Zheng Haifeng, Zhou Lin, Shi Yaru, Tian Jie, Wang Fu
Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi'an, Shaanxi 710071, China.
Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi'an, Shaanxi 710071, China; CAS Key Laboratory of Molecular Imaging, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China.
Mol Ther Nucleic Acids. 2018 Jun 1;11:34-40. doi: 10.1016/j.omtn.2018.01.008. Epub 2018 Feb 13.
MicroRNAs (miRNAs) are a class of negative regulators of gene expression and play critical roles in various biological processes. Conventional approaches for detecting miRNAs, such as northern blotting, microarray, and real-time PCR, usually require the lysis of cell samples and could not provide the in vivo information about miRNAs in living organisms. Here, we designed a tyrosinase (TYR)-based reporter to monitor miR-9 expression that is regulated by DNA methylation in living cells and animals. During DNA methylation of A549 cells treated by 5-aza-2'-deoxycytidine (5-Aza-dC), the CMV/TYR-3xTS reporter-transfected cells demonstrated a gradual decrease in melanin content, TYR activity, and photoacoustic signal because of the gradual activation of miR-9 expression. The miR-9-regulated repression of TYR activity also resulted in a significant decrease in photoacoustic signal from the flank of mice with 5-Aza-dC treatment, whereas the bioluminescence signal from internal control had no obvious change. The TYR-based miRNA reporter may serve as a new imaging probe for monitoring the dynamic expression of miRNAs during various cellular or disease progression in cells and living animals.
微小RNA（miRNA）是一类基因表达的负调控因子，在各种生物学过程中发挥关键作用。检测miRNA的传统方法，如Northern印迹、微阵列和实时PCR，通常需要裂解细胞样本，无法提供活生物体中miRNA的体内信息。在此，我们设计了一种基于酪氨酸酶（TYR）的报告基因，用于监测活细胞和动物中受DNA甲基化调控的miR-9表达。在用5-氮杂-2'-脱氧胞苷（5-Aza-dC）处理A549细胞的DNA甲基化过程中，转染了CMV/TYR-3xTS报告基因的细胞由于miR-9表达的逐渐激活，黑色素含量、TYR活性和光声信号逐渐降低。miR-9对TYR活性的调控抑制也导致5-Aza-dC处理的小鼠胁腹光声信号显著降低，而来自内部对照的生物发光信号没有明显变化。基于TYR的miRNA报告基因可作为一种新的成像探针，用于监测细胞和活体动物在各种细胞或疾病进展过程中miRNA的动态表达。
