Research On The Inhibitive Effect Of Htert And Trf2 Protein Expression And Cell Growth In Mcf-7 Cells By Interference Htert And Trf2 Gene

Objectives: To construct the adenovirus-mediated RNA interference expression vector aimed at hTERT and TRF2 gene ,explore the relativity of hTERT and TRF2 protein expression in MCF-7 cells,and the therapy cooperativity on breast cancer by combinate hTERT and TRF2 gene ,which could be provide experimental evidence for interference combination hTERT and TRF2 gene as a new effective way of tumor gene therapy .Methods: Designed RNA interference target sequence aimed at hTERT and TRF2 gene, then synthesize two strands of oligonucleotides including a hairpin structure,than the double-strand DNA was inserted downstream from the U6 promoter of the linear pGenesil-1 vector by T4 DNA ligase and the recombination expression plasmid pGenesil-shRNA-hTERT and pGenesil-shRNA-TRF2 was constructed. Then the expression cassette of U6-hTERT-shRNA and U6-TRF2-shRNA were subcloned into entry vector pENTRTM1A. Followed by homologous recombination technique ,the expression frame(U6-shRNA-hTERT,U6-shRNA-TRF2)was integrated into adenovirus expression plasmid pAd/PL-DEST, and the recombinant adenovirus were packaged in HEK293 cells,gained rAd-hTERT and rAd-TRF2 .At the same time, constructed the negative control recombinant adenovirus rAd-HK that did not aim at any gene,blank adenovirus controlr Ad-blank,rAd–EGFP that express green fluorescence protein by the same method. MCF-7 cells that were in good condition were plated in 6-well cell culture plates,and transfected rAd–EGFP into MCF-7 cells to judge optimal MOI and transfection time. Then set up six experimental groups, they were blank control group,rAd-blank group,rAd-HK group,rAd-hTERT group,rAd-TRF2 group and rAd-hTERT/rAd-TRF2 group.They were transfected into MCF-7 cells separately at 50MOI . At 48 hours after transfection, the total protein from each well of cells was isolated for Western blot to detect the relative expression of hTERT and TRF2 protein in every group. At the same time,at 1～6d after transfection, the inhibition effects of the cell growth were detected by MTT assay;and the cell cycle was observed by means of flow cytometry FCM by PI staining after gathered every group cells at 48h after transfection . Results:①The recombinant plasmids pGenesil-shRNA-hTERT and pGenesil-shRNA -TRF2 were digested with xbal to agarose gel electrophoresis and sequence analysis ,showed that the target sequence had inserted into the predicted site precisely and the recombining plasmids were successfully constructed.②Recombinant adenovirus rAd-hTERT and rAd-TRF2 were digested with PI-SceI and I-CeuI to agarose gel electrophoresis and PCR analysis ,showed that recombinant adenovirus rAd-hTERT and rAd-TRF2 were successfully constructed.③Observe the MCF-7 cells under the fluorescence microscope 48h after transfection rAd–EGFP at 50MOI, transfection efficiency was about 99%.④The result of analysis of the western blottong showed that the relative expression quantities of hTERT protein at rAd-hTERT group and rAd-hTERT+ TRF2 group was about 82% lower than the others groups,but the difference between rAd-hTERT group and rAd-hTERT+ TRF2 group had no statistical significance (P>0.05);the relative expression quantities of TRF2 protein at rAd-TRF2 group and rAd-hTERT+ TRF2 group was about 83% lower than the others groups,but the difference between rAd-TRF2 group and rAd-hTERT+ TRF2 group had no statistical significance (P>0.05).⑤The result of analysis of MTT showed that rAd-hTERT group,rAd-TRF2 group and rAd-hTERT/rAd -TRF2 group MCF-7 cells growth velocity were step down, and proliferation were inhibited significantly at 1～6d after transfection, but rAd-hTERT/rAd -TRF2 group MCF-7 cells proliferation inhibition rate were higher than rAd-hTERT group or rAd-TRF2 group,and the persistence time of MCF-7 cells proliferation inhibition peak at rAd-hTERT/rAd-TRF2 group were longer than rAd-hTERT group or rAd-TRF2 group.⑥Detection of cell cycle distribution by FCM at 48h after transfection,showed that MCF-7cell cycle was arrested at G0/G1 phase and cell proliferation was inhibited at rAd-hTERT group,rAd-TRF2 group and rAd-hTERT/rAd-TRF2 group,but the effect of cell cycle stoppage and cell proliferation inhibition were more significant at rAd-hTERT/rAd-TRF2 group .Conclusions:①Adenovirus-mediated RNA interference expression vector aimed at hTERT and TRF2(rAd-hTERT and rAd-TRF2)were successfully constructed.②The target sequence of RNA interference which aimed at hTERT and TRF2 gene were designed efficiently,hTERT and TRF2 protein expression were inhibited significantly at 48h after rAd-hTERT and rAd-TRF2 were transfected into MCF-7 cells,but protein expression of hTERT and TRF2 have no relativity.③MCF-7 cell cycle was arrested at G0/G1 phase,cell proliferation was inhibited when rAd-hTERT or rAd-TRF2 were transfected into MCF-7 cells separately,but MCF-7 cell proliferation inhibition rate are more significant and persistence time are langer when combinate rAd-hTERT and rAd-TRF2,show that it could be possibly a new effective way of tumor gene therapy that interference combinate hTERT and TRF2 gene.