-Egr1-EGFP and construct pcDNA3.1-HRE/Egr1-EGFP, and call it pcHEE, it was identificated by PCR, restriction enzyme digestion and sequencing; shTRAIL were gotten from pshuttle-shTRAIL which digested by restrition enzyme, inserted it to pcDNA3.1 vector and construct pcDNA3.1-shTRAIL plasmid, and call it pshT, it was identificated by PCR, restriction enzyme digestion and sequencing; CMV promoter was cut from pcDNA3.1-CMV-shTRAIL, then to replace it with Egr1, to construct pcDNA3.1-Egr1-shTRAIL and call it pcEshT, it was identificated by PCR, restriction enzyme digestion and sequencing; pcDNA3.1-Egr1-shTRAIL plasmid was digested by restriction enzyme again, conneting HRE upstream to Egr1 and construct pcDNA3.1-HRE/Egr1-shTRAIL, and call it pcHEshT, it was identificted by PCR, restriction enzyme digestion and sequencing. In the study, the five recombinant plamid is fully consistent with the predication, indicates that construction is right.2 Radiation inducing character of Egr1pEE plasmid was transfected into lung adenocarcinoma A549 cells by liposome, under nomaxic and hypoxic conditions, these cells were radiated by 1,2,4,6,8 and 10 Gy X-rays,0,3,8,12,24 and 36 h later, the green fluorescent protein expression were detected by flow cytometry. The results show that the normal oxygen conditions, after pcEE plamid transfection into A549 cells, the green fluorescent protein expression increase with time prolongation and dose increasing, and it reachs peak 12 h after 8 Gy irradiation, but they reach peak 8 h after other dose X-rays irradiation, then they all begin to fall after 24 h. Under hypoxic condition, when oxygen concentration were different, green fluorescent protein expression was inhibited by the same dose irradiation, inhibition was obvious when the oxygen concentration was less than 2.5%, with the lower oxygen concentration the more inhibition, and then the oxygen concentration is higher than 2.5%, green fluorescent protein expression inhibtion is not large with the relationship between oxygen concentration. The expression difference 8 h after different dose irradiation between normoxic condition and hypoxic condition was signifinant (P<0.05, P <0.01). This shows that Egrl has radiation inducing expression enhancement character, and hypoxia can inhibit Egrl pormoter features.3 The influence of HRE on Egrl radiation inducing enhancement characterAfter pEE plasmid was transfected into lung adenocarcinoma A549 cells, under normaxic and hypoxic conditions, these cells were irradiated by 0,2,4,6,8 and 10 Gy, after 0,3,8,12,24 and 36 h, green fluorescent protein expression was measured by flow cytometry, results showed that green fluorescent protein expression in cells transfected by pEE plasmid at different time after various dose irradiation was basically same under the normoxic condition. But under hypoxic condition of 0.1%-2.5% of the oxygen concentration, EGFP expression significantly increased, and increased with oxygen concentration and radiation dose increase, and gradually reached the maximum in 1% oxygen concentration, the expression has significant difference with those in cells 8 h after different dose irradiation under normaxiccondition (P< 0.05, P<0.01). under the condition of 5%-10% oxygen concentration. EGFP expression was basically similar with the expression under normaxic condition. This indicated that HRE has the feature to enhance radiation inducing properties of Egrl under hypoxic condition, but has not under normoxic condition.4 Radiation inducing expression regularity of the recombinant plasmid in A549 cellsAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, cells irradiated by 6 Gy. then TRAIL mRNA and TRAIL protein expression were measured, results showed that the TRAIL mRNA expression increased in A549 cells transfected by pcshT and pcEshT plasmid after 6 Gy irradiation and 6 Gy irradiation + hypoxia of 1% oxygen concentration, and had significant difference with expression in control cells, but the expression was more under 6 Gy irradiation + hypoxia of 1% oxygen concentration condition (P<0.05); and the TRAIL mRNA expression increased in A549 cells transfected by pcHEshT plasmid after 1% oxygen concentration hypoxia,6 Gy irradiation and 6 Gy irradiation +1% oxygen concentration hypoxia, it had significant difference compared with control group(P <0.05), and expression was more under radiation condiation than hypoxic condition, and the expression was more obvious under radiation and hypoxic condition (P <0.05). The shTRAIL protein expression regularity was basically same with mRNA expression regularity, those indicated that radiation could induce shTRAIL expression enhancement in A549 cells transfected by 3 plasmids. and under hypoxic condition the enhancement effect was more obvious.5 Inhibition effect of recombinant plasmid on A549 cells5.1 The recombinant plasmid inhibited A549 cell proliferationAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, A 549 cells were irradiated by 6Gy, then counted the living cells number by trypan blue dye at 0,2,4,6 and 8 d respectively and ploted the cell growth curve, the results showed that