Inhibition Effects Of RNAi On Telomerase Activation In ECA109 Cells

Telomere, the specific DNA-protein complex on the end of eukaryotic chromosomes, can maintain the stableness and completion of chromosomes. It becomes shorter gradually when cells divided until the loss, fusion, recombination, and degradation of chromosomes and cells apoptosis, so it is named the"biological clock"of cell division. Telmerase, a kind of reverse transcriptase, can use its own RNA template to synthesize the repeated sequences (5-T T A G G G-3)n of telomere DNA, and telomerase , a ribonucleoprotein enzyme , utilizes it's own RNA as a template to add the hexanucleotide to the ends of replicating chromosomes. Telomerase is an attractive molecular target towards cancer therapeutic agents because telomerase activity is expressed in approximately 85% tumors, but undetectable in normal somatic cells. Telomerase is composed of two major components: the catalytic subunit(hTERT) and the template RNA (hTR). The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. HTERT is the most critical component that influences the telomerase activity. HTERT is the limiting component necessary for restoration of telomerase activity in these cells. HTERT seems the idea target of choice as this protein is the rate limiting factor for telomerase activity. Inhibition of reverse transcriptases falls into two major classes: antisense oligonucleotides/ribozymes; inhibiting hTERT transcription. RNA interference (RNAi) is the process of sequence specific ,post transcriptional gene silencing initiated by double-stranded RNA(dsRNA) . Recent studies showed small interfering RNAs(siRNA) generated by Dicer from longer dsRNA specifically suppressed expression of genes in somatic and embryo cells. An RdRP activity might provide amplification by replication of long trigger dsRNAs or copying of short siRNAs in a primer independent manner. The siRNA primed RdRP reaction converts target mRNA into dsRNA ,as well as possibly replicating trigger dsRNA.Both products then serve as Dicer substrates , initiating the RdRP chain reaction.Objective: The aim of this theme was to investigate the effects of RNA interference (RNAi) on telomerase and hTERT-mRNA expression in ECA109 cells .To develop RNAi technology that inhibits the activity of the telomerase carcinoma cell line ECA109 in order to suppress the proliferation of the cells .Methods: According to nucleotide sequence of hTERT in Gene bank and refering to design strategies of siRNA ,through gene blast , three siRNA sequences of target hTERT gene were chosen:Ⅰ:TTGCAAAGCATTGGAATCA,Ⅱ:AGAACGTTCCGCAGAGAA,Ⅲ:GTACAGGTTTCACGCATGT.Carcinoma cells of the line ECA109 were cultured, mRNA interfering double-stranded DNA vectorⅠ,Ⅱ,Ⅲtargeting the mRNA of human telomerase reverse transcriptase (hTERT) and the control vector were constructed respectively and then were tranfected into the ECA109 cells.The expression of hTERT mRNA of the transfected cells was determined by RT-PCR and the activity of telomerase was determined by telomeric repeat amplification-ELISA(TRAP-ELISA) after incubate cells at 37℃in a co2 incubator for 24-48 hours.Results: 1 Identification of interfering vectorⅠ,Ⅱ,Ⅲand the control vector :digested by BamHⅠand HindⅢ,an inserted fragment of interfering vector was about 110bp, an inserted fragment of control vector was about 70bp .2 Green fluorescent cells could be seen in each transfection group through fluorescence microscope after transfected for 24,48 hours and the number of green fluorescent protein (GFP) positive cell about 50%-60%.3 Exponential growth and satisfactory growth ECA109 cells were treated with interfering vectorⅠ,Ⅱ,Ⅲand control vector for 24h,48h respectively ,then cells were collected.Telomerase activation was measured according to the manual of the kit bought from Roche company.The Telomerase activation means were 0.417±0.023, 0.189±0.025,0.435±0.034 respectively after 24h and 0.435±0.032,0.177±0.020,0.395±0.021 respectively after 48h. Interfering vectorⅠ,Ⅱ,Ⅲand control vector had statistical difference(P<0.05). TRAP-ELISA showed that the inhibition rate of interfering vectorⅠ,Ⅱ,Ⅲon the telomerase activation were 49%,79%,53% and the maximum inhibition rate was interfering vectorⅡ,which would be chosen for the next experiment.4 Agarose gel electrophroesis of PCR amplified product of hTERT andβ–actin primer was 198 bp and 621 bp,the same as designed. Interfering vectorⅡtransfected into ECA109 cells down regulated the mRNA expression level of hTERTgene,as compared with the control group.Quantitation of RT-PCR results by scanning densitomery of DNA bands ratio hTERT/β-actin of interfering groupⅡwas 0.5167±0.01922 ,ratio hTERT/β-actin of control group was 0.9323±0.0155 , Interfering vectorⅡand control vector had statistical difference (P<0.05, n=4).Conclusions:The siRNA expresson plasmid was successfully constructed and it can inhibit the expression of hTERT-mRNA and telomerase activity in ECA109 cells,which may be beneficial in searching new gene therapy of the tumors.