Establishment Of RNA Interference Targeting Against HTERT And Research For Its Mechanism On Proliferation And Apoptosis Of Cancer Cells

ObjectiveThe human telomerase reverse transcriptase(hTERT) is the catalytically active component of the telomerase complex.hTERT catalyzes the telomere elongation by reverse transcription,using RNA component of telomerase as template.Its expression correlates with telomerase activity and is restricted to germ cells,stem cells and to more than 90%of human cancers,whereas most normal human somatic cells have no hTERT expression.As close relations with the development and progression of many malignant tumors,telomerase and hTERT were in the spotlight of research.With research going deeply,some new phenomenas and results were revealed and indicated that telomerase has important functions in anti-apoptosis and cell proliferation.To further study the new biological functions of hTERT,The main contents of this research are as following:The first part:To investigate the efficency of construct the small interferening RNA(siRNA)of suppress the expression of hTERT in HeLa cell line after knocked down hTERT gene by siRNA synthesized in chemosynthesis;The second part:To study the effects of silencing of hTERT on transcriptional profile of cancer associated genes in HeLa cells;The third part:To investigate the molecular mechanism of induction of apoptosis by siRNA targeting hTERT in HeLa Cells.MethodsThe first part:Five double-stranded siRNAs targeting coding of hTERT gene were designed by full length gene targeting technique or selected randomly.RNAi-Mate mediated transient transfection was conducted to transmit the siRNA into HeLa cells.Five pairs of specific targeted sequence were selected in the coding region of hTERT gene.mRNA and protein levels of hTERT were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.Proliferation inhibition rate of HeLa cells was analyzed by MTT assay.The second part:Total RNA were extraeted from untreated control group,S₃ group and S₄ group,respectively,with Trizol one-step method,RNAs were further purified with QIANGEN Rneasy Kit.Agarose gel electrophoresis was used to analyze the intensity ratio between 28s and 18s to evaluate the quality of total RNA.cDNA probes were reversely transeribed from mRNA according to the methods bySehena et al.Cy5-dUTP and Cy3-dUTP were used to label mRNA probes from differeni tissues.After the degeneration of hybridization soluteion of mixed probes and microarray,hybridization soluteion was dropped onto the sampling area of microarray,covered with a cover sliP,placed into the hybridization cabin,sealed with parafilm,and then hybridized in 42℃water bath for 16h.The microarrays were scanned with confocal fluorescence scanner and the data was analyzed by ImaGene software to plot the value of Cy3 and Cy5.The third part:To detect apoptosis rate by flow cytometer after transfection for 48h;Measurement of relative Caspase-3 and Caspase-8 activity by colorimetric assay;Cytoplasm Cyt c were detected by Western blotting.ResultsThe first part:Sequence specific siRNAs targeting hTERT down-regulated mRNA expression significantly,protein expression level was decreased after transfection of siRNA-hTERT by means of western blot.After treatment of siRNA,proliferation inhibition rate of HeLa cells were increased(P＜0.01).The second part:The hybridized microarray was scanned.The analysis for the date revealed that there were 3 down-regulated gene(EGFR,VEGF and bcl-2) and 1 up-regulated gene(TRAIL). Wester blot analysis demonstrated that the expression change of bcl-2,EGFR,VEGF and TRAIL was in accord with chip analytic result.The third part:After transfection for 48h,annexin-V and PI double-staining FCM analysis showed that the cell apoptosis rate in two RNAi-hTERT groups was noticeably higher than in three control groups(P＜0.01),and there was no significant difference on the cell apoptosis rate between three control groups(P＞0.05).After transfection for 48h,the difference of Caspase-8 enzymatic activity ratio between each groups was of no significance(P＞0.05),but the Caspase-3 enzymatic activity ratio in two RNAi-hTERT groups was noticeably higher than in three control groups(P＜0.01).Wester blot analysis demonstrated that the level of cytosolic Cyt c was significantly increased after RNAi-hTERT.Conclusion1.Specific siRNA targeting hTERT could effectively inhibit hTERT expression and HeLa cell proliferation.2.Specific siRNA targeting hTERT could up-regulate the expression of TRAIL,and down-regulate the expression of EGFR,VEGF and bcl-2.3.Specific siRNA targeting hTERT induces apoptosis of HeLa cells via activating mitochondrial signal transduction pathway.