Real-time RT-PCR quantification of human telomerase reverse transcriptase splice variants in tumor cell lines and non-small cell lung cancer
Real-time RT-PCR quantification of human telomerase reverse transcriptase splice variants in tumor cell lines and non-small cell lung cancer
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DOI：
10.1373/clinchem.2006.073015
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发表时间：
2007-01-01
影响因子：
9.3
通讯作者：
Lianidoui, Evi S.
中科院分区：
文献类型：
--
作者：
Mavrogiannou, Eleni;Strati, Areti;Lianidoui, Evi S.
背景：我们开发并验证了一种实时逆转录 (RT)-PCR，用于对肿瘤细胞系和非小细胞肺癌 (NSCLC) 中 4 种单独的人端粒酶逆转录酶 (TERT) 剪接变体（α + β +、α - β +、α + β -、α - β -）进行定量。方法：我们使用计算机设计的引物和通用引物。 TaqMan 探针用于高度特异性扩增每个 TERT 剪接变体，PCR 转录物特异性 DNA 外部标准品作为校准物，以及用于方法开发和验证的 MCF-7 细胞系。然后，我们对 6 个肿瘤细胞系中的 TERT 剪接变异体以及来自 28 名 NSCLC 患者的癌性和配对非癌性组织样本中的端粒酶活性和 TERT 剪接变异体表达进行了定量。 结果：在大多数肿瘤细胞系中，我们观察到 TERT 剪接变异体比例几乎没有变化。 α+β-剪接变体表现出最高的表达，而α-β+和α-β-表现出最低的表达。对 NSCLC 和周围非肿瘤组织中 4 个 TERT 剪接变体的定量显示，在 NSCLC 和邻近非肿瘤组织样本中，α+β- 变体的表达百分比最高，其次是 α+β+，其中 α-β+ 和 α-β- 剪接变体的表达量最低。在NSCLC肿瘤中，α+β+变异体的表达量高于其他剪接变异体，其表达与端粒酶活性、总生存期和无病生存期相关。结论：实时RT-PCR定量是一种特异、灵敏、快速的方法，可以阐明TERT剪接变异体在肿瘤发生和进展中的生物学作用。我们的结果表明，TERT α+β+ 剪接变体的表达可能是 NSCLC 患者的独立负面预后因素。 (c) 2007 年美国临床化学协会
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 (alpha + beta +, alpha - beta +, alpha + beta -, alpha - beta -) 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 alpha+beta- splice variant showed the highest expression and alpha-beta+ and alpha-beta- the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the alpha+beta- variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by alpha+beta+, with the alpha-beta+ and alpha-beta- splice variants having the lowest expression. In the NSCLC tumors, the alpha+beta+ 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 alpha+beta+ splice variant may be an independent negative prognostic factor for NSCLC patients. (c) 2007 American Association for Clinical Chemistry