Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions.
Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions.
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DOI：
10.1007/s12149-014-0901-2
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发表时间：
2014-12
影响因子：
2.6
通讯作者：
Saga, Tsuneo
中科院分区：
文献类型：
--
作者：
Takeuchi, Yasuto;Inubushi, Masayuki;Jin, Yong-Nan;Murai, Chika;Tsuji, Atsushi B.;Hata, Hironobu;Kitagawa, Yoshimasa;Saga, Tsuneo
HIF-1/HRE通路是一个很有前途的靶点，用于难治性恶性肿瘤的成像和治疗（HIF-1;缺氧诱导因子1，HRE;缺氧反应元件）。我们的研究目的是：（1）评估在各种缺氧条件下不同数量的HRE导致的基因激活水平，（2）评估多个HRE的双向活性，以及（3）确认多个HRE是否可以诱导体内基因表达。用含有萤火虫荧光素酶报告基因和不同数量（2、4、6、8、10和12）的HRE（nHRE+、nHRE-）的构建体瞬时转染人结肠癌HCT 116细胞。在不同缺氧持续时间（6、12、18和24 h）、O2浓度（1、2、4、8和16%）和不同浓度的甲磺酸去铁胺（20、40、80、160和320 µg/mL生长培养基）下测量相对荧光素酶活性。在nHRE两侧含有萤火虫和海肾荧光素酶报告基因的构建体（双-luc-nHRE）中检查HRE的双向基因激活水平。最后，为了测试含有12 HRE和NIS报告基因的构建体（12 HRE-NIS）是否可以在体内诱导基因表达，在小鼠异种移植模型中进行SPECT成像。(1)HRE的基因激活水平随HRE拷贝数的增加而增加，但在HRE拷贝数大于6或8的构建体中观察到饱和效应，（2）HRE的基因激活水平在缺氧6-12 h期间显著增加，但不超过12 h，（3）HRE的基因激活水平随O2浓度的增加而降低，但即使在16%O2的轻度缺氧下也可以检测到，（4）证实了HRE的双向成比例活性，而不管缺氧的严重程度如何，和（5）在体内SPECT成像上可以观察到由响应缺氧的HRE的12个串联拷贝驱动的NIS表达。本研究的结果将有助于了解和评估缺氧条件下多个HRE的活性，并成为未来缺氧靶向成像和治疗的基础。
HIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo. Human colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE−). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model. (1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6–12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging. The results of this study will help in the understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.
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影响因子：
5.1
作者：
Greco, O;Joiner, MC;Scott, SD
通讯作者：
Scott, SD
影响因子：
11.2
作者：
Li, Xiao-Feng;Carlin, Sean;O'Donoghue, Joseph A.
通讯作者：
O'Donoghue, Joseph A.
影响因子：
9.3
作者：
Hsieh, Chia-Hung;Kuo, Jung-Wen;Liu, Ren-Shyan
通讯作者：
Liu, Ren-Shyan
单光子发射计算机断层扫描显示稳定表达人钠/碘同向转运蛋白报告基因的原位植入人结肠癌细胞系的自发性肝转移。
影响因子：
3.2
作者：
Inubushi M;Jin YN;Murai C;Hata H;Kitagawa Y;Saga T
通讯作者：
Saga T
DOI：
10.1007/s00259-007-0438-x
发表时间：
2007-06-01
影响因子：
9.1
作者：
Inubushi, Masayuki;Tamaki, Nagara
通讯作者：
Tamaki, Nagara