Effect On Telomerase Activity By 5'Regulatory Sequence Methylation Profile And SiRNA

Marek's disease is a malignant lymphoproliferative disease of domestic chickens caused by MD virus. To date, MD is still a major problem in the poultry industry, which has been listed as one of the most serious diseases.Telomerase activity has been detected in 90% human tumor and MD while it is undetectable in most of the normal somatic cells,which suggested telomerase may play an important role in tumorigenesis. Since MDV is the first herpesvirus proved to be oncogenic by experiment and prevented by DNA tumorvirus, exploring the role that telomerase play in tumor formation is crucial to understand the mechanism of MD development, as well as contribute to promote the research of the human tumor. .DNA methylation,as the main epigenetic modification,often occurs in human tumorigenesis.Aberrant methylation of cancer-related genes,including hyper- methylation of anti-oncogene and hypomethylation of proto-oncogenesis, play an important role in tumorigenesis.As the number of genes known to be aberrantly methylated in cancer is growing,the detection of of chTERT regulatory sequence methylation profile will help us make further research in the field of chTERT transcriptional control.RNA interference (RNAi) refers to the phenomenon of post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. It can result in highly specific suppression of gene expression,which is mediated by 21-23 nucleotide small RNA that is homologous in the sequence to the target gene. For tumor therapy,RNAi has been intensively used as a new powerful tool to supress the expression of anti-oncogenes or tumorigenesis related genes.Chickens might be more useful than other animals as a model for discover the role telomere play in human tumorigenesis.For chickens live much longer,they live for up to30 years,and some of their telomeres are reported to be similar in length to humans telomeres.Moreover,primary cultures of chicken cells resemble those of human cells in that they undergo a clear-cut replicative senescence and exhibit very low rates of spontaneous immortalization.Thus,we used MDCC-MSB1 as the model to detect the methylation peofile of chTERT regulatory sequence and suppress the expression of chTERT mRNA by siRNA, as well as contribute to the comparative analysis of chTERT expression and regulation from the transcriptional and post-transcriptional level respectively.ChTERT 5′regulatory sequence was cloned,sequenced and analyzed. The sequence was 997bp,and the ratio of G+C in the Genome was 50.78%. The homology of chTERT promoter region to that of sequences (AY505015) was 99.22% in the nucleotide.There were only 3 nucleotide mutation in the 530bp upstream of the first ATG coden,which covered the core promoter of chTERT. Compared the transcription factor binding sites in the chTERT 5′regulatory sequence to that of hTERT 5′regulatory sequence,several c-Myb sites were found associated with chTERT only and c-Ets-2 and WT1 were associated with hTERT only.Both have one E-box at -250bp,and the other transcription factor binding sites are similar to each other.Results presented here should promote structure-function studies of chTERT expression and regulation.ChTERT regulatory region possesses an extensive CpG island,extending well into the coding sequence,along which over 80 transcription factor binding sites have been identified.We detect the methylation profile of the six CG sequences by methylation-sensitve restriction enzyme and methylation-specific PCR respectively.The results that the sites of MDCC-MSB1 are unmethylated versus of CEF suggest that the methylation profile of CpG island may be the limmiting-step for chTERT expression,which coincide with telomerase activity of the cells.In this research small interfering RNAs(siRNAs) obtained by in vitro transcriptional methods were applied to suppress the expression of chTERT mRNA in MDCC-MSB1 cells by relative quantitative real time RT-PCR, and to evaluate the biological consequences of chTERT down-regulation on MDCC-MSB1 cells growth. Three small siRNAs(siRNA; siRNA-1, siRNA-2, siRNA-3)specific for different target sites of chTERT(chicken TERT) mRNA were designed and obtained by using T7 RNA polymerase in vitro transcription. These siRNAs were transfected into MDCC-MSB1 cells to knockdown chTERT mRNA expression. Of the three siRNAs, only siRNA-1 and siRNA-3 strongly suppressed the expression of chTERT mRNA in MDCC-MSB1 cells. The silencing efficiency is 68% and 81%, respectively, accompanied by cell growth inhibition from G1 to S phage. The siRNA-2,exerted no effect on chTERT mRNA expression, which indicated that the inhibition of chTERT mRNA expression can effectively suppress the growth of MDCC-MSB1 cells. The siRNA can strongly inhibit chTERT mRNA expression in MDCC-MSB1cells, and the down-regulation of chTERT mRNA can effectively suppress the progression of MDCC-MSB1cells, which is crucial to characterize the roles chTERT may play in the mechanism of MD development.Three chTERT mRNA-specific-shRNAs, which were expressed by using DNA-vector-based RNAi technology, target on chTERT mRNA, and one control shRNA expressing plasmids was constructed and obtained. All of these plasmids were transfected into MDCC-MSB1 cell lines to knockdown chTERT mRNA expression. The results demonstrated that 72 hours after transfection, chTERT expression was effectively suppressed for cells transfected with shRNA-1 and shRNA-3, the prohibition rates being 66% and 89% respectively; compared with the control group, the tolemerase activities were distinctly reduced, and the S period apparently falls for the shRNA-3 cells (P<0.01). This shows that the fall of chTERT mRNA can effectively reduce telomerase activity, and hence prohibit the transition of MDCC-MSB1 from the G1 to the S period.Our results suggest that the methylation profile of CpG island may play an important role for chTERT expression. RNAi is a powerful tool to effectively suppress the expression of chTERT mRNA. and the down-regulation of chTERT mRNA can effectively suppress the progression of MDCC-MSB1cells, which is crucial to characterize the roles chTERT may play in the mechanism of MD development.