Evidence for transcriptional interference in a dual-luciferase reporter system

• Published in: Scientific reports 2015-12, Vol.5 (1), p.17675-17675, Article 17675
• Main Authors: Wu, Guo-Qing, Wang, Xiao, Zhou, Hong-Ying, Chai, Ke-Qun, Xue, Qian, Zheng, Ai-Hong, Zhu, Xiu-Ming, Xiao, Jian-Yong, Ying, Xu-Hua, Wang, Fu-Wei, Rui, Tao, Xu, Li-Yun, Zhang, Yong-Kui, Liao, Yi-Ji, Xie, Dan, Lu, Li-Qin, Huang, Dong-Sheng
• Format: Article
• Language: English
• Subjects: 3' Untranslated regions; 3' Untranslated Regions - physiology; 631/337/572/2102; 631/45/500; 631/61/185; Cell Line, Tumor; Curcumin; Cytomegalovirus; Enhancer of Zeste Homolog 2 Protein; Flavopiridol; Genes, Reporter; Herpes simplex; Histone methyltransferase; Humanities and Social Sciences; Humans; Luciferases - biosynthesis; Mammals; miRNA; multidisciplinary; Polycomb Repressive Complex 2 - biosynthesis; Polycomb Repressive Complex 2 - genetics; Promoter Regions, Genetic - physiology; Promoters; Protein-tyrosine kinase; Science; Thymidine; Thymidine kinase; Transcription; Transcription, Genetic - physiology
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep17675

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3′untranslated region (3′UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3′UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.