Expression Of HTERT In Nephroblastoma And Its' Clinical Significance

PrefaceNephroblastoma is a kind of malignant and embryonic tumor commonly occurred in children. It is arising from metanephric balstema, which typically exhibits triphasic epithelial,balstemal,and stromal differentiation. Because it has both embryonic genetic change and body cell mutation, it becomes a suitable model for studying genetic tumorigenesis. Previous study proved that nephroblastoma has high express of telomerase activity . Study showed that telomerase is expressed in up to 85% of human tumor cell and immortal cell line and its activity was not detected in normal somatic cell.Telomerase is a ribouncleoprotein that synthesizes telomeric DNA onto chromosomal ends using a segment of its RNA component as a template. It has been shown that the expression pattern of hTERT ( telomerase reverse transcriptase, it is a catalytic protein subunit which is components of human telomerase ) is closely associated with telomerase activity.To explore the expression of hTERT in nephroblastoma and its' clinical significance , in this study, the expression levels of hTERT were evaluated by using RT - RCR in 30 nephroblastoma tissues, 10 paracancer renal tissues. Its correlations with the clinical stage, pathology type, prognosis were evaluated in posi-tivecase.Methodin our university from 1999 to 2003. These samples were staged according to NWTS4, which is 13stage I , 6stage II , lOstagelfl, 1 stage IV. pathology type were confirmed among these sample that is 6blastema, 10epithelium% 5stromax 9mixture . lOSampes from tumor - adjacent tissues were collected after operation. All of samples are stored in - 80T after bathing by water. RT - RCR kit are obtained from Takara Shuzo Co. Ltd. hTERT - F, hTERT - R and B - actin primer are obtained from Takara; TRIZOL from GEBCOBRL. Expression levels of hTERT mRNA were determined by RT -PCR,Total RNA was refine according to kit directions, RNA concentration and purity were tested by Ultraviolet spectrum. 2ul of RNA were used for each 20 - ul RT - cDNA reaction. The reaction under the following condition;65CC for lmin ,301! for 5min ,65X1 for 30min, 98^ for5min, 5X. for 5min.For hTERT mRNA detection,3ul of RT - cDNA product were amplified u-sing the primers hTERT - F 5' - TAT GCC GTG GTC CAG AAGG - 3' and hTERT - R 5' - GCG TGG GET AGG TGA GGT GT - 3' . Each 25 - ul reaction contained dd. H2O17. lul 10 x buffer 2.5ul dNTPs 2ul Taq - E 0.2ul hTERT -F 0. lul hTERT - R 0. lul . each cycling conditions were as follows; 94X1 for 3min ,32cycles of 94H45S, 57.6Tllmin, and 72^ lmin, followed by one cycle of 7211 for 7min. The primers of B - actine as inside contrast take part in the reaction.ResultThere were over expression of hTERT mRNA in nephroblastoma. The positive rate in the nephroblastoma was ditinctively different from the paracancer renal tissues ( p <0.05 ) . Our data indicate that hTERT transcript levels do not correlate with clinical stage, pathology type in nephroblastoma. The disease free survival rate in hTERT overexpression case was significantly lower than that of the lowexpression case ( p<0.05 ) .DiscussionTelomeres are specialized structures at the ends of eukaryotic chromosomes that appear to function in chromosome protection, positioning, and replication. In vertebrates, telomeres consist of hundreds to thousands of tandem repeats of the sequence TTAGGG and associated proteins. In normal cells, analysis of chromosome terminal restriction fragments has shown that the chromosomes lose about 200 to 300 nucleotides of telomeric sequence per cell division. In contrast, all immortal cells examined to date show no loss of telomere length or sequence with cell division, suggesting that maintenance of telomeres is required for cells to escape from replicative senescence and proliferate indefinitely.Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends using a segment of its RNA component as a template. There are three molecules, including hTERTxhTR and TP1/TLP1, which compose telomerase.Human telomerase consists of a catalytic telomerase reverse transcriptase (hTERT) that synthesizes a sequence ( TTAGGG) at the ends of chromosomes by using an RNA template encoded by the telomerase RNA component gene. In human cells and tissues, the presence of telomerase activity correlates well with the level of TERT gene transcription. hTERT gene expression is limited mostly to embryonic tissues and activated lymphocytes.Previous study showed that human blastocyst - drived pluripotent cell lines are described that have normal karyotypes express high levels of telomerase activity, and express cell surface makers that characterize primate embryonic stem cell but do not characterize other early lineages, these cell lines maintained the developmental potential to form trophoblast and derivatives of all three embryonic germ layers including endoderm, mesoderm and ectoderm. Its maintained the activity of telomerase and associated with malignant tumor progression. Nephro-blastoma is a kind of malignant and embryonic tumor commonly occurred in children. It has both embryonic genetic change and body cell mutation. Previous study proved that nephroblastoma is expressed in up to 80% of telomerase activi-ty. To explore the expression of hTERT in nephroblastoma and its'clinical significance , in this study, the expression levels of hTERT were evaluated by using RT - RCR in 30 nephroblastoma tissues, 10 paracancer renal tissues. We analyzed the gene expression of thecatalytic subunit of telomerase (hTERT) in 30 nephroblastoma, differing in stage and grade, and in 10 paracancer renal tissues. There were over expression of hTERT mRNA in nephroblastoma whereas paracancer renal tissues were all negative. The positive rate in the nephroblastoma was ditinctively different from the paracancer renal tissues ( p <0.05 ) .Our data indicate that hTERT transcript levels do not correlate with clinical stage,pathology type in nephroblastoma. The reason is that a number of factors, including both embryonic genetic change and body cell mutation, such as WT1 gene abnormal expression , inactivation of suppressor gene and elimination of printing gene etc, can be invoke to explain the lack of relationship between them.All the patients were followed up range 15. 6 to 73. 9 months. The survival rate in hTERT overexpression case was significantly lower than that of the lowex-pression case ( p < 0. 05 ) . Kaplan - meier analysis shower that hTERT expression was associated with shorter disease - free survival (p =0.0073 ). Multi-variate analysis confirmed this independent prognostic value of hTERT expression. The overexpression of hTERT mRNA was significantly higher in the nephroblastoma. It might serve as a prognostic marker for estimating the biologic characteristics of the nephroblastoma.In conclusion, our findings indicate that tumor hTERT mRNA expression level correlates with outcome in patients with nephroblastoma. A larger study will be necessary to determine whether hTERT mRNA expression is predictive of outcome independent of clinical stage ,pathology type in nephroblastoma.