the hypoxia of 1% oxygen concentration inhibited A549 cells growth. in which A549 cells transfected by pcHEshT plasmid were inhibited most:and 6 Gy X-rays also inhibited A549 cells growth transfected by 3 plasmid,which cells transfected by pcEshT and pcHEshT plasmid were inhibited most obviously; cells transfected by 3 plasmids all were inhibited very strongly under 6 Gy and 1% oxygen concentration, particularly cells transfected by pcHEshT were inhibited most strongly. At the same time, After pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration. A 549 cells were irradiated by 6 Gy, the cell obsorbance value (A) at 0,12,24,48 and 72 h was detected by MTT, results showed that the A value of A549 cells transfected by 3 plasmids decreased after 1% oxygen concentration hypoxia, in which A value in the cells transfected by pcHEshT decreased greatest; but the A value of A549 cells transfected by 3 plasmids decreased after irradiation by 6 Gy decreased, in which A value in cells transfected by pcEshT decreased greatest; the A value in cells transfected by pcHEshT after 1% oxygen concentration and 6 Gy irradiation decreased greastest at 48 h. These results showed that the inhibition was most strong in cells transfected by pcHEshT after 1% oxygen concentration and 6 Gy X-rays irradiation.5.2 Effect on cell phage and apoptosis of recombinant plasmidAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, A 549 cells were irradiated by 6Gy, cell apoptosis was detected by flow cytometry with PI sigle staining, results showed that after transfected 3 plasmids cell percentage of G0/G1 phase increased under hypoxia of 1% oxygen concerntration and 6 Gy irradiation condition, and cell percentage of G2/M phage decreased, but cell percentage of S phage did not change basically, at same time, and cell percentage of apoptosis increased.At same time, cell apoptosis was measured by TUNEL, cell apoptosis was enhanced after 3 plasmids transfection from radiation and hypoxia. apoptosis change of cell transfected by pcshT and pcEshT plasmid from hypoxia was not obvious, and apoptosis change of cell transfected by pcHEshT was obvious: apoptosis change of cell transfected by pcshT and pcEshT plasmid from hypoxia was obvious, in which apoptosis change of cell transfected by pcEshT and pcHEshT was most obvious, hypoxia and radiation had common promotion on apoptosis change of cell transfected by pcshT, and had more promotion on apoptosis change of cell transfected by pcEshT, and had most promotion on apoptosis change of cell transfected by pcHEshT. This is basically the same as the results of flow cytometry. indicating after transfection by three kinds of plasmids. hypoxia and radiation can induce G0/G1 cell cycle arrest and apoptosis increase.5.3 Effect on radiation sensitivity of recombinant plasmidNormal A549 cells and cells transfected by pcshT, pcEshT and pcHEshT plasmid irradiated by 0,2,4,6.8 and 10 Gy under nomoxic and hypoxic condition, cells were cultured for 14 days, then counted the clones and calculated survival fraction, in accordance with multi-hit single-target model for curve fitting to calculate the Do value, analysised the radiation sensitivity difference among normal cells, cells transfected by pcshT, pcEshT and pcHEshT plasmid. Results showed that under normoxic condition. Do value of 4 cells was 3.26,2.12,1.91 and 1.89 respectively, this indicated that radiation sensitivity in cells transfected by pcEshT and pcHEshT plasmid was maximum;but under hypoxic condition, the Do value was 5.81.6.12,4.24 and 1.13 respectively, this indicated that hypoxia could decrease radiation sensitivity of normal cell and cell transfected by pcshT and pcEshT. and enhance radiation sensitivity of cell transfected by p cHEshT plasmid.5.4 Effect on DR4, DR5 and caspase-3 of recombinant plasmidAfter pcshT, pcEshT and pcHEshT three kinds of plasmid transfected into A549 cells, under normoaxic and/or hypoxic condition of 1% oxygen concentration, cells irradiated by 6 Gy. then DR4 and DR5 mRNA were measured by RT-PCR, results showed that hypoxia effect little on DR4 mRNA in cell transfected by pcshT and pcEshT plasmid, but could induce DR4 mRNA increase in cell transfected by pcHEshT plasmid; but hypoxia could induce DR5 mRNA increase, in which the increase of DR5 mRNA in cell transfected by pcHEshT was most obvious:6 Gy radiation could induce DR4 and DR5 mRNA increase in cell transfected by 3 plasmids;hypoxia and radiation combination could induce increased DR4 and DR5 mRNA, in which DR5 mRNA increase in cells transfected by pcHEshT was most obvious. In addition, DR4, DR5 and caspase-3 protein expression was detected by Western blot, results showed that DR4 and DR5 protein expression regularity were similar with their mRNA, this indicated that cells transfected by 3 plasmids could induce DR4 and DR5 increase under hypoxia and/or radiation.After 3 plasmids transfection, hypoxia and radiation could induce caspase-3, caspase-3 expression in cells transfected by pcHEshT was most obvious under hypoxia and randiation combination, caspase-3 expression regularity was similar with apoptosis regularity.