Du Ting-Ting, Fu Yan-Fang, Dong Mei, Wang Lei, Fan Hong-Bo, Chen Yi, Jin Yi, Chen Sai-Juan, Chen Zhu, Deng Min, Huang Qiu-Hua, Liu Ting Xi
Shanghai Institute of Hematology, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, China.
Biochem Biophys Res Commun. 2009 Apr 17;381(4):688-93. doi: 10.1016/j.bbrc.2009.02.122. Epub 2009 Feb 28.
MicroRNAs (miRNAs) are endogenous small non-protein coding RNAs that play important regulatory roles in animals and plants by binding to target transcripts for cleavage or translational repression. Despite increasing number of genes has been predicted to be miRNA targets by bioinformatics analysis and luciferase-based reporter assay in vitro (RA-In Vitro), few of them have been experimentally validated in physiological context. Using transient reporter assay in vivo (TRA-In Vivo), we identified hydroxymethylbilane synthase b (hmbsb) and Krüppel-like transcription factor d (klfd) as potential target gene for miR-451 and miR-144, respectively. Although hmbsb, miR-451, klfd and miR-144 are all co-expressed in the developing erythroid progenitors during zebrafish erythropoiesis, only klfd can be validated as a bona fide physiological target of miR-144 using a transgene-based physiological reporter assay in vivo (PRA-In Vivo). Thus, failure to verify hmbsb as miR-451 target in physiological context raises a crucial question as to how to determine bona fide target of miRNA. The results address the importance of using multiple approaches combined with Western blot analysis to validate the physiological target of a given miRNA.
微小RNA（miRNA）是内源性的非蛋白质编码小RNA，通过与靶转录本结合以进行切割或翻译抑制，在动植物中发挥重要的调控作用。尽管通过生物信息学分析和基于荧光素酶的体外报告基因检测（RA-In Vitro）预测了越来越多的基因是miRNA的靶标，但其中很少有在生理环境中得到实验验证。通过体内瞬时报告基因检测（TRA-In Vivo），我们分别鉴定出羟甲基胆色素原合酶b（hmbsb）和类Krüppel转录因子d（klfd）作为miR-451和miR-144的潜在靶基因。尽管hmbsb、miR-451、klfd和miR-144在斑马鱼红细胞生成过程中的发育中的红系祖细胞中均共表达，但使用基于转基因的体内生理报告基因检测（PRA-In Vivo），只有klfd可以被验证为miR-144真正的生理靶标。因此，在生理环境中未能验证hmbsb是miR-451的靶标，这就提出了一个关键问题，即如何确定miRNA真正的靶标。这些结果说明了结合蛋白质印迹分析使用多种方法来验证特定miRNA生理靶标的重要性。
