Quantification of human telomerase RNA(hTR)and human telomerase reverse transcriptase(hTERT)mRNA in testicular tissue of infertile patients

Aim:To evaluate the quantitative detection of human telomerase RNA(hTR)and human telomerase reverse tran-scriptase(hTERT)mRNA as diagnostic parameters in the workup of testicular tissue specimens from patients presentingwith non-obstructive azoospermia.Methods:hTR and hTERT mRNA expression were quantified in 38 cryopre-served testicular tissue specimens by fluorescence real-time reverse transcription-polymerase chain reaction(RT-PCR)in a LightCycler(r).This was paralleled by conventional histological workup in all tissue specimens and additionalsemithin sectioning preparation in cases with maturation arrest(n=12)and Sertoli-cell-only syndrome(n=12).Re-sults;The average normalized hTERT expression(N_(hTERT))was 131.9±48.0 copies(mean±SD)in tissue speci-mens with full spermatogenesis,N_(hTERT)=51.2±17.2 copies in those with maturation arrest and N_(hTERT)=2.7±2.4copies in those with Sertoli-cell-only syndrome(SCOS).The discriminant analysis showed that detection of N_(hTERT)(N_(hTR))had a predictive value of 86.8%(55.3%)for correct classification in one of the three histological subgroups.Conclusion;Our results demonstrate that quantitative detection of hTERT mRNA expression in testicular tissue en-ables a molecular-diagnostic classification of gametogenesis.Quantitative detection of hTERT in testicular biopsies isthus well suited for supplementing the histopathological evaluation.

Gain of human telomerase RNA gene is associated with progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ

Background The 3q26 chromosome region, where the human telomerase RNA gene （hTERC） is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade Ⅰ or Ⅱ （CIN-Ⅰ and -Ⅱ, respectively） to CIN-Ⅲ and cervical cancer. Methods Liquid-based cytological samples from 54 patients with CIN-Ⅰ or CIN-Ⅱ lesions were enrolled in this study. Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-Ⅰ or CIN-Ⅱ. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe （labeled red） and the control, the chromosome 3 centromere-specific probe （labeled green）.Results All patients whose lesions progressed from CIN-Ⅰ or CIN-Ⅱ to CIN-Ⅲ displayed a gain of the hTERC gene, whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-Ⅲ or cervical cancer. The signal ratio pattern per cell was recorded as N：N （green： red）. The numbers of cells with the signal ratio pattern of 4：4 or N：≥5 in patients whose lesions progressed to CIN-Ⅲ were significantly higher than those whose lesions did not progress. Significantly, none of the patients with a 4：4 signal ratio pattern regressed spontaneously.Conclusions In conclusion, measurement of hTERC gene gain in CIN-Ⅰ or CIN-Ⅱ patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions. In addition, a 4：4 or N：≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-Ⅰ or CIN-Ⅱ lesions.

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA （siRNA） has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA （hTR） can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells （HRCCs）.METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined.RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed.CONCLUSION siRNA agains： the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.

Expression of Telomerase Subunits in Gastric Cancer

To detect the expression of telomerase subunits （human telomerase reverse transcriptase, human telomerase associated protein 1 and human telomerase RNA） in gastric cancer and to examine the role that different telomerase subunits play in the gastric carcinogenesis, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect telomerase suhunits messenger RNA in 24 samples of gastric cancer and corresponding non-cancerous tissue. The results showed that the positive rate of hTERT mRNA from gastric cancer and corresponding non-cancerous tissues was 100% and 25 %, respectively. The former was significantly higher than the latter （X^2 = 26.4, P〈0.01）. The positive rate of hTEP1 mRNA from gastric cancer and corresponding non-cancerous tissues was 100 % and 91.7%, respectively and no significant difference was found between them （X^2 =2.1, P〉0.05）. The positive rates of hTR for gastric cancer and corresponding non-cancerous tissues were both 100 % and no significant difference existed between them. It is concluded that in contrast to hTEP1 and hTR, the up-regulation of hTERT mRNA expression may play a more important role in the development of gastric cancer.