Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements.
Robust cancer-specific gene expression by a novel cassette with hTERT and CMV promoter elements.
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DOI：
10.3892/or.2017.5710
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发表时间：
2017-08
期刊：
影响因子：
4.2
通讯作者：
M. Sakaguchi;T. Sadahira;H. Ueki;R. Kinoshita;H. Murata;Ken‐ichi Yamamoto;J. Futami;Y. Nasu;K. Ochiai;H. Kumon;N. Huh;Masami Watanabe
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作者：
M. Sakaguchi;T. Sadahira;H. Ueki;R. Kinoshita;H. Murata;Ken‐ichi Yamamoto;J. Futami;Y. Nasu;K. Ochiai;H. Kumon;N. Huh;Masami Watanabe
我们开发并验证了一种新的hTERT/CMV启动子元件驱动的基因表达盒，可以稳健地增强癌症特异性基因表达。以下基因表达元件串联位于质粒构建体内：[hTERT核心启动子、巨细胞病毒（CMV）最小化启动子、RU 5 '序列、插入基因、BGH polyA、hTERT增强子];这在下文中称为hT/Cm-R-hT构建体。使用各种人癌细胞系和正常细胞，通过蛋白质印迹和荧光显微镜检查绿色荧光蛋白（GFP）基因的癌症特异性转录。与其他对照hT/Cm驱动的构建体相比，在hT/Cm-R-hT质粒中稳健地实现了癌症特异性基因表达。值得注意的是，在HEK 293细胞中，在hT/Cm-R-hT驱动的构建体中观察到的GFP表达水平上级于具有常规CMV启动子的对照质粒，已知HEK 293细胞具有比正常细胞更高的hTERT活性。我们接下来检查了hT/Cm-R-hT在检测来自人外周血单核细胞（PBMC）的表达靶GFP的癌细胞中的可用性。hT/Cm-R-hT质粒成功地诱导了癌症特异性基因表达;在靶HeLa癌细胞中观察到GFP的稳健表达，而GFP在正常PBMC中没有明显表达。该质粒允许在含有高达10000倍以上的PBMC的混合细胞培养物中选择性地观察活的HeLa癌细胞。这些发现表明hT/Cm-R-hT表达系统是检测与正常细胞混合的活癌细胞的有价值的工具。因此，本系统可应用于体内血液和其他类型体液中散布的癌细胞的体外检测。由于目前的系统也可以应用于其他类型的载体，包括病毒载体，这种方法使用的hTERT启动子为基础的构建体有望成为一个有价值的工具，用于增强癌症特异性基因表达。
We developed and validated a novel hTERT/CMV promoter element-driven gene expression cassette that can robustly enhance cancer-specific gene expression. The following gene expressional elements were located in tandem within the plasmid construct: [hTERT core promoter, cytomegalovirus (CMV) minimized promoter, RU5' sequence, an inserted gene, BGH polyA, hTERT enhancer]; this is hereafter referred to as the hT/Cm-R-hT construct. Using various human cancer cell lines and normal cells, the cancer-specific transcription of the green fluorescent protein (GFP) gene was examined by western blotting and fluorescence microscopy. Cancer-specific gene expression was robustly achieved in the hT/Cm-R-hT plasmid in comparison to the other control hT/Cm-driven construct. Notably, the expression level of GFP observed in the hT/Cm-R-hT-driven construct was superior to that of the control plasmid with the conventional CMV promoter in HEK293 cells, which are known to possess higher hTERT activity than normal cells. We next examined the availability of hT/Cm-R-hT in detecting the target GFP expressing cancer cells from human peripheral blood mononuclear cells (PBMCs). The hT/Cm-R-hT plasmid successfully induced cancer-specific gene expression; the robust expression of GFP was observed in target HeLa cancer cells, whereas GFP was not visibly expressed in normal PBMCs. The plasmid allowed for the selective visualization of viable HeLa cancer cells in mixed cell cultures containing up to 10000-fold more PBMCs. These findings indicate that the hT/Cm-R-hT expressional system is a valuable tool for detecting viable cancer cells mixed with normal cells. The current system can therefore be applied to the in vitro detection of cancer cells that are disseminated in the blood and other types of body fluid in vivo. Since the current system can also be applied to other types of vectors, including virus vectors, this approach using the hTERT promoter-based construct is expected to become a valuable tool for enhancing cancer-specific gene expression.