Research On Cell Proliferation Inhibition And Apoptosis In MCF-7 Cells By Combining HTERT And PUMA Gene

Objectives:To construct the RNA interference expression vector for hTERT gene and promoting expression vector for PUMA gene, study on the effects of MCF-7 breast cancer cell proliferation inhibition and apoptosis when hTERT and PUMA gene were transfected separately and cooperately, which could provide experimental basis for multi-gene therapy for tumor. Methods:Designed RNA interference target sequence GGAACACCAAG AAGTTCATCT which aimed at hTERT gene, then synthesize two strands of oligonucleotides including a short hairpin structure, then with T4 DNA ligase the double-strand DNA was inserted downstream from the U6 promoter of the linear pYr-2.1-hU6 vector and the recombination expression plasmid pYr-2.1-hTERT-shRNA was constructed. Nextly the recombinant was transformed into susceptible cell E.coli DH5a, then choosing the positive colony to amplify and extracting the plasmid, and the extracted recombinant was constructed successfully by enzyme digestion and sequence analysis. At the same time, the negative control recombinant pYr-2.1-HK that did not aim at any gene was constructed with the same method. In addition, the expression sequence of PUMA gene was inserted into pUC57 vector and was analyzed. Then, the pUC57-PUMA and pYr-adshuttle-1 were digested with BamHI and EcoRI, combined them with T4 DNA ligase, and pYr-adshuttle-1-PUMA was constructed, and the extracted recombinant was constructed successfully by enzyme digestion. MCF-7 cells were cultured, and those in good condition were plated in 6-well cell culture plates on the day before transfection, then transfected pYr-2.1-EGFP and pYr-adshuttle-1-RFP into MCF-7 separately and cooperately, at 48h post transfection, observed the ratio of green and red fluorescence cells under the fluorescence microscope to judge transfection rate. Set up six experimental groups, they were hTERT group, PUMA group, hTERT/PUMA group, HK group, pYr-adshuttle-1 group and blank control group, then recombinant was transfected into those groups except blank group. At 48h after transfection, the relative expression of hTERT and PUMA protein was detected by western blot, and cell apoptosis and cell cycle distribution was detected by FCM. At 1～4d post transfection, the inhibition effects of the cell growth were detected by CCK-8 assay. Results:1. Recombinant plasmids pYr-2.1-hTERT-shRNA and pYr-adshuttle-1-PUMA were identified by enzyme digestion and sequence analysis confirmed that the purposed sequence had been inserted into proper points, and recombining plasmids were constructed successfully.2. Observe the MCF-7 cells under the fluorescence microscope 48h after transfection and judge the transfection rate that were about 60% basing on the proportion of cells shining fluorescence.3. The results of western blot showed that in hTERT group the relative expression quantities of hTERT protein was about 54.4% lower than blank group, and in hTERT/PUMA group the relative expression quantities of hTERT protein was about 52.3% lower than blank group; in PUMA group the relative expression quantities of PUMA protein was about 168.4% higher than blank group, and in hTERT/PUMA group the relative expression quantities of PUMA protein was about 158.6% higher than blank group.4. The results of analysis of CCK-8 showed that in hTERT group,PUMA group and hTERT/PUMA group MCF-7 cells growth velocity were declined, and the proliferation were significantly inhibited, and for hTERT/PUNA group the rate of MCF-7 cells proliferation inhibition was highter significantly than hTERT group and PUMA group.5. Detection of cell apoptosis and cell cycle distribution by FCM showed that in hTERT group,PUMA group and hTERT/PUMA group MCF-7 cell apoptosis rate were 9.87%,19.02% and 31.46%, in hTERT group and hTERT/PUMA group cell cycle was blocked at G0/G1 phase, and the effect of cell cycle stoppage was more significant in hTERT/PUMA group, but in PUMA group cell cycle had no change. Conclusions:1. The RNA interference expression vector pYr-2.1-hTERT-shRNA aimed at hTERT was successfully constructed.2. The promoting expression vector pYr-adshuttle-1-PUMA targeted at PUMA was successfully constructed.3. The effects of pYr-2.1-hTERT-shRNA and pYr-adshuttle-1-PUMA on MCF-7 cell proliferation inhibition were significantly higher than them separately, so it had showed that the effect on inhibiting on breast cancer MCF-7 cells by combining hTERT with PUMA was more efficacious than that by single gene, and there were synergistic effect between hTERT and PUMA, there is a useful option for cancer gene therapy by combining hTERT and PUMA.