Effective Inhibition Of Telomerase Activity In Breast Cancer Cell T47D By SiRNA

Backgroud&ObjectiveTelomere which locates at the end of chromosome is composed of repeated nucleic acid,and it can protect DNA from being destroyed.Telomerase is a special enzyme with reverse transcriptase activity and it is made up of nucleic acid and protein.The activation of telomerase can prolong the telomere of cells with repeated sequences. As a result,cells can escape from senescence and apoptosis.That's the crucial factor during the course that many carcinomas come into being.The telomerase activity of tumor cells and other immortal cells are notably high.It is reported that nearly 85% tumors express telomerase,while normal cells(germ cell and embryonic cell excluded)seldom express it.So the telomerase must play an important role during the formation and development of tumors.It is showed that the telomerase are composed of three subunits.They are telomerase RNA which is used as template(hTR), telomerase reverse transcriptase(hTERT)and telomerase proteinl(TP1).The hTERT is supposed to be the catalytic subunit of telomerase and it is crucial to telomerase activity.But the other two subunits can not be taken as the symbol of telomerase activation.RNAi is a widely used new technology which can knock down the target gene efficiently and exclusively.In order to find a new way for anti-cancer therapy, we used RNAi to block the hTERT of the breast cancer cell line T47D which highly expresss telomerase in our study and examine the telomerase acitivity and growth ability of those cellsMethods:The breast cancer cell T47D which highly expresses the gene hTERT was taken as target cell.And the plasmid pBAsi-hU6-neo(BamHI/HindⅢ)(TaKaRa)was chosen as vector for expressing siRNAs.First,four pairs of strands with 18 bps were selected from the gene sequence of hTERT and one pair was taken as negative control after its sequence was re-arranged randomly.Then five pairs of DNA strands were designed and synthesized.Double strands formed after annealing and then they were connected to generate five plasmids,pBAsi-hU6-neo/siRNA1,pBAsi-hU6-neo/siRNA2, pBAsi-hU6-neo/siRNA3,pBAsi-hU6-neo/siRNA4 and pBAsi-hU6-neo/siRNA2 negative.These five plasmids were transformed into Ecoli DH5 a.After being cloned,selected and amplified,plasmids were extracted and validated by DNA sequencing.The five plasmids were transfected into cell T47D respectively.With the work of promoter U6,these plasmids can produce shRNAs in cells.G418 was used to select those cells which were successfully transfected.RT-PCR and western blot were adopted to investigate the expression of hTERT mRNA and protein respectively; TRAP-ELISA was used to test the cells' telomerase activity;MTT was taken to draw the growth curve and flowcytometry was used to examine the changes of cell cycles.Results:1.Five plasmids that can express siRNAs were successfully constructed.The sequence of the strands inserted into plasmids was correct by DNA sequencing.2.The five plasmids were transfected into breast cancer cell T47D respectively. After selected by G418,five kinds of cells which were successfully transfected were taken out.They were T47D/siRNA1,T47D/siRNA2,T47D/siRNA3,T47D/siRNA4 and T47D/siRNA2negative.3.The results of RT-PCR and western blot showed that the expression of hTERT mRNA and hTERT protein in T47D/siRNA2,T47D/siRNA3 and T47D/siRNA4 was much lower than T47Dcontrol(P＜0.05);while there was no significant difference among T47D/siRNA1,T47D/siRNA2negative and T47Dcontrol.4.The TRAP-ELISA test showed that:when compared to T47Dcontrol,the telomerase activity of T47D/siRNA2,T47D/siRNA3 and T47D/siRNA4 was significantly reduced(P＜0.05);while there was no significant difference among T47D/siRNA1,T47D/siRNA2negative and T47Dcontrol.5.The growth curve drawn by MTT revealed that the growing activity of T47D/siRNA1,T47D/siRNA2,T47D/siRNA3 and T47D/siRNA4 was much lower than T47D control,while T47D/siRNA2 negative nearly had the same growing activity with T47D control.6.The results of flowcytometry showed that the cell cycles of T47D/siRNA1, T47D/siRNA2,T47D/siRNA3 and T47D/siRNA4 changed when compared with T47Dcontrol:the ratio of stages G0/G1 rose,while the ratio of stage S decreased.But the difference of cell cycles between T47D/siRNA2negative and T47D control was of no significance.Conclusions:1.hTERT siRNA can significantly reduce the expression of hTERT in the breast cancer cell T47D.2.The decreased expression of hTERT in breast cancer cell can bring down the cell's telomerase activity.3.Breast cancer cell's growing activity decreased and its cell cycles changed as a result of the decreased telomerase activity.