Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

摘要

Background: We developed and validated a real-time reverse transcription (RT)–PCR for the quantification of 4 individual human telomerase reverse transcriptase ( TERT ) splice variants (α+β+, α?β+, α+β?, α?β?) in tumor cell lines and non–small cell lung cancer (NSCLC).Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript–specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients.Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The α+β? splice variant showed the highest expression and α?β+ and α?β? the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the α+β? variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by α+β+, with the α?β+ and α?β? splice variants having the lowest expression. In the NSCLC tumors, the α+β+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival.Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT α+β+ splice variant may be an independent negative prognostic factor for NSCLC patients.