Experimental Research On Anti-Lungcancer Effect Of SEA Gene Modulated By Tumor-Specific Promoter

ObjectiveFirstly, to construct 3 eukaryotic expression plasmids which has Red Fluorescin reporter gene (Red) and been modulated by hTERT promoter from hepG2, hTERT promoter from A549 and surviving promoter respectively, and compare their transcriptional activities in human lung cancer cells. To construct pGL-S-SEA eukaryotic expression plasmid modulated by surviving promoter which has highest transcriptional activities in lung cancer A549 cells. To identify the specific expression of sea gene of pGL-S-SEA plasmid in A549 by RT-PCR. To study the superantigen effect of recombinant SEA expressed in A549 by stimulation test of PBMC and MTT test, and to detect the anti-A549 effect of PBMC induced by recombinant SEA expressed by pGL-S-SEA plasmid in A549. It would lay the groundwork for the exploring strategy and mechanism of transcriptional targeting of lung cancer and other cancer gene therapy.Methods1.By PCR and gene cloning technique, 3 eukaryotic expression plasmids (pGL-H-RED, pGL-LH-RED and pGL-S-RED) were constructed, which has Red Fluorescin reporter gene (Red) and been modulated by hTERT promoter from hepG2, hTERT promoter from A549 and survivin promoter respectively.2.These recombinant plsmids were transfected into A549 cells and MRC-5 cells with liposome. After 72 hours, the expressed red fluorescin target protein was observed under fluorescence microscope, then the expression level of red fluorescin modulated by 3 promoters at the same time was analyzed by Imagepro-Plus6.0 software. 3.The sea gene was amplified by PCR from genome of staphylococcus aureus ATCC25923, then choice the survivin promoter as modulating sequence which has highest transcriptional activities in lung cancer A549 cells, and replace the"Red"gene of pGL-S-RED with sea gene to construct the pGL-S-SEA. The recombinant plsmid was transfected into A549 cells. After 72 hours, extract the whole RNA of transfected A549 and identify the specific expression of sea gene of pGL-S-SEA plasmid in A549 by RT-PCR.4.The pGL-S-SEA plasmid was transfected into A549 cells. After 72 hours, gather the supernatant and lysate of transfected A549 and prepare the human PBMC, then do the stimulation test of PBMC and MTT test, and analyze the SI (Stimulation index) to study the superantigen effect of recombinant SEA expressed in A549.5.Prepare the PBMC induced by the supernatant and lysate of transfected A549, and cultivate the A549 and the induced PBMC together. After 24 hours, by doing MTT test and analyzing the cell-killing rate, study the anti-A549 effect of PBMC induced by recombinant SEA expressed by pGL-S-SEA plasmid in A549.Results1.The pGL-H-RED, pGL-LH-RED and pGL-S-RED plasmids were constructed successfully, which promeoter sequence and"Red"gene were confirmed by restriction enzyme analysis and DNA sequencing. After the 3 plasmids were transfected into A549 cells and MRC-5 cells, the red fluorescin target protein was observed in A549 cells, but there has no red fluorescence in MRC-5 control cells. Analyzing the level of red fluorescence by"Imagepro-Plus6.0 software", the pGL-S-RED has highest expression activity in A549 cells and the strength rate (IOD) of red fluorescence of A549 cells transfected by pGL-S-RED, pGL-LH-RED and pGL-H-RED is 201.17, 171.70 and 136.34 respectively.2.The pGL-S-SEA plasmid was constructed successfully, which inserted sea gene was confirmed by restriction enzyme analysis and DNA sequencing. After being transfected in A549 cells, the sea gene was transcribed successfully, which was confirmed by RT-PCR, and the transcription strength rate of sea gene and internal control gene (GADPH gene) is 65.96%.3.The lysate of A549 cells transfected by pGL-S-SEA plasmid can stimulate the PBMC activity and the supernatant has no this function, which confirmed by MTT test. The SI of lysate group, supernatant group and control group is 1.29, 0.95 and 1.58 respectively.4.After the PBMC was induced by the lysate of transfected A549 cells, the PBMC has significantly increased killing activity on A549 cells. The killing rate of lysate group and control group is 31.965% and 21.116% respectively.Conclusions1.The pGL-H-RED, pGL-LH-RED and pGL-S-RED plasmids were constructed successfully, and these 3 promoters all are tumor-specific, and the surviving promoter has highest transcriptional activities in lung cancer A549 cells.2.The pGL-S-SEA plasmid was constructed successfully, which sea gene was modulated by surviving promoter. RT-PCR confirmed that the sea gene was transcribed successfully.3.The expression product of A549 cells can induce the PBMC activation, and the activated PBMC has significantly increased killing effect on lung cancer A549 cells.