Tu Yingfeng, Wan Lin, Zhao Dongliang, Bu Lihong, Dong Dandan, Yin Zheyu, Cheng Zhen, Shen Baozhong
Radiology Department and Molecular Imaging Center, The Fourth Hospital of Harbin Medical University, Harbin, Heilongjiang, 150001, People's Republic of China,
Eur J Nucl Med Mol Imaging. 2014 May;41(5):972-84. doi: 10.1007/s00259-013-2596-3. Epub 2014 Feb 7.
Growing evidence suggests that microRNAs (miRNAs) play key roles in cardiac hypertrophy. To measure the expression of endogenous miRNAs is very conducive to understanding the importance of miRNAs in cardiac hypertrophy. However, current methods to monitor endogenous miRNA levels, such as Northern blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and microarrays cannot provide real-time information on miRNA biogenesis in vivo.
We constructed a miRNA reporter imaging system to monitor miR-22 expression in isoproterenol-induced cardiac hypertrophy repetitively and noninvasively. There were three copies of the antisense of miR-22 (3×PT_miR-22) cloned into the 3' untranslated region (UTR) of the Gaussia luciferase (Gluc) reporter genes under the control of the cytomegalovirus (CMV) promoter in this miRNA reporter system (CMV/Gluc/3×PT_miR-22). CMV/firefly luciferase (Fluc) was used as a positive control for imaging of miR-22 expression. Meanwhile, quantifications of miR-22 in cardiomyocyte hypertrophy and in mouse cardiac hypertrophy induced by isoproterenol stimulation were measured by qRT-PCR. Furthermore, we used this miRNA reporter imaging system to appraise the antihypertrophic effect of antagomir-22 in vitro and in vivo.
The bioluminescence signals of the CMV/Gluc/3×PT_miR-22 were gradually decreased with prolongation of isoproterenol intervention in vitro and in vivo. Overexpression of miR-22 was observed in cardiac hypertrophy, and markedly administration of antagomir-22 could reverse the upregulation of miR-22 and its prohypertrophic effects. Furthermore, knockdown of miR-22 by antagomir-22 could markedly reverse the repressed Gluc activities in vitro and in vivo. However, the Fluc activity of CMV/Fluc was not affected with isoproterenol treatment.
This study elucidates the feasibility of using our constructed miRNA reporter imaging system to monitor the location and magnitude of expression levels of miR-22 in cardiac hypertrophy in vitro and in vivo.
越来越多的证据表明，微小RNA（miRNA）在心肌肥大中起关键作用。检测内源性miRNA的表达非常有助于理解miRNA在心肌肥大中的重要性。然而，目前监测内源性miRNA水平的方法，如Northern印迹法、定量实时聚合酶链反应（qRT-PCR）和微阵列分析，无法提供体内miRNA生物合成的实时信息。
我们构建了一个miRNA报告基因成像系统，用于重复且无创地监测异丙肾上腺素诱导的心肌肥大中miR-22的表达。在这个miRNA报告基因系统（CMV/Gluc/3×PT_miR-22）中，将三个拷贝的miR-22反义序列（3×PT_miR-22）克隆到在巨细胞病毒（CMV）启动子控制下的高斯荧光素酶（Gluc）报告基因的3'非翻译区（UTR）中。CMV/萤火虫荧光素酶（Fluc）用作miR-22表达成像的阳性对照。同时，通过qRT-PCR检测异丙肾上腺素刺激诱导的心肌细胞肥大和小鼠心肌肥大中miR-22的表达量。此外，我们使用这个miRNA报告基因成像系统在体外和体内评估抗miR-22的抗肥大作用。
在体外和体内，随着异丙肾上腺素干预时间的延长，CMV/Gluc/3×PT_miR-22的生物发光信号逐渐降低。在心肌肥大中观察到miR-22的过表达，而明显给予抗miR-22可以逆转miR-22的上调及其促肥大作用。此外，抗miR-22敲低miR-22可以在体外和体内明显逆转被抑制的Gluc活性。然而，CMV/Fluc的Fluc活性不受异丙肾上腺素处理的影响。
本研究阐明了使用我们构建的miRNA报告基因成像系统在体外和体内监测心肌肥大中miR-22表达水平的位置和大小的可行性。
