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Research on the Inhibitive Effect of hTERT and TRF2 Protein Expression and Cell Growth in MCF-7 Cells by Interference hTERT and TRF2 Gene

Author: ChenShaoKun
Tutor: ShuiQingLin
School: Luzhou Medical College
Course: Chinese and Western medicine combined with the basis
Keywords: RNA interference siRNA expression vectors Adenovirus hTERT gene TRF2 gene Gene therapy Tumor
CLC: R737.9
Type: Master's thesis
Year: 2008
Downloads: 7
Quote: 0
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Abstract


Objective: To build adenovirus-mediated human telomerase reverse transcriptase (hTERT) gene and telomeric repeat binding factor 2 (TRF2) gene RNA interference (RNAi) expression vector, to explore hTERT and TRF2 protein in MCF-7 cells in expression, as well as hTERT and TRF2 double of gene interference synergies for the treatment of breast cancer and cancer gene therapy application of a new strategy for joint interference hTERT and TRF2 gene as to provide experimental basis. Methods: RNA interference target sequence design for hTERT and TRF2 gene, synthetic oligonucleotides having the target sequence short hairpin structure, annealed to form a double-stranded DNA by T4 DNA ligase, connected to the linearized pGenesil-1 plasmid U6 downstream of the promoter recombinant expression plasmid pGenesil-shRNA-hTERT and pGenesil-shRNA-TRF2. Subsequently cloned into the U6-hTERT-shRNA and U6-TRF2-shRNA expression cassettes the entry vector pENTRTM1A, by LR vitro homologous recombination expression cassette (U6-shRNA-hTERT U6-shRNA-TRF2) transferred to pAd/PL- DEST adenoviral expression plasmid and packaging in HEK293 cells with the ability to infect virus particles rAd-hTERT and rAd-TRF2. Build with the law is not directed against any gene shRNA negative control rAd-HK, empty virus control rAd-blank adenovirus expressing green fluorescent protein (EGFP) rAd-EGFP. Cultured MCF-7 cells, inoculated exponential growth phase cells in 6-well plates, transfected with rAd-EGFP to determine the optimal intensity of infection and the best infection time. Then set the blank group, empty virus group, negative control group, rAd-hTERT group, rAd-TRF2 group, rAd-hTERT/rAd-TRF2 combination group, respectively, to 50MOI transfected human breast cancer MCF-7 cells. 48 h after transfection, cracking cells in each group to extract total protein detected by western blot in each group hTERT and TRF2 protein relative expression level. 1 ~ 6d after transfection, MTT assay was used to detect cell proliferation; PI staining flow cytometry 48h after transfection cell cycle distribution. Results: ① the recombinant plasmid pGenesil-shRNA-hTERT and pGenesil-shRNA-TRF2 Xbal restriction enzyme digestion and sequencing confirmed that the target sequence is accurately inserted into the expected loci, the recombinant plasmid was constructed successfully. ② packaging successfully confirmed by restriction endonuclease and PCR identification of recombinant adenovirus rAd-hTERT and rAd-TRF2. To 50MOI rAd-EGFP transfected MCF-7 cells after 48h fluorescence microscopy observation, the transfection efficiency of about 99%. ④ Western blot analysis showed that: the rAd-of hTERT the group and rAd-hTERT/rAd-TRF2 joint group relative hTERT protein expression compared with the other four groups were decreased by about 82%, while the rAd-hTERT group with rAd-hTERT/rAd- of the between TRF2 joint group of hTERT protein expression differences significantly (P gt; 0.05); the rAd-TRF2 group and the rAd-hTERT/rAd-TRF2 combined group TRF2 protein relative expression level decreased compared with the other four groups of about 83% rAd-TRF2 the group and rAd-hTERT/rAd-TRF2 joint group between TRF2 protein expression was no significant difference (P gt; 0.05). ⑤ MTT Experimental results show that: the rAd-of hTERT group, rAd-TRF2 group as well rAd-hTERT/rAd-TRF2 of the Joint Group proliferation was significantly inhibited cell growth slowed in the range of 1 ~ 6d; of but rAd-hTERT/rAd-TRF2 joint group proliferation of MCF-7 cells was significantly stronger than the group of rAd-hTERT group and rAd-TRF2, and inhibition of cell proliferation peak was significantly longer than the duration of rAd-hTERT group and rAd-TRF2 group. ⑥: the rAd-of hTERT Group, rAd-TRF2 group and rAd-hTERT / rAd-TRF2 combined group after transfection 48h MCF-7 cell cycle arrest in G0/G1 phase cell cycle distribution by flow cytometry. proliferation index (PI) decreased significantly; which rAd-hTERT/rAd-TRF2 combination group than rAd-hTERT group and rAd-TRF2 cell cycle arrest more significant cell proliferation index (PI) decreased more significantly. Conclusion: ① successfully constructed the adenovirus-mediated RNA interference targeting hTERT and TRF2 gene eukaryotic expression vector of rAd-hTERT rAd-TRF2. (2) RNAi target sequences for hTERT and TRF2 gene design effective rAd-hTERT and rAd-TRF2 transfected human breast cancer MCF-7 cells after 48h significantly inhibit hTERT protein TRF2 protein expression, hTERT protein TRF2 protein-free correlation. ③ rAd-hTERT transfected alone and rAd-TRF2 in human breast cancer MCF-7 cells after cell cycle arrest in G0/G1 phase, inhibition of cell proliferation, but co-transfected with rAd-hTERT and rAd-TRF2 cell proliferation inhibition more significantly longer duration, and joint interference the hTERT and TRF2 genes available the more effective tumor gene therapy.

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