Experimental Study On Anti-breast Cancer Activity By The Tumor-targeted TRAIL-expressing Vector In Vitro

Objective:To detect the tumor-specific transcriptional activity of the hTERT promoter in hTERT-positive MCF-7/ADR cells by Dual-Luciferase Reporter Assay. To construct the TRAIL gene expression vector under the hTERT promoter and SV40 enhancer by recombinant DNA technology. Then to observe its tumor-specific expression by RT-PCR, Western blotting and FCM analysis and to detect its specific apoptosis-inducing effects in MCF-7/ADR cells by morphologic changes, PI stained flow cytometry (FCM) and AnnexinV-FITC/PI stained FCM. Methods:1 . Measurement of targeting transcriptional activity of the hTERT promoter by luciferase assayThe plasmid phTERTlucl with luciferase gene under the hTERT promoter, positive control plasmid pGL3-Control, and the negative control plasmid pGL3-Basic were respectively co-transfected with pRL-TK, an internal control into MCF-7/ADR cells and HELF cells. The specific transcriptional activity of the hTERT promoter in telomerase-positive cells was determined by Dual-Luciferase^TM Reporter Assay System. 2. Construction of the recombinant vector phTERT-TRAIL and the positive control vector pSV40-TRAIL2.1 Design of primers and amplification of the TRAIL gene by PCRSpecific forward and reverse primers for the TRAIL gene were designed and both of them contained restrictive enzyme site (Hindlll and Nhel, respectively) at the 5' end. Then the TRAIL gene(862bp) was specifically amplified using pEGFP-TRAIL as a template.2.2 Construction of the recombinant vetor phTERT-TRAIL and the positive control vector pSV40-TRAILDigested with this two restrictive enzymes Hindlll and Nhel, the PCR-amplified product was then extracted and orientationly ligated into phTERTlucl, digested with Hindlll and Xbal, an isoaudamer of Nhel. Ligation product was transformed into E. Coli expression host JM109. To screen and identify the positive recombinants, plasmids from positive clones were digested by restrictive enzymes, sequenced and analyzed by BLAST search. A positive control vector pSV40-TRAIL was constructed after digesting phTERT-TRAIL and pGL3^Control with the same restrictive enzymes Hindlll and BamHI in the same way.3. Evaluation of transfection efficiency mediated by lipofectamine MCF-7/ADR and HELF cells were transfected with pEGFP-C3, which encodesGFP gene, using lipofectamine 2000. 24 h later, cells were photographed on a fluorescence microscope and the percentage of GFP expressing cells within the live cell population was determined by FCM.4. Detection of targeting expression of the TRAIL gene driven by hTERT promoter in MCF-7/ADR cellsThe expression of the TRAIL gene was determined in each of the four groups as follows: non-transfected cells, a mock control group; Cells transfected with phTERTlucl, a negative control group; Cells transfected with pSV40^TRAIL, a positive control group; Cells transfected with phTERT-TRAIL, an experimental group.4.1 Semiquantitative RT-PCR analysis for expression of TRAIL mRNA inall groupsThe cells in each group were harvested 48 h after transfection. Total RNA of each group was extracted. The TRAIL gene was amplified by PCR using cDNA templates produced from mRNA by reverse transcription. The amplified products were fractionated on an agrose gel(containing EB); gels were photograghed and quantitatively scanned using the image software for analysis of the TRAIL gene expression in each group.4. 2 Western blotting analysis for the expression of TRAIL protein in all groupsA total 5X lO*1 cells in each group were harvested 48 h after transfection. Cells were then lysed with Cell Lysis Buffer plus 1 mM PMSF and an equal amount of proteins for each sample was loaded on a SDS-PAGE, blotted against nitrocellulose filter, incubated with anti-TRAIL antibody and peroxidase-lablled anti-rabbit IgG secondary antibody, and finally the TRAIL protein in each group was analyzed by using DAB staining.4.3 Flow cytometry assay for the expression of TRAIL protein in all groupsThe cells in each group were harvested 48 h after transfection. After incubation with anti-TRAIL antibody and fluorescence-lablled second antibody, the expression of TRAIL protein in each group was determined by FCM.5. Measurement of targeting apoptosis induced by the TRAIL gene under the hTERT promoterThe morphology of the cells was observed and photographed 24 h and 48 h after transfection under optical microscope.The apoptotic rates were detected at different time points post transfection using PI stained FCM and Annexin-V FITC/PI stained FCM. Results: 1. The significant transcriptional activity of the hTERT promoter onlyin hTERT-positive MCF-7/ADR cellsThe result of luciferase assay confirmed that the significant transcriptional activity of the hTERT promoter was only observed in hTERT-positive MCF-7/ADR cells, while it cannot be effectively activated in hTERT-negative HELF cells.2. Successful construction of recombinant vector phTERT-TRAIL and the positive control vector pSV40-TRAILPositive recombinants were screened and identified by restrictive enzymes digestion before and after ligation, DNA sequencing and BLAST search after plasmid isolation, which confirmed that recombinant vectors phTERT-TRAIL and pSV40-TRAIL were constructed succesfully.3. Transfection efficiency mediated by lipofectaminMCF-7/ADR and HELF cells were transfected with pEGFP-C3 using lipofectamine 2000. 24 h later, the strong GFP fluorescence was observed in about 70 percent of MCF-7/ADR cells and about 40 percent of HELF cells under a fluorescence microscope. FCM analysis showed the transfection rates were 72. 6% and 42% in MCF-7/ADR cells and HELF cells, respectively.4. Targeting expression of phTERT-TRAIL in MCF-7/ADR cellsResults of semiquantitative RT-PCR, Western-blotting and FCM all showed the TRAIL gene was expressed obviously in MCF-7/ADR cells transfected with phTERT-TRAIL, while there was very low expression level of TRAIL in HELF cells.5. Apoptosis of MCF-7/ADR cells targetedly induced by TRAILThe observation of cell morphology under optical microscope, and results of PI stained FCM and Annexin-V FITC/PI stained FCM all showed effective apoptosis induced by the recombinant vector phTERT-TRAIL in MCF-7/ADR cells, while not in HELF cells. Conclusion:In the present study, by construction of the nonviral recombinant vectorphTERT-TRAIL, verification of its specific expression and inducing apoptosis activity in MCF-7/ADR cells, a safe, specific, effective therapy against breast tumor was preliminarily established, which would provide reliable experimental data for future study in vivo.