The inhibition of hTERT and TRF2 gene expression and inducing cells apoptosis by adenovirus-mediated hTERT/TRF2 RNA interference in MCF-7 cells / 中国癌症杂志

ABSTRACT

Background and purpose: High expression of telomerase and telomere stability are two common features in tumor cell. hTERT is a catalytic subunit of telomerase, TRF2 is extremely important to maintain the length and stability of telomerase. This study was to construct the recombinant adenovirus mediated shRNA to hTERT and TRF2, and to investigate the inhibitory effects of the vector by solo-inhibiting and connect-inhibiting in the MCF-7 cells, in order to present a new approach to the gene therapy for breast cancer. Methods: rAd-hTERT and rAd-TRF2 were constructed and the expression of hTERT mRNA and TRF2 mRNA were tested by FQ-PCR 48 hours after transfecting in MCF-7 cells. Apoptosis was observed by flow cytometry 1 to 6 days after transfection. Results: ①At 48 hours after transfection, the results of FQ-PCR showed that compared to PBS group, the expression of hTERT in rAd-hTERT group was obviously decreased and the inhibition ratio was about 86%, but TRF2 had not been obviously inhibited (P>0.05);the expression of TRF2 in rAd-TRF2 group was obviously decreased and the inhibition ratio was about 80%, but hTERT had not been obviously inhibited (P>0.05);in rAd-hTERT/rAd-TRF2 group, the inhibition ratio of hTERT and TRF2 were about 88% and 85%. Comparing rAd-hTERT/rAd-TRF2 group with rAd-hTERT group and rAd-TRF2 group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). Otherwise, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences of inhibition ratio between hTERT gene and TRF2 gene(P>0.05). ②The result of flow cytometry showed that apoptosis was induced at the first day after transfecting in rAd-hTERT group and rAd-TRF2 group, the most obvious apoptosis was in the 3rd to 5th days,at the peak in the 5th day, and decreased in the 6th day after transfection. The apoptosis ratio of rAd-hTERT group was 46.2%, rAd-TRF2 group was 43.5%. The apoptosis ratio of rAd-hTERT/rAd-TRF2 group was 46.2% at first day, 68.5% at the second day, the most obvious apoptosis was in the 3rd to 6th days and was 77.6% in the 6th days in rAd-hTERT/rAd-TRF2 group. There were significant differences in apoptosis ratio in solo-inhibiting and connect-inhibiting(P<0.05). In addition, comparing rAd-HK group, rAd-blank group with PBS group, there were no significant differences in apoptosis ratio(P>0.05). Conclusion: ①Target sequence of RNAi which aimed at hTERT gene and TRF2 gene was designed efficiently, and the RNAi expression vectors were seen in vivo study efficiently and specifically inhibited the correspond gene expression and promoted cell apoptosis in MCF-7 cells. ②rAd-hTERT vector and rAd-TRF2 vector have no synergistical effect and antagoinstical effect on inhibiting hTERT gene and TRF2 gene mRNA expressing in MCF-7, but there was synergistical effect in terms of the induction of apoptosis. So association-RNAi-technique targeting to the genes of telomere length and stability can effectively promote tumor cell into apoptosis and inhibit breast cancer cell growth. RNAi technique of connecting correlation genes is a more effective gene therapy strategy.