Zeng Liang, Wang Yue-Li, Wang Fa, Cui Shi-Quan, Hu Liang, Huang Di-Nan, Hou Gan
Department of Basic Medicine, Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, Guangdong 524023, P.R. China.
Department of Clinical Biochemistry, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China.
Oncol Lett. 2017 Dec;14(6):7232-7240. doi: 10.3892/ol.2017.7127. Epub 2017 Oct 3.
By using human genomic DNA as a template to clone protection of telomere 1 (POT1) promoter gene segments and construct the POT1 promoter luciferase report gene vector (pGL3-Control-POT1-promoter), the association between POT1, and the regulation of telomerase and telomere length was investigated. In the present study, two recombinant luciferase report gene vectors were constructed, which included different regions of the POT1 promoter. The plasmids were transformed into DH5α and the positive clones were obtained. The two plasmids termed as pGL3-Control-POT1-promoter-1 and pGL3-Control-POT1-promoter-2, were confirmed using restriction enzyme analysis and sequencing. They were separately and transiently transfected into four types of human tumor cells (A549, H460, HepG2 and HeLa). The transcriptional activities of the POT1 promoter were verified using the dual-luciferase assay. The relative expression of POT1 and human telomerase reverse transcriptase (hTERT), and telomere length were analyzed using quantitative polymerase chain reaction in the four types of non-transfected tumor cells. Using SPSS software, correlations between POT1 promoter activity, and POT1 expression, hTERT expression and telomere length were analyzed. Two POT1 promoter fragments (POT1-promoter-1 and -2) were successfully constructed into the pGL3-Control luciferase report gene vector. POT1-promoter-1 exhibited significantly stronger transcription activity compared with POT1-promoter-2. The results of the partial correlation and linear regression analyses were similar: POT1 promoter activity was identified to be significantly and positively correlated with POT1 expression and telomere length (partial correlation coefficients, both P<0.05; linear regression, both P<0.01). However, POT1 promoter activity and hTERT expression were significantly negatively correlated (both P<0.05). The results obtained in the present study suggest that the POT1 promoter influences telomere length. Furthermore, these data indicated that POT1 promoter activity and POT1, as well as telomere length, may be a useful biomarker for tumor detection and future patient prognosis.
以人基因组DNA为模板克隆端粒保护蛋白1（POT1）启动子基因片段，并构建POT1启动子荧光素酶报告基因载体（pGL3-Control-POT1-promoter），研究POT1与端粒酶调控及端粒长度之间的关联。在本研究中，构建了两个包含POT1启动子不同区域的重组荧光素酶报告基因载体。将质粒转化至DH5α中，获得阳性克隆。经限制性内切酶分析和测序确认这两个质粒分别为pGL3-Control-POT1-promoter-1和pGL3-Control-POT1-promoter-2。将它们分别瞬时转染至四种人肿瘤细胞（A549、H460、HepG2和HeLa）中。采用双荧光素酶检测法验证POT1启动子的转录活性。运用定量聚合酶链反应分析四种未转染肿瘤细胞中POT1和人端粒酶逆转录酶（hTERT）的相对表达以及端粒长度。使用SPSS软件分析POT1启动子活性与POT1表达、hTERT表达及端粒长度之间的相关性。成功将两个POT1启动子片段（POT1-promoter-1和-2）构建到pGL3-Control荧光素酶报告基因载体中。与POT1-promoter-2相比，POT1-promoter-1表现出显著更强的转录活性。偏相关分析和线性回归分析结果相似：POT1启动子活性与POT1表达和端粒长度呈显著正相关（偏相关系数，均P<0.05；线性回归，均P<0.01）。然而，POT1启动子活性与hTERT表达呈显著负相关（均P<0.05）。本研究结果表明，POT1启动子影响端粒长度。此外，这些数据表明POT1启动子活性、POT1以及端粒长度可能是肿瘤检测和未来患者预后的有用生物标志物。